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Quantitative image analysis of protein expression and colocalisation in skin sections
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.ORCID iD: 0000-0001-9280-9832
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
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2018 (English)In: Experimental dermatology, ISSN 0906-6705, E-ISSN 1600-0625, Vol. 27, no 2, p. 196-199Article in journal, Letter (Refereed) Published
Abstract [en]

Immunofluorescence (IF) and in situ proximity ligation assay (isPLA) are techniques that are used for in situ protein expression and colocalisation analysis, respectively. However, an efficient quantitative method to analyse both IF and isPLA staining on skin sections is lacking. Therefore, we developed a new method for semi-automatic quantitative layer-by-layer measurement of protein expression and colocalisation in skin sections using the free open-source software CellProfiler. As a proof of principle, IF and isPLA of ichthyosis-related proteins TGm-1 and SDR9C7 were examined. The results indicate that this new method can be used for protein expression and colocalisation analysis in skin sections.

Place, publisher, year, edition, pages
2018. Vol. 27, no 2, p. 196-199
National Category
Biomedical Laboratory Science/Technology Medical Image Processing
Research subject
Computerized Image Processing
Identifiers
URN: urn:nbn:se:uu:diva-338544DOI: 10.1111/exd.13457ISI: 000423679700014OAI: oai:DiVA.org:uu-338544DiVA, id: diva2:1172480
Projects
Inherited skin disorders
Funder
Swedish Research Council, K2013-57x-22309-3Available from: 2018-01-09 Created: 2018-01-10 Last updated: 2019-02-21Bibliographically approved
In thesis
1. Congenital Recessive Ichthyosis: Studies of Gene Expressions Related to Keratinocyte Differentiation and Skin Barrier Repair
Open this publication in new window or tab >>Congenital Recessive Ichthyosis: Studies of Gene Expressions Related to Keratinocyte Differentiation and Skin Barrier Repair
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Autosomal recessive congenital ichthyosis (ARCI) is a rare monogenetic disorder characterized by a defective skin barrier, hyperkeratosis, and dry, scaly skin. It affects keratinocyte differentiation and is caused by mutations in any of at least 12 genes believed to control the formation of ω-O-acylceramide and the corneocyte lipid envelope (CLE): ABCA12, ALOXE3, ALOX12B, CERS3, CYP4F22, ELOVL4, LIPN, NIPAL4, PNPLA1, SDR9C7, SLC27A4, and TGM1.

Studies of keratinocyte differentiation and gene expression in ARCI may help us better understand the pathobiology of skin barrier formation. One way to verify that ARCI-related gene products are operating in a chain of events essential for lipid barrier formation is to use immunofluorescence and in situ proximity ligation assays to demonstrate the proteins’ colocalizations in the epidermis. In paper I, a new method for the objective quantitative image analysis of protein expression and colocalization in different epidermal layers of skin sections was developed using free, open-source software, CellProfiler. Using this method and microarray analyses of skin biopsies from ARCI patients with TGM1 mutations (n = 5) compared with those of healthy controls (n = 4), many ARCI-related genes were found to be upregulated in patient epidermis (paper II). Because many other genes involved in keratinocyte differentiation and immune/inflammatory response, including PPARD, were also induced in the patients’ microarrays, the effects of a ligand-dependent transcription factor, PPARδ, encoded by PPARD, were studied on ARCI-related gene expression in cultured keratinocytes, usually showing the pronounced upregulation by PPARδ agonists (paper III). Furthermore, using previous array data obtained from cultured differentiated keratinocytes and from skin biopsies of patients with TGM1 mutations, nine novel candidate markers of differentiation were identified, and the upregulation was verified by qPCR of mRNA from cultured keratinocytes and skin biopsies. These transcripts were also induced by PPARδ agonists in cultured proliferating keratinocytes (paper IV).

To conclude, the upregulation of other ARCI-related genes in patients with TGM1 mutations might reflect a feedback mechanism in ω-O-acylceramide biosynthesis, which, however, is unable to restore the patients’ skin barrier. In theory, substitution therapy with ω-O-acylceramide and recombinant TGm-1 may be required. Because PPARδ activation appears involved in upregulating ARCI-related genes and nine novel differentiation marker genes, all potentially important for barrier repair, this approach could become a treatment option for several types of ichthyosis and wound healing.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 58
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1541
Keywords
genodermatoses, oligoarray, trancriptomics, transglutaminase-1, cornified envelope, peroxisome proliferator-activated receptor δ, all-trans retinoic acids.
National Category
Dermatology and Venereal Diseases
Research subject
Dermatology and Venerology
Identifiers
urn:nbn:se:uu:diva-377557 (URN)978-91-513-0578-3 (ISBN)
Public defence
2019-04-11, Fåhraeussalen, Rudbeck Laboratory, C5, entréplan, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2019-03-20 Created: 2019-02-21 Last updated: 2019-05-07

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Zhang, HanqianVirtanen, MarieWählby, CarolinaVahlquist, AndersTörmä, Hans
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