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Image analysis to quantify histological and immunofluorescent staining of ex vivo skin and skin cell cultures

Image processing steps and analysis techniques were developed for the quantification of photomicrographs obtained from light and fluorescence microscopy. The substrates examined were either skin cell cultures, such as normal human keratinocytes (NHK) or fibroblasts, or ex vivo skin sections. Examples of the analyses are provided for the comparison of skincare active ingredient treated samples vs. placebo to demonstrate the utility of the methods to quantify and provide numerical data for a procedure that is typically qualitative in nature and based on observations by a histologist. Quantifiable experiments that are discussed include: Fontana Masson staining for melanin expression; Nile red staining to detect cellular lipid droplets; nuclei staining with diamidino‐phenylindole (DAPI); and immunofluorescent staining of protein expression with a primary antibody directed against the protein (antigen) and a secondary antibody tagged with a fluorescent dye (Alexa Fluor 488) against the primary antibody.

Impact of AQP3 inducer treatment on cultured human keratinocytes, ex vivo human skin and volunteers.

One of the main functions of the skin is to protect the organism against environmental threats, such as thermal stress. Aquaporin‐3 (AQP3) facilitates water and glycerol transport across cell membranes and therefore regulates osmotic balance in different situations of stress. This mechanism seems to be particularly important for the resistance of different organisms to cold stress. Consequently, we were interested in investigating the effect of cold and osmotic stress on AQP3 expression in normal human keratinocytes. We developed a new active ingredient to stimulate aquaporins in skin and demonstrated the partial restoration of AQP3 expression in keratinocytes transfected with AQP3 siRNA. Moreover, we examined the effect of cold stress on cell morphology and the impact of a pre‐treatment with the active ingredient. Our results indicated that induction of AQP3 helped maintain a correct organization of the actin cytoskeleton, preserving cell morphology and preventing cells from rounding. Immunofluorescent staining revealed cytoplasmic localization of AQP3 and its translocation to the cell membrane following osmotic stress. Histological ex vivo studies of skin under different conditions, such as cold environment and tape‐stripping, indicated that increase in AQP3 expression appears to be involved in skin protection and showed that the pattern of AQP3 expression was more enhanced in the active ingredient‐treated samples. In vivo confocal microscopy by Vivascope showed a generally healthier appearance of the skin in the treated areas. These results attest to the potential value of the active ingredient in optimizing environmental stress resistance and protecting the skin from stratum corneum damage.

Microarray profiling of gene expression in human keratinocytes suggests a new protective activity against UV-induced DNA damage for a compound previously known to interact with SCF-KIT signalling pathway

The stem cell factor (SCF) and its protein–tyrosine kinase receptor KIT are together implicated in the regulation of diverse biological processes and particularly in melanogenesis. Indeed, this signalling pathway controls melanoblast migration from the neural crest during embryogenesis and allows the communication between keratinocytes and melanocytes in the adult. In melanocytes, the binding of SCF to its transmembrane receptor leads to the activation of signalling pathways implicating protein kinases which finally control the expression of pigmentation‐related genes. We have developed a biological compound called IV09.007, which we previously described as a modulator of the SCF/KIT signalling pathway with a pro‐pigmenting effect. In the present work, we have studied the expression and localization of both SCF and KIT mRNAs and proteins in the skin or skin‐derived cell lines. Then, we explored with a microarray approach the ability of IV09.007 to modulate the expression of genes in human keratinocytes and melanocytes in culture. Thereby, we observed the regulation of genes implicated in DNA repair, mainly related to base/nucleotides excision pathways. A modulated transcriptional response was also observed for some genes implicated in the response against oxidative stress, in apoptosis inhibition and in lowering inflammatory immune response. These microarray results predicted a conferred protective effect of IV09.007 and we verified this hypothesis by performing comet assays on UVB‐irradiated keratinocytes or melanocytes, to demonstrate the efficacy of IV09.007 on preventing DNA damage.

Maintenance of the ubiquitin-proteasome system activity correlates with visible skin benefits

Researches on longevity and anti‐ageing molecules have clearly evidenced the potential to increase lifespan of the cells. These recent scientific data raise interests and questions on the capacity of the cells to live longer and maintain their fundamental mechanisms of protection, reparation or degradation of abnormal proteins to maintain their capital of healthy and functional cellular activity. In this concern, this study was focused on the ubiquitin–proteasome system as an essential cellular tool to maintain the pool of functionally active proteins allowing renewal of proteins and degradation of damaged proteins. As the proteasome keeps the ‘cells health capital’, it should be particularly interesting to associate the maintenance of the proteasome activity with increasing longevity. Indeed, although oxidative stress damage increases with ageing leading to collagen and cellular membrane alterations, it also leads to a reduction in the proteasome activity which is critical for the cells. The aim of this study was to better understand the cellular role of the proteasome and to provide new data showing the skin beneficial effects in activating the overall system of ubiquitination and proteasomal degradation. For this purpose, in vitro, ex vivo and in vivo experiments were performed to evaluate the effects of maintaining the ubiquitin–proteasome activity in basal and stress conditions on young versus aged cells. Experiments have included evaluation of a newly developed dimerized tripeptide targeting specifically the ubiquitin–proteasome pathway. Our results have demonstrated that maintenance of this essential mechanism that participates in abnormal protein elimination and protein renewal allows maintaining cellular integrity that correlates with visible skin benefits.

Offline Monitoring of Hydroxyethyl Methacrylate and 3-Dimethylaminopropyl Methacrylamide Copolymerization: Correlation Between FTIR and GC Quantifications

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy is classically used to monitor homopolymerizations. In this article, this analytical technique was extended to monitor the synthesis of VIVIPRINT 300, which is a copolymer of 2-hydroxyethyl methacrylate (HEMA) and N-[3-dimethylaminopropyl]methacrylamide (DMAPMA). The calibration curves devised for this study were based on the two homopolymers P(HEMA) and P(DMAPMA). A good correlation was realized between the FTIR absorbance intensities observed respectively at 1300 cm−1 (polymerized C-O ester bond) and 1230 cm−1 (polymerized C-N amide bond) and the level of residual HEMA and DMAPMA monomers determined by GC. Application of these calibration curves to the copolymerization also exhibited a good correlation of data relating to residual monomer determination by FTIR and GC, validating the success of this spectroscopic in situ technique.