GARD – Genomic Allergen Rapid Detection. A Testing Strategy for Assessment of Chemical Sensitizers

Doctoral thesis defense date 2013-06-05 09:00     ISBN978-91-7473-519-2 Johansson, Henrik LU Abstract (Swedish) Popular Abstract in Swedish Vi lever i en tid och i ett samhälle där kemikalier har blivit en naturlig del av vår industrialiserade vardag. Till stor del ligger kemikalieindustrin bakom flera av de stora tekniska framsteg som gör att västvärlden har […]

Doctoral thesis
defense date 2013-06-05 09:00     ISBN978-91-7473-519-2

Johansson, Henrik LU

Abstract (Swedish)

Popular Abstract in Swedish

Vi lever i en tid och i ett samhälle där kemikalier har blivit en naturlig del av vår industrialiserade vardag. Till stor del ligger kemikalieindustrin bakom flera av de stora tekniska framsteg som gör att västvärlden har den levnadsstandard den idag har. Tyvärr har den stora spridningen av icke naturligt förekommande kemikalier ibland också negativa effekter både på miljö och hälsa. Ett stort antal tungmetaller har visat sig ackumuleras i naturliga näringskedjor, för att slutligen lagras i mänsklig vävnad, med ännu ej helt fastställda konsekvenser. Flera kemiska bekämpningsmedel slår hårt mot naturliga ekosystem, men har också visat sig påverka människors hormonella balans, vilket bland annat… (More)

Abstract

The modern world is increasingly dependent of the use of chemicals. The chemical industries have greatly contributed to the high standard of living in industrialised societies, and chemical compounds surround us in everyday life. Unfortunately, a vast number of chemicals cause adverse effects on the environment and human health. One such concern is chemical hypersensitivity, which is a state caused by the human immune system. Upon exposure of certain chemical substances, the body will in some instances mount immunologic responses, giving rise to clinical symptoms such as irritation and damage on skin and impaired function of the respiratory tract.

In order to limit the usage of chemical compounds that induce…more 

The GARD assay for assessment of chemical skin sensitizers.

Toxicol In Vitro. 2013 Apr;27(3):1163-9. doi: 10.1016/j.tiv.2012.05.019. Epub 2012 Sep 29. Johansson H., Albrekt A.S., Borrebaeck C.A.K., Lindstedt M Abstract Allergic contact dermatitis is a skin disease caused by an immunologic reaction to low molecular weight compounds, so called haptens. These substances are commonly present in products used by humans in daily life, such as […]

Toxicol In Vitro. 2013 Apr;27(3):1163-9. doi: 10.1016/j.tiv.2012.05.019. Epub 2012 Sep 29.

Johansson H., Albrekt A.S., Borrebaeck C.A.K., Lindstedt M

Abstract

Allergic contact dermatitis is a skin disease caused by an immunologic reaction to low molecular weight compounds, so called haptens. These substances are commonly present in products used by humans in daily life, such as in cosmetics and fragrances, as well as within chemical industry and in pharmaceuticals. The frequent usage of these compounds in different applications has led to increasing incidences of allergic contact dermatitis, which has become a substantial economic burden for society. As a consequence, chemicals are routinely tested for their ability to induce skin sensitization, using animal models such as the murine Local Lymph Node Assay. However, recent legislations regulate the use of animal models within chemical testing. Thus, there is an urgent need for in vitro alternatives to replace these assays for safety assessment of chemicals. Recently, we identified a signature of predictive genes, which are differentially regulated in the human myeloid cell-line MUTZ-3 when stimulated with sensitizing compounds compared to non-sensitizing compounds. Based on these findings, we have formulated a test strategy for assessment of sensitizing compounds, called Genomic Allergen Rapid Detection, GARD. In this paper, we present a detailed method description of how the assay should be performed.

Pattern rules: biomarker signatures for sensitization as an alternative to animal testing

Biomarkers in Medicine, December 2011 ,Vol. 5, No. 6 , Pages 809-811 (doi: 10.2217/bmm.11.82) Lindstedt M., Borrebaeck C.A.K. “Identification of relevant endpoints and biomarkers is essensial for development of alternative assays predicting sensitisation. Omics-based techniques have recently generated very promising biomarker patterns, as well as pathways, which will be used in the identification of sensitising agents” […]
Biomarkers in Medicine, December 2011 ,Vol. 5, No. 6 , Pages 809-811
(doi: 10.2217/bmm.11.82)
Lindstedt M., Borrebaeck C.A.K.
“Identification of relevant endpoints and biomarkers is essensial for development of alternative assays predicting sensitisation. Omics-based techniques have recently generated very promising biomarker patterns, as well as pathways, which will be used in the identification of sensitising agents”

A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests.

BMC Genomics. 2011 Aug 8;12:399. doi: 10.1186/1471-2164-12-399. Johansson H., Albrekt A.S., Lindstedt M., Borrebaeck C.A.K Abstract BACKGROUND: Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current […]

BMC Genomics. 2011 Aug 8;12:399. doi: 10.1186/1471-2164-12-399.

Johansson H., Albrekt A.S., Lindstedt M., Borrebaeck C.A.K

Abstract

BACKGROUND:

Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power.

RESULTS:

We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization.

CONCLUSIONS:

A gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests.

Article on line

Functional and transcriptional profiling of MUTZ-3, a myeloid cell line acting as a model for dendritic cells

Immunology. 2006 Feb; 117(2): 156–166. doi:  10.1111/j.1365-2567.2005.02274.x Larsson K., Lindstedt M., Borrebaeck C.A.K. ABSTRACT The incidence of allergy is steadily increasing, but the molecular mechanisms involved in the allergic immune response are still not fully understood. In particular, further investigations focusing on dendritic cells, which are central in orchestrating the immune response, are needed. The […]
Immunology. 2006 Feb; 117(2): 156–166.

Larsson K., Lindstedt M., Borrebaeck C.A.K.

ABSTRACT

The incidence of allergy is steadily increasing, but the molecular mechanisms involved in the allergic immune response are still not fully understood. In particular, further investigations focusing on dendritic cells, which are central in orchestrating the immune response, are needed. The objective of this study was to investigate the ability of myeloid leukaemia-derived cell lines, such as KG-1, THP-1 and MUTZ-3, to serve as in vitro models for dendritic cells. The ability of these cell lines to mature into functional dendritic cells, expressing costimulatory molecules, was assessed by functional and transcriptional profiling and compared with that of monocyte-derived dendritic cells, which are now used as a standard source of dendritic cells. High-density microarray analysis was utilized to study the transcriptional activity and kinetics of activation of the differentiated MUTZ-3 cell line, in response to a cocktail of inflammatory cytokines. The data obtained clearly demonstrate that MUTZ-3 cells have the ability to induce antigen-independent proliferation in CD4+CD45RA+ T cells, whereas KG-1 and THP-1 only induced a marginal response. Furthermore, MUTZ-3 displayed the phenotypic and transcriptional profiles of immature dendritic cells, after differentiation with granulocyte–macrophage colony-stimulating factor and interleukin-4. Upon activation with inflammatory cytokines, MUTZ-3 matured phenotypically and exhibited a gene induction similar to that of monocyte-derived dendritic cells. This delineation of the cellular and transcriptional activity of MUTZ-3, in response to maturational stimuli, demonstrates the significance of this cell line as a model for functional studies of inflammatory responses.

Keywords: dendritic cells, myeloid cell line, high-density microarray, inflammation, allergy