A. El Ghalbzouri

Leiden reconstructed human epidermal model as a tool for the evaluation of the skin corrosion and irritation potential according to the ECVAM guidelines

In the ECVAM validation studies two common skin protocols have been developed, the skin corrosion and skin irritation protocol. Both protocols include next to general and functional conditions that the skin model must meet, also the correct prediction of the activity of certain reference chemicals. For the skin corrosion protocol, the OECD TG 431 defined 12 reference chemicals that should be correctly predicted by the epidermal skin model. For skin irritation 20 test substances should meet the defined criteria. In this study we aimed to subject our Leiden human epidermal (LHE) model to both common protocols according to the ECVAM guidelines. The LHE model generated in this study has been fully characterized and shows very high similarities with the native skin. After minor technical changes in both protocols, corrosion classifications were obtained in concordance with those reported for the validated human skin models EpiSkin and EpiDerm. The results obtained with the common skin irritation protocol were very similar to that of earlier studies with the SkinEthic, EpiSkin and EpiDerm models. This means that the protocols and prediction models developed during the validation studies with a specific skin model can be used with other similar skin models. This study demonstrates that reconstructed human skin equivalents have been proven to be efficient and reliable alternatives to animal testing.

Reduced filaggrin expression is accompanied by increased Staphylococcus aureus colonization of epidermal skin models

Atopic dermatitis is an inflammatory skin disease that is characterized by a reduced skin barrier function, reduced filaggrin (FLG) expression as well as increased colonization by Staphylococcus aureus.

ECM1 interacts with fibulin-3 and the beta 3 chain of laminin 332 through its serum albumin subdomain-like 2 domain

The extracellular matrix protein 1 (ECM1) is an 85 kDa secreted glycoprotein, comprising four variants and playing a pivotal role in endochondral bone formation, angiogenesis, and tumour biology. A computational model for the three-dimensional structure of ECM1a was determined to identify the potential and/or concealed region(s) for binding with candidate partners in human skin. Multiple alignments for the secondary structure of ECM1a and b revealed similarity with serum albumin. The N-terminal domain of ECM1a consists mainly of alpha-helices (alphaD1), while the remaining three domains, namely serum albumin subdomain-like (SASDL) domains 2-4, were topologically comparable with the subdomain of the third serum albumin domain. Yeast-two-hybrid screening of a human foreskin cDNA library using both full-length ECM1a and the hot spot region for ECM1 gene mutations in lipoid proteinosis, an autosomal recessive genodermatosis (complete SASDL2 and the linker to SASDL3: aa177-aa361), as bait, isolated seven extracellular proteins. The site-specific interaction of ECM1a with two of these candidate binders, laminin 332 beta-3 chain and fibulin-3, was confirmed by in vitro and in vivo co-immunoprecipitation experiments. Immunohistologically both binders co-localized with ECM1 in human skin. Together, ECM1 is a multifunctional binding core and/or a scaffolding protein interacting with a variety of extracellular and structural proteins, contributing to the maintenance of skin integrity and homeostasis. Hence, disruption of the ECM1 function may cause the failure of multi-communication among the surrounding skin interstitial molecules, as seen in lipoid proteinosis pathology.

Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model

Background & Aim The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. Results All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. Conclusion Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

Rapamycin impairs UV induction of mutant‐p53 overexpressing cell clusters without affecting tumor onset

Because of its antitumor effect, the immunosuppressant rapamycin holds great promise for organ transplant recipients in that it may lower their cancer risk. In a mouse model, we showed previously that rapamycin inhibits the outgrowth of primary skin carcinomas induced by UV radiation. However, the tumors that did grow out showed an altered p53 mutation spectrum. Here, we investigated whether this shift in p53 mutations already occurred in the smallest tumors, which were not affected in onset. We found that rapamycin did not alter the mutational spectrum in small tumors and in preceding microscopic clusters of cells expressing mutant‐p53. However, rapamycin did reduce the number of these cell clusters. As this reduction did not affect tumor onset, we subsequently investigated whether rapamycin merely suppressed expression of mutated p53. This was not the case, as we could demonstrate that switching from a diet with rapamycin to one without, or vice versa, did not affect the number of existing mutant‐p53 expressing cell clusters. Hence, rapamycin actually reduced the formation of mutant‐p53 cell clusters. In wild‐type and p53‐mutant mice, we could not measure a significant enhancement of UV‐induced apoptosis, but we did observe clear enhancement in human skin equivalents. This was associated with a clear suppression of HIF1α accumulation. Thus, we conclude that rapamycin reduces the formation of mutant‐p53‐expressing cell clusters without affecting tumor onset, suggesting that tumors grow out of a minor subset of cell clusters, the formation of which is not affected by rapamycin.