RESUMO
BACKGROUND: Allergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions. OBJECTIVE: We show the molecular details and the effects of three non-specific lipid transfer proteins (nsLTPs; Mal d 3, Cor a 8 and Pru p 3) upon epithelial cell uptake and transport. METHODS: We used fluorescent imaging, flow cytometry, proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines. RESULTS: NsLTPs are transported across the epithelium without affecting cell membrane stability or viability and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis (CME). Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent upon these ligands in the protein complex.nsLTPs were found to initiate cellular signaling via TLR2 but not the CD1d receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the three allergens induced intracellular stress signaling through activation of the NOD2 pathway. CONCLUSIONS: Our work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. Additionally, we have developed a versatile toolbox to extend these investigations to other allergens and cell types.
RESUMO
Phospholipids are versatile formulation compounds with high biocompatibility. However, no data on their effect on skin in combination with UVA radiation exist. Thus, it was the aim of this work to (i) develop o/w nanoemulsions (NEs) differing in surfactant type and to investigate their physicochemical stability at different storage temperatures, (ii) establish a standardized protocol for in vitro phototoxicity testing using primary human skin cells and (iii) investigate the phototoxicity of amphoteric phospholipids (S45, S75, E80, S100, LPC80), sodium lauryl ether sulfate (SLES) and polysorbate 80 (PS80). Satisfying systems were developed with all surfactants except S100 due to low zeta potential (-21.4 mV ± 4.69). SLES and PS80-type NEs showed the highest stability after eight weeks; temperature-dependent variations in storage stability were most noticeable for phospholipid surfactants. For phospholipid-based NEs, higher phosphatidylcholine content led to unstable formulations. Phototoxicity assays with primary skin fibroblasts confirmed the lack of UVA-related phototoxicity but revealed cytotoxic effects of LPC80 and SLES, resulting in cell viability as low as 2.7 % ±0.78 and 1.9 % ±1.57 compared to the control. Our findings suggest that surfactants S45, S75 and PS80 are the most promising candidates for skin-friendly emulsifiers in sensitive applications involving exposure to UV light.
Assuntos
Dermatite Fototóxica , Tensoativos , Humanos , Tensoativos/química , Polissorbatos/farmacologia , Raios Ultravioleta , Fosfolipídeos , Emulsões/farmacologia , PeleRESUMO
The World Health Organization (WHO) declared certain fungal pathogens as global health threats for the next decade. Candida auris (C. auris) is a newly emerging skin-tropic multidrug-resistant fungal pathogen that can cause life-threatening infections of high mortality in hospitals and healthcare settings. Here, we address an unmet need and present novel native ex vivo skin models, thus extending previous C. auris-host interaction studies. We exploit histology and immunofluorescence analysis of ex vivo skin biopsies of human adult and fetal, as well as mouse origin infected with C. auris via distinct routes. We demonstrate that an intact skin barrier efficiently protects from C. auris penetration and invasion. Although C. auris readily grows on native human skin, it can reach deeper layers only upon physical disruption of the barrier by needling or through otherwise damaged skin. By contrast, a barrier disruption is not necessary for C. auris penetration of native mouse skin. Importantly, we show that C. auris undergoes morphogenetic changes upon skin penetration, as it acquires pseudohyphal growth phenotypes in deeper human and mouse dermis. Taken together, this new human and mouse skin model toolset yields new insights into C. auris colonization, adhesion, growth and invasion properties of native versus damaged human skin. The results form a crucial basis for future studies on skin immune defense to colonizing pathogens, and offer new options for testing the action and efficacy of topical antimicrobial compound formulations.
Assuntos
Candida auris , Candidíase , Animais , Humanos , Camundongos , Candidíase/microbiologia , Modelos Animais de DoençasRESUMO
In previous studies, lecithin-based nanoemulsions (NEs) have been shown to be skin friendly drug carrier systems. Due to their nontoxic properties, NEs might also be suitable as wound healing agents. Hence, different O/W NEs based on lecithin Lipoid® S 75 and plant oils or medium chain triglycerides were produced and characterised. Two lipophilic natural wound healing agents, a betulin-enriched extract from birch bark (BET) and a purified spruce balm (PSB), were successfully incorporated and their effects on primary human skin cells were studied in vitro. MTT, BrdU and scratch assays uncovered the positive influence of the drug-loaded NEs on cell viability, proliferation and potential wound closure. Compared to control formulations, the NEs loaded with either BET or PSB led to higher cell viability rates of fibroblasts and keratinocytes. Higher proliferative activity of keratinocytes and fibroblasts was observed after the treatment, which is a prerequisite for wound closure. Indeed, in scratch assays NEs with PSB and notably BET showed significantly ameliorated wound closure rates than the negative control (unloaded NEs) and the positive control (NEs with dexpanthenol). Our findings suggest that BET and PSB are outstanding wound healing drugs and their incorporation into lecithin-based NEs may represent a valid strategy for wound care.
Assuntos
Lecitinas/farmacologia , Óleos de Plantas/farmacologia , Pele/citologia , Pele/efeitos dos fármacos , Triglicerídeos/farmacologia , Cicatrização/efeitos dos fármacos , Betula , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Emulsões , Humanos , Técnicas In Vitro , Picea , Triterpenos/farmacologiaRESUMO
Octenidine dihydrochloride (OCT) is a widely used antiseptic molecule, promoting skin wound healing accompanied with improved scar quality after surgical procedures. However, the mechanisms by which OCT is contributing to tissue regeneration are not yet completely clear. In this study, we have used a superficial wound model by tape stripping of ex vivo human skin. Protein profiles of wounded skin biopsies treated with OCT-containing hydrogel and the released secretome were analyzed using liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA), respectively. Proteomics analysis of OCT-treated skin wounds revealed significant lower levels of key players in tissue remodeling as well as reepithelization after wounding such as pro-inflammatory cytokines (IL-8, IL-6) and matrix-metalloproteinases (MMP1, MMP2, MMP3, MMP9) when compared to controls. In addition, enzymatic activity of several released MMPs into culture supernatants was significantly lower in OCT-treated samples. Our data give insights on the mode of action based on which OCT positively influences wound healing and identified anti-inflammatory and protease-inhibitory activities of OCT.