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1.
Br J Dermatol ; 189(1): 91-102, 2023 07 07.
Article in English | MEDLINE | ID: mdl-36972303

ABSTRACT

BACKGROUND: Ichthyosis defines a group of chronic conditions that manifest phenotypically as a thick layer of scales, often affecting the entire skin. While the gene mutations that lead to ichthyosis are well documented, the actual signalling mechanisms that lead to scaling are poorly characterized; however, recent publications suggest that common mechanisms are active in ichthyotic tissue and in analogous models of ichthyosis. OBJECTIVES: To determine common mechanisms of hyperkeratosis that may be easily targeted with small-molecule inhibitors. METHODS: We combined gene expression analysis of gene-specific short hairpin RNA (shRNA) knockdowns in rat epidermal keratinocytes (REKs) of two genes mutated in autosomal recessive congenital ichthyosis (ARCI), Tgm1 and Alox12b, and proteomic analysis of skin scale from patients with ARCI, as well as RNA sequencing data from rat epidermal keratinocytes treated with the Toll-like receptor 2 (TLR2) agonist Pam3CSK4. RESULTS: We identified common activation of the TLR2 pathway. Exogenous TLR2 activation led to increased expression of important cornified envelope genes and, in organotypic culture, caused hyperkeratosis. Conversely, blockade of TLR2 signalling in keratinocytes from patients with ichthyosis and our shRNA models reduced the expression of keratin 1, a structural protein overexpressed in ichthyosis scale. A time course of TLR2 activation in REKs revealed that although there was rapid initial activation of innate immune pathways, this was rapidly superseded by widespread upregulation of epidermal differentiation-related proteins. Both nuclear factor kappa B phosphorylation and GATA3 upregulation was associated with this switch, and GATA3 overexpression was sufficient to increase keratin 1 expression. CONCLUSIONS: Taken together, these data define a dual role for TLR2 activation during epidermal barrier repair that may be a useful therapeutic modality in treating diseases of epidermal barrier dysfunction.


Subject(s)
Ichthyosis , Toll-Like Receptor 2 , Animals , Rats , Ichthyosis/genetics , Keratin-1/genetics , Mutation , Phenotype , Proteomics , RNA, Small Interfering , Toll-Like Receptor 2/genetics
2.
Int J Mol Sci ; 20(14)2019 Jul 12.
Article in English | MEDLINE | ID: mdl-31336867

ABSTRACT

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. METHODS: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. RESULTS: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. CONCLUSIONS: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Squamous Cell/pathology , Disease Models, Animal , Drug Evaluation, Preclinical/methods , Skin Neoplasms/pathology , Animals , Biomarkers, Tumor , Biopsy , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Gene Expression Profiling , Humans , Immunohistochemistry , Keratinocytes/drug effects , Keratinocytes/metabolism , Male , Mutation , Neoplasm Metastasis , Neoplasm Staging , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Xenograft Model Antitumor Assays
3.
J Am Heart Assoc ; 7(2)2018 01 22.
Article in English | MEDLINE | ID: mdl-29358198

ABSTRACT

BACKGROUND: Transplantation of adventitial pericytes (APCs) promotes cardiac repair in murine models of myocardial infarction. The aim of present study was to confirm the benefit of APC therapy in a large animal model. METHODS AND RESULTS: We performed a blind, randomized, placebo-controlled APC therapy trial in a swine model of reperfused myocardial infarction. A first study used human APCs (hAPCs) from patients undergoing coronary artery bypass graft surgery. A second study used allogeneic swine APCs (sAPCs). Primary end points were (1) ejection fraction as assessed by cardiac magnetic resonance imaging and (2) myocardial vascularization and fibrosis as determined by immunohistochemistry. Transplantation of hAPCs reduced fibrosis but failed to improve the other efficacy end points. Incompatibility of the xenogeneic model was suggested by the occurrence of a cytotoxic response following in vitro challenge of hAPCs with swine spleen lymphocytes and the failure to retrieve hAPCs in transplanted hearts. We next considered sAPCs as an alternative. Flow cytometry, immunocytochemistry, and functional/cytotoxic assays indicate that sAPCs are a surrogate of hAPCs. Transplantation of allogeneic sAPCs benefited capillary density and fibrosis but did not improve cardiac magnetic resonance imaging indices of contractility. Transplanted cells were detected in the border zone. CONCLUSIONS: Immunologic barriers limit the applicability of a xenogeneic swine model to assess hAPC efficacy. On the other hand, we newly show that transplantation of allogeneic sAPCs is feasible, safe, and immunologically acceptable. The approach induces proangiogenic and antifibrotic benefits, though these effects were not enough to result in functional improvements.


Subject(s)
Cell- and Tissue-Based Therapy/methods , Myocardial Infarction/surgery , Myocardial Reperfusion Injury/surgery , Myocardium/pathology , Neovascularization, Physiologic , Pericytes/transplantation , Ventricular Function, Left , Ventricular Remodeling , Aged , Aged, 80 and over , Allogeneic Cells , Animals , Cells, Cultured , Disease Models, Animal , Female , Fibrosis , Heterografts , Humans , Male , Middle Aged , Myocardial Contraction , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardial Reperfusion Injury/pathology , Myocardial Reperfusion Injury/physiopathology , Recovery of Function , Stroke Volume , Sus scrofa , Transplantation, Homologous
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