Genomic allergen rapid detection in-house validation-a proof of concept.

Toxicol Sci. 2014 Jun;139(2):362-70. doi: 10.1093/toxsci/kfu046. Epub 2014 Mar 27. Johansson H., Rydnert F., Kuehnl J., Schepky A., Borrebaeck C.A.K., Lindstedt M. Abstract Chemical sensitization is an adverse immunologic response to chemical substances, inducing hypersensitivity in exposed individuals. Identifying chemical sensitizers is of great importance for chemical, pharmaceutical, and cosmetic industries, in order to prevent […]

Toxicol Sci. 2014 Jun;139(2):362-70. doi: 10.1093/toxsci/kfu046. Epub 2014 Mar 27.

Johansson H., Rydnert F., Kuehnl J., Schepky A., Borrebaeck C.A.K., Lindstedt M.

Abstract

Chemical sensitization is an adverse immunologic response to chemical substances, inducing hypersensitivity in exposed individuals. Identifying chemical sensitizers is of great importance for chemical, pharmaceutical, and cosmetic industries, in order to prevent the use of sensitizers in consumer products. Historically, chemical sensitizers have been assessed mainly by in vivo methods, however, recently enforced European legislations urge and promote the development of animal-free test methods able to predict chemical sensitizers. Recently, we presented a predictive biomarker signature in the myeloid cell line MUTZ-3, for assessment of skinsensitizers. The identified genomic biomarkers were found to be involved in immunologically relevant pathways, induced by recognition of foreign substances and regulating dendritic cell maturation and cytoprotective mechanisms. We have developed the usage of this biomarker signature into a novel in vitro assay for assessment of chemical sensitizers, called Genomic Allergen Rapid Detection (GARD). The assay is based on chemical stimulation of MUTZ-3 cultures, using the compounds to be assayed as stimulatory agents. The readout of the assay is a transcriptional quantification of the genomic predictors, collectively termed the GARD Prediction Signature (GPS), using a complete genome expression array. Compounds are predicted as either sensitizers or nonsensitizers by a Support Vector Machine model. In this report, we provide a proof of concept for the functionality of the GARD assay by describing the classification of 26 blinded and 11 nonblinded chemicals as sensitizers or nonsensitizers. Based on these classifications, the accuracy, sensitivity, and specificity of the assay were estimated to 89, 89, and 88%, respectively.

KEYWORDS:

GARD; allergic contact dermatitis; chemical sensitizers; in vitro assay; predictive assay; skin sensitization

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Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles

Immunology. 2014 Jun;142(2):279-88. doi: 10.1111/imm.12252. Lundberg K., Rydnert F, Greiff L, Lindstedt M. Abstract Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human […]

Immunology. 2014 Jun;142(2):279-88. doi: 10.1111/imm.12252.

Lundberg K., Rydnert F, Greiff L, Lindstedt M.

Abstract

Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c(+) , CD141(+) and CD16(+) myeloid DCs and CD123(+) plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141(+) DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization.

KEYWORDS:

C-type lectin receptors; Toll-like receptors; human dendritic cell subsets; pattern recognition receptors

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Evaluation of high throughput gene expression platforms using a genomic biomarker signature for prediction of skin sensitization.

BMC Genomics. 2014 May 16;15:379. doi: 10.1186/1471-2164-15-379. Forreryd A., Johansson H., Albrekt A.S., Lindstedt M Abstract BACKGROUND: Allergic contact dermatitis (ACD) develops upon exposure to certain chemical compounds termed skin sensitizers. To reduce the occurrence of skin sensitizers, chemicals are regularly screened for their capacity to induce sensitization. The recently developed Genomic Allergen Rapid Detection […]

BMC Genomics. 2014 May 16;15:379. doi: 10.1186/1471-2164-15-379.

Forreryd A., Johansson H., Albrekt A.S., Lindstedt M

Abstract

BACKGROUND:

Allergic contact dermatitis (ACD) develops upon exposure to certain chemical compounds termed skin sensitizers. To reduce the occurrence of skin sensitizers, chemicals are regularly screened for their capacity to induce sensitization. The recently developed Genomic Allergen Rapid Detection (GARD) assay is an in vitro alternative to animal testing for identification of skinsensitizers, classifying chemicals by evaluating transcriptional levels of a genomic biomarker signature. During assay development and biomarker identification, genome-wide expression analysis was applied using microarrays covering approximately 30,000 transcripts. However, the microarray platform suffers from drawbacks in terms of low sample throughput, high cost per sample and time consuming protocols and is a limiting factor for adaption of GARD into a routine assay for screening of potential sensitizers. With the purpose to simplify assay procedures, improve technical parameters and increase sample throughput, we assessed the performance of three high throughput gene expression platforms–nCounter®, BioMark HD™ and OpenArray®–and correlated their performance metrics against our previously generated microarray data. We measured the levels of 30 transcripts from the GARD biomarker signature across 48 samples. Detection sensitivity, reproducibility, correlations and overall structure of gene expression measurements were compared across platforms.

