Genes involved in the induction and resolution of eczema using the Atopy Patch Test as an in vivo model for atopic dermatitis

Atopic dermatitis (AD) is a chronic inflammatory skin disease, characterised by
high levels of circulating IgE and skin infiltration of immune cells in both
lesional and nonlesional skin. Skin T cells have been found to express a
predominant Th2 type cytokine expression pattern. Nonlesional skin is suggested
to be a 'pre activated' state resulting from the presence of increased numbers
of inflammatory cells compared to 'healthy' skin. Immune cells from nonlesional
AD skin react with a lower threshold to antigens. Reaction of immune cells with
antigens results in the release of cytokines, chemokines and other inflammatory
markers resulting in eczema. Little is known about the markers/intracellular
pathways that play a role in the induction of AD.

The atopy patch test (APT) is a human in vivo model for AD. By application of
allergens (e.g. house dust mite) to the skin for 24-48-72-96 hours an
eczematous reaction that is very similar to lesional AD develops. The APT is
used as a model to investigate genes involved in the induction and resolution
of eczema lesions in AD patients. A biopsy at 24 hours is appropriate to study
induction markers, a biopsy at 48 hours reveals which markers have an extended
elevated expression but also reveals which factors decline in comparison to a
24 hrs biopsy. A biopsy at 72 and 96 hours shows which markers play a role in
the resolution of the APT. We have previously shown several genes that may be
involved in the induction and resolution of eczema (see attachments). However,
the latter study lacked power. A sample size calculation (corrected for
multiple testing) revealed to extend the number of patients with n=10. To
confirm our hypothesis that these markers are really involved in the induction
and resolution of eczema (not based on 'statistical coincidence* and to correct
for multiple testing), and to create a reliable selection of the genes of
interest, we propose to extend the number of patients with n=10 AD patients.
This additional study, will provide more insight concerning the induction and
resolution of eczema, and may reveal new therapeutic targets.

Note: the 72 and 96 hours biopsies are included to study the resolution of the
APT. Furthermore, the translation from genes to protein takes several hours.
Therefore, a 72-96 hrs biopsy reflects the protein concentrations of 48 hours
biopsies that show a high gene expression at 48 hours. The same applies to the
48 hrs biopsies compared to 24 hours biopsies in terms of protein measurments
(but not gene expression)

To investigate biomarkers that play a role in the induction and resolution of
AD.

Secondary objective: to develop therapeutic interventions (pharmacologically)
based on the revealed biomarkers.

- The APT is applied to the skin and biopsies are harvested after 24-48-72-96
hours after the induction of AD. Biopsies from nonlesional, lesional skin, and
a control biopsy after the application of petrolatum are additionally harvested.
- from the harvested biopsies, mRNA measurments of the markers will be
performed with qPCR. Localization of markers will be performed with IHC.

Participants will undergo an APT and seven skin punch biopsies (4 mm diameter)
in four sessions. Performing a biopsy entails a slight risk of haemorrhage and
infection. Over the past years, no SAE's were observed in patients that were
included in the Biobank. A small scar at the site of biopsy will gradually fade
in colour. No biopsies will be taken from the face or neck. Biopsies will only
be taken from the arms and legs.
The specified number of biopsies is necessary to perform the proposed analyses.
There is no direct benefit for the participants. An APT will induce erythema
and pruritus with sometimes papules and vesicles. This reaction usually
disappears after 72-96 hours. Erythema, pruritus, papules and vesicles normally
disappear after 72-96 hours, but this process can be enhanced by application of
topical steroids.