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The crème de la crème of contact allergy tests goes in vitro

The cosmetics industry is desperately looking for ways to test products without the need for animal testing. As of 2013, all cosmetic products containing a chemical with inherent skin sensitisation potential must be tested without the traditional animal testing. Prof. Dr. Stefan Martin from the Department of Dermatology at the University of Freiburg Medical Centre and his partners in the EU-funded “Sens-it-iv” research project have over the past five years been looking for alternatives to animal testing and have come up with the most specific in vitro test there has ever been. The researchers involved in the project have also been awarded a prize for their achievement by the Baden-Württemberg government. What can T cells tell us?

Eczemas can result from contact allergies. © Prof. Dr. Stefan Martin

Chemicals in household detergents, jewellery containing nickel, volatile substances in perfumes or skin lotions are just some of the around four thousand substances that are known to cause allergic contact dermatitis. Typical symptoms include rashes, irritation or weeping skin lesions. If the allergy becomes chronic, the people who are affected need to avoid any contact with the allergen, which in many cases means giving up their job. Allergens have an increasing impact on occupational health, as people tend to develop allerigc contact dermatitis due to regular exposure to chemicals at their workplace.

Academic researchers and industry are working hard to identify contact allergens and reduce their presence in the environment in order to avoid such problems. “To date, the identification and evaluation of unknown sensitisers relies on animal testing as no validated alternatives exist,” said Prof. Dr. Stefan Martin from the Allergy Research Group in the Department of Dermatology at the Freiburg University Medical Centre. “However, in 2009, the EU banned the sale of cosmetics and toiletries containing ingredients tested on animals,” Martin added.

The search has begun

The 2009 ban caused major anxiety in the cosmetics industry: how will it be possible to test new perfumes and shampoos for the presence of allergenic substances and obtain market authorisation for a new drug without using animals? “Tests involving cell cultures are excellent alternatives to animal testing,” Martin said. “However, the development of such tests had long been put on the backburner. I believe that this was partially due to the fact that we have only found out more about the development of contact allergies in the last four to five years. Another reason might be that there was also no compelling need to develop alternatives to animal tests and that financial support for research into alternatives was rather scarce.”

This said, in vitro tests are not only required for ethical reasons. They are also cheaper than experiments involving rats and mice. Since 2005, researchers from industry and basic research have intensified their efforts to identify contact allergens in culture dishes: in 2005, the EU initiated the project “Sens-it-iv”, in which around 30 European research teams joined forces with the cosmetics industry between 2005 and 2011 with the aim of developing non-animal tests to assess the allergenic potential of chemicals and develop new tests.

Dendritic cells from the skin (green-yellow) have invaded the T-cell region of the lymph node where they can activate T cells (red). © Prof. Dr. Stefan Martin

Numerous approaches are being pursued. The molecular and cellular processes that lead to contact allergies are well known; Prof. Martin’s research group played a decisive role in these investigations. It is now known that contact allergens such as nickel ions or other low-molecular substances that penetrate the skin interact with endogenous proteins located on the surface of cells, for example. This step is crucial, as it causes many cell types to trigger specific molecular responses.

For example, Martin and his team have been able to show that one of the major responses of the body is related to the maturation of the dendritic cells of the immune system. Upon contact with an allergen, dendritic cells enter local lymph nodes where they “activate” immature T cells. “This is the crucial step in the development of contact allergies,” said Martin. “The activated T cells produce receptor proteins on their surface that are specific for individual contact allergens.”  

The most specific test ever

These receptor proteins function like probes. The activated T cells migrate back into the skin where they can be stimulated by the respective contact allergens and trigger an immune system response. A T cell subpopulation, so-called memory T cells, remains in the skin tissue and can recognise foreign invaders during a second infection. These “experienced” T cells secrete inflammatory mediators such as interferon gamma and TNF alpha that attract even more cells than during the first encounter of the immune system with an allergen; the immune system reacts more rapidly and more aggressively to further contact with the specific allergen, even if it is only present in tiny quantities.

The “Sens-it-iv” researchers have investigated numerous steps in the development of contact allergies in greater detail. They have developed a T-cell-based assay that imitates the complex series of events that occur in vivo. If an unknown substance leads to a reaction, then this particular substance might be a contact allergen. However, many of these intermediary steps also occur when substances that are not true contact allergens enter the tissue. For example, dendritic cells might also be stimulated by irritants, i.e. molecules that only trigger an acute skin reaction without leading to a true allergic reaction. Viruses and bacteria can trigger similar reactions.

The primary activation of T cells by allergen-presenting dendritic cells is the crucial step in developing a contact allergy. © Prof. Dr. Stefan Martin

“None of the in vitro tests is 100 % specific for contact allergens. This is due to the complex immunological mechanisms involved in sensitisation,” Prof. Martin said. However, the innovative in vitro T cell test, developed by Prof. Martin, his co-worker Dr. Philipp Esser and their cooperation partners Dr. Lisa Dietz and PD Dr. Hermann-Josef Thierse from the Mannheim University Hospital and other “Sens-it-iv” project partners, imitates the most specific step in the immune system’s response to contact allergens in vitro. The researchers have chosen a process for the assay that plays a decisive role as to whether a contact allergy develops or not: the activation of specific T cells in the lymph nodes by dendritic cells.

The test is based on naïve T cells isolated from the blood of donors and on dendritic cells produced from blood cells from the same donor. The researchers cultivate these two cell types in a cell culture dish and bring them into contact with the substance suspected of having a sensitising potential. Does the sensitiser lead to the maturation of dendritic cells and the activation of T cells? Do the activated T cells secrete inflammatory mediators such as interferon gamma and TNF alpha upon the second encounter with the sensitiser? Do interferon gamma and TNF alpha alarm the immune system? Do these events only happen if the cells are exposed to a specific sensitising substance?

Award-winning research

The test had previously only been used in mice and was recognised as the gold standard. The researchers thus had information that they could put to good use for their innovative in vitro test. However, the know-how had to be transferred to human cell cultures, which required the researchers to develop new laboratory protocols relating to the cultivation of T cells and dendritic cells, amongst others. In addition, Prof. Martin and his colleagues had to find a way to keep the test substances in solution and exclude potential toxic side effects. Working with the researchers from Mannheim, they developed a kind of shuttle. By conjugating the test substance to proteins such as human serum albumin, which is the most abundant protein in human blood plasma, they succeeded in avoiding these problems and bringing the test substance to its site of action, i.e. the surface of dendritic cells.

Three of the tests developed under the “Sens-it-iv” project will now be validated and tested by the authorities. Five other tests, including the one developed by Prof. Martin’s team and their cooperation partners, have been standardised and over the next few years will be further developed and validated. Although the project ended in 2011, Martin and his team have found a sponsor, which enables them to continue their work. In cooperation with the cosmetics industry consortium Cosmetics Europe, they are working with partners from Lyon (France) with the aim of making the test more sensitive. However, the ultimate goal is to establish a quantitative component. “The cosmetics industry is highly interested in being able to test chemicals for their allergic potency,” Prof. Martin said. Is it a strong or a weak contact allergen? This is a question that might decide whether a chemical can be used in cosmetics products or not, where threshold limits have to be observed. The cosmetics industry is desperately looking for in vitro tests that allow them to classify chemicals according to their allergic potency.

As the development of the test makes an important contribution to the protection of animals, the Baden-Württemberg Ministry for Rural Areas, Food and Consumer Protection decided to award the Baden-Württemberg Animal Welfare Price to Martin, Esser, Dietz and Thierse. The researchers are delighted with the award and will use the 25,000 euros for new projects.

Further information:
Prof. Dr. rer. nat. Stefan Martin
Allergy Research Group
Department of Dermatology and Venerology
Freiburg University Medical Centre
Hauptstraße 7
Freiburg
Tel.: +49 (0)761/ 270 - 6738
E-mail: stefan.martin(at)uniklinik-freiburg.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/the-cr-me-de-la-cr-me-of-contact-allergy-tests-goes-in-vitro