RESULTS:

Gene expression data from all of the evaluated platforms could be used to classify most of the sensitizers from non-sensitizers in the GARD assay. Results also showed high data quality and acceptable reproducibility for all platforms but only medium to poor correlations of expression measurements across platforms. In addition, evaluated platforms were superior to the microarray platform in terms of cost efficiency, simplicity of protocols and sample throughput.

CONCLUSIONS:

We evaluated the performance of three non-array based platforms using a limited set of transcripts from the GARD biomarker signature. We demonstrated that it was possible to achieve acceptable discriminatory power in terms of separation between sensitizers and non-sensitizers in the GARD assay while reducing assay costs, simplify assay procedures and increase sample throughput by using an alternative platform, providing a first step towards the goal to prepare GARD for formal validation and adaption of the assay for industrial screening of potential sensitizers.

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Prediction of skin sensitizers using alternative methods to animal experimentation.

Basic Clin Pharmacol Toxicol. 2014 Jul;115(1):110-7. doi: 10.1111/bcpt.12199. Epub 2014 Feb 18. Johansson H., Lindstedt M. Abstract Regulatory frameworks within the European Union demand that chemical substances are investigated for their ability to induce sensitization, an adverse health effect caused by the human immune system in response to chemical exposure. A recent ban on the […]

Basic Clin Pharmacol Toxicol. 2014 Jul;115(1):110-7. doi: 10.1111/bcpt.12199. Epub 2014 Feb 18.

Johansson H., Lindstedt M.

Abstract

Regulatory frameworks within the European Union demand that chemical substances are investigated for their ability to induce sensitization, an adverse health effect caused by the human immune system in response to chemical exposure. A recent ban on the use of animal tests within the cosmetics industry has led to an urgent need for alternative animal-free test methods that can be used for assessment of chemical sensitizers. To date, no such alternative assay has yet completed formal validation. However, a number of assays are in development and the understanding of the biological mechanisms of chemical sensitization has greatly increased during the last decade. In this MiniReview, we aim to summarize and give our view on the recent progress of method development for alternative assessment of chemical sensitizers. We propose that integrated testing strategies should comprise complementary assays, providing measurements of a wide range of mechanistic events, to perform well-educated risk assessments based on weight of evidence.

Skin sensitizers differentially regulate signaling pathways in MUTZ-3 cells in relation to their individual potency.

BMC Pharmacol Toxicol. 2014 Feb 11;15:5. doi: 10.1186/2050-6511-15-5. Albrekt A.S., Johansson H., Borrebaeck C.A.K., Lindstedt M. Abstract BACKGROUND: Due to the recent European legislations posing a ban of animal tests for safety assessment within the cosmetic industry, development of in vitro alternatives for assessment of skin sensitization is highly prioritized. To date, proposed in vitro […]

BMC Pharmacol Toxicol. 2014 Feb 11;15:5. doi: 10.1186/2050-6511-15-5.

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

Abstract

BACKGROUND:

Due to the recent European legislations posing a ban of animal tests for safety assessment within the cosmetic industry, development of in vitro alternatives for assessment of skin sensitization is highly prioritized. To date, proposed in vitro assays are mainly based on single biomarkers, which so far have not been able to classify and stratify chemicals into subgroups, related to risk or potency.

METHODS:

Recently, we presented the Genomic Allergen Rapid Detection (GARD) assay for assessment of chemical sensitizers. In this paper, we show how the genome wide readout of GARD can be expanded and used to identify differentially regulated pathways relating to individual chemical sensitizers. In this study, we investigated the mechanisms of action of a range of skin sensitizers through pathway identification, pathway classification and transcription factor analysis and related this to the reactive mechanisms and potency of the sensitizing agents.

RESULTS:

By transcriptional profiling of chemically stimulated MUTZ-3 cells, 33 canonical pathways intimately involved in sensitization to chemical substances were identified. The results showed that metabolic processes, cell cycling and oxidative stress responses are the key events activated during skin sensitization, and that these functions are engaged differently depending on the reactivity mechanisms of the sensitizing agent. Furthermore, the results indicate that the chemical reactivity groups seem to gradually engage more pathways and more molecules in each pathway with increasing sensitizing potency of the chemical used for stimulation. Also, a switch in gene regulation from up to down regulation, with increasing potency, was seen both in genes involved in metabolic functions and cell cycling. These observed pathway patterns were clearly reflected in the regulatory elements identified to drive these processes, where 33 regulatory elements have been proposed for further analysis.

CONCLUSIONS:

This study demonstrates that functional analysis of biomarkers identified from our genomics study of human MUTZ-3 cells can be used to assess sensitizing potency of chemicals in vitro, by the identification of key cellular events, such as metabolic and cell cycling pathways.

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.

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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