Reproducing the scalp microbiota community: co-colonization of a 3D reconstructed human epidermis with C. acnes and M. restricta.

OBJECTIVE: A 3D reconstructed human epidermis (RHE) model colonized with specific microbial strains was developed to model the complex interactions between strains of the human scalp hair. METHODS: Reconstructed human epidermis was colonized with Cutibacterium acnes and Malassezia restricta for 72 h. The epidermal model was characterized in terms of morphology, using immune-labelling targeting biomarkers for barrier structure, proliferation, differentiation and anti-microbial defence. The barrier function was assessed by transepithelial electrical eesistance (TEER) measurements. In order to study the microorganisms on the epidermal model, viable counts and phenotype ultrastructure analysis were performed by scanning electron microscopy (SEM). RESULTS: The RHE colonized with C. acnes did not lead to severe modifications of the physiological barrier integrity and viability, though it shows aggregates. M. restricta formed large aggregates by a close interaction with the RHE, thus causing both a strong decrease in barrier function and structure degradation and an increased human beta defensin 2 (HBD2) expression. The co-colonized model resulted in barrier depletion, but the overall damage was less severe, respecting the single colonization with M. restricta. The developed 'scalp model' allowed to identify morphological modifications leading to uncontrolled epidermal renewal. CONCLUSION: This study shows a pre-clinical model that recapitulates the interactions that can occur between site-specific microbial strains and keratinocytes in dandruff condition. The model can be applied to assess ingredients and products' mechanism of action.

OBJECTIF: Un modèle d'épiderme humain reconstruit a été colonisé par des souches microbiennes spécifiques du cuir chevelu pour étudier les interactions complexes entre les microorganismes et l'épiderme. MÉTHODES: Les épidermes humains reconstruits ont été colonisés par Cutibacterium acnes et Malassezia restricta pendant 72 h, puis caractérisés morphologiquement et par immunomarquages pour suivre les marqueurs de la différenciation kératinocytaire pour la fonction barrière, de prolifération et de défense antimicrobienne. La fonction barrière a également été évaluée par des mesures de résistance électrique transépithéliale (TEER). Afin d'étudier les microorganismes sur le modèle épidermique, des numérations des microorganismes viables et une analyse de l'ultrastructure phénotypique par microscopie électronique à balayage ont été effectuées. RÉSULTATS: Les modèles colonisés par C. acnes n'ont pas conduit à des modifications conséquentes de l'intégrité et de la viabilité de la barrière physiologique, bien que cette souche forme des agrégats. M. restricta a formé de gros agrégats par une interaction étroite avec l'épiderme, provoquant ainsi à la fois une forte diminution de la fonction barrière, une dégradation de la structure et une augmentation de l'expression de la bêta-défensine 2 humaine. Les modèles co-colonisés ont montré une altération de la fonction barrière, mais les dommages globaux étaient moins drastiques que lors de la simple colonisation par M. restricta. Ce « modèle de cuir chevelu ¼ développé a permis d'identifier des modifications morphologiques conduisant à un renouvellement épidermique incontrôle. CONCLUSION: Cette étude propose un modèle préclinique qui mime les interactions qui peuvent se produire entre les souches microbiennes spécifiques de ce site anatomique et les kératinocytes du scalp en condition pelliculaire. De plus, ce modèle peut être utiliser pour screener ingrédients et produits formulés et ainsi accéder à leurs mécanismes d'action.

Development of a standardized method to evaluate the protective efficiency of cosmetic packaging against microbial contamination.

Doubts surrounding the potential adverse effects of antimicrobial preservatives have modified the demand of consumers, who increasingly insist on the production of low-level and even preservative-free cosmetics. Protection of the product against microbial contamination is therefore focused on the packaging. This has prompted the emergence of a highly diverse array of so-called "protective", "overprotective", and "barrier" packaging. However, these designations are not normalized and the choice of the right packaging adapted to each cosmetic product is still essentially empirical, hazardous, and time consuming. The Cosmetic Valleys cluster has launched a commission to define a complete and experimentally-validated method to classify the level of protection of cosmetic packaging against microbial contamination. As reported herein, this required the development a specific bacteriostatic medium that can be used for 7 days and an in vitro procedure that reproduces in-use contamination and consumer practices. Based on tests performed on over 800 packages of different origin and performance characteristics, we propose a classification, divided into six grades, to differentiate the protective efficiency of cosmetic packaging. This work can be considered as a first step towards a regulatory text.

Mammalian antioxidant defenses are not inducible by H2O2.

As an approach to understanding how mammals regulate H(2)O(2) toxicity, intracellular concentration to prevent its we analyzed the genome-wide mRNA profile changes of human cells after treatment with a non-toxic H(2)O(2) concentration. We identified a large and essentially late H(2)O(2) response of induced and repressed genes that unexpectedly comprise few or no antioxidants but mostly apoptosis and cell cycle control activities. The requirement of the p53 regulator for regulating about a third of this H(2)O(2) stimulon and the lack of an associated enhancement of total cellular H(2)O(2) scavenging activity further suggest that H(2)O(2) elicits a stress antiproliferative/repair response that does not increase antioxidant defenses. We conclude that mammalian antioxidant defenses are constitutive, a finding that contrasts with the oxidