A new tool to test active ingredient using lactic acid in vitro,a help to understand cellular mechanism involved in stinging test: An example using a bacterial polysaccharide (Fucogel®)

The stinging test is an in vivo protocol that evaluates sensitive skin using lactic acid (LA). A soothing sensation of cosmetics or ingredients can be also appreciated through a decrease in stinging score. To predict the soothing sensation of a product before in vivo testing, we developed a model based on an LA test and substance P (SP) release using a co‐culture of human keratinocytes and NGF‐differentiated PC12 cells. A bacterial fucose‐rich polysaccharide present in Fucogel^® was evaluated as the soothing molecule in the in vivo stinging test and our in vitro model. Excluding toxic concentrations, the release of SP was significant from 0.2% of lactic acid for the PC12 cells and from 0.1% of lactic acid for the keratinocytes. When the pH was adjusted to approximately 7.4, LA did not provoke SP release. At these concentrations of LA, 0.1% of polysaccharide showed a significant decrease in SP release from the two cellular types and in co‐cultures without modifying the pH of the medium. In vivo, a stinging test using the polysaccharide showed a 30% decrease in prickling intensity vs the placebo in 19 women between the ages of 21 and 69. Our in vitro model is ethically interesting and is adapted for cosmetic ingredients screening because it does not use animal experimentation and limits human volunteers. Moreover, Fucogel^® reduced prickling sensation as revealed by the in vivo stinging test and inhibits the neurogenic inflammation as showed by our new in vitro stinging test based on SP release.     

Staphylococcus epidermidis protease EcpA can be a deleterious component of the skin microbiome in atopic dermatitis

1. Download : Download high-res image (400KB)
2. Download : Download full-size image

     

Amniotic membrane stimulates cell migration by modulating transforming growth factor‐β signalling

Keratinocyte migration is a mandatory aspect of wound healing. We have previously shown that amniotic membrane (AM) applied to chronic wounds assists healing through a process resulting in the overexpression of c‐Jun at the wound's leading edge. We have also demonstrated that AM modifies the genetic programme induced by transforming growth factor‐ß (TGF‐ß) in chronic wounds. Here we used a scratch assay of mink lung epithelial cells (Mv1Lu) and a spontaneously immortalized human keratinocyte cell line (HaCaT) cells to examine the influence of AM application on the underlying signalling during scratch closure. AM application induced c‐Jun phosphorylation at the leading edge of scratch wounds in a process dependent on MAPK and JNK signalling. Strikingly, when the TGF‐ß‐dependent Smad‐activation inhibitor SB431542 was used together with AM, migration improvement was partially restrained, whereas the addition of TGF‐ß had a synergistic effect on the AM‐induced cell migration. Moreover, antagonizing TGF‐ß with specific antibodies in both cell lines or knocking out TGF‐ß receptors in Mv1Lu cells had similar effects on cell migration as using SB431542. Furthermore, we found that AM was able to attenuate TGF‐ß‐Smad signalling specifically at the migrating edge; AM treatment abated Smad2 and Smad3 nuclear localization in response to TGF‐ß in a process dependent on mitogen‐activated protein kinase kinase 1 (MEK1) activation but independent of EGF receptor or JNK activation. The involvement of Smad signalling on AM effects on HaCaT keratinocytes was further corroborated by overexpression of either Smad2 or Smad3 and the use of Smad phosphorylation‐specific inhibitors, revealing a differential influence on AM‐induced migration for each Smad. Thus, AM TGF‐ß‐Smad signalling abating is essential for optimal cell migration and wound closure.