faetal on 31/1/2014 at 16:23

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(by request)

Basically, my PhD is part of an industry-wide push to develop alternatives to animal testing for screening novel chemicals for use in cosmetic and toiletry products.

Allergic contact dermatitis happens when a chemical penetrates into the skin, modifies self-proteins making them look foreign and inducing an adaptive immune response (swelling, itching, blister, bleeding, scarring if regular enough).

My aims were broadly to develop a cell line derived from keratinocytes to use as an in vitro model for human skin, then use a model sensitizer called dinitrochlorobenzene (DNCB) to modify proteins within these cells, compare those modifications to ones in ex vivo skin from mastectomies, then use those modified proteins to elicit lymphocyte proliferation in the white cells taken from the blood of people sensitized to DNCB.

After this, I fractionated the modified proteins from the cells using something called isoelectric focussing, which uses a liquid pH gradient with current applied to move the proteins through a gel until they reach the part of the gradient at which they are electrically neutral, at which point they come out of the gel into solution and can then be extracted and analysed by mass spectrometry to see which ones are modified and hence potentially immunogenic.

After that, I looked at the modification kinetics of DNCB by incubating different concentrations of it for different lengths of time with the model protein human serum albumin and the used mass spectrometry to identify which amino acid residues had been modified and as such, derived some of the protein modifying properties of DNCB by which order the amino acid rsidues appeared to be modified by dose and time. The beauty part of this one was using a stable isotope labelled version of DNCB alongside the normal equivalent, meaning that we could set the mass spec to be more sensitive than usual and weed out false positives by only accepting modification signatures which were doublets separated by the 3 Da difference between the labelled and non-labelled.

Lastly is showed a novel process in the way that antigen processing cells (APCs, the cells which munch up macromolecules for presentation to the acquired immune system) treat modifications to a specific set of amino acids, I did this by showing that peptides (protein fragments) which were too small to be presented by APCs but had a modification on some amino acid residues, could still induce a response and that this happened by the modification being transferred to other proteins during processing. I then presented a protein which unfolds other proteins as a candidate for this process happen in living systems.

Then I began the process of weaning myself off of caffeine.

Apologies for the format of this, it's a brain vomit in as close to lay terms as I could get without being a professional science writer.

Happy to field any questions.

faetal on 31/1/2014 at 16:42

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(http://ec.europa.eu/consumers/sectors/cosmetics/animal-testing/index_en.htm) MANDATORYYYYYY

faetal on 31/1/2014 at 16:59

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I agree that it's not practical to replace animal models with what we know so far. But my work was cool regardless of that. It's worth knowing how shit works whether we test on animals or not.

Next I have a post doc starting in April in Lyon. Time for some of that good eating :)

Queue on 31/1/2014 at 19:22

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There are plenty of unwanted children running around. Just stick a couple in bunny costumes and go to town.

Remember, pink-eye is contagious...Mary Kay Radiant Skin Creme is not.




... after all, wasn't modern civilization built on finding new and creative ways to be awful toward our offspring?

faetal on 31/1/2014 at 22:31

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It's true, just look up mice v nickel studies. The human toll like receptor is specifically a trigger for nickel contact allergy. Mice with human tlr spliced in exhibit contact allergy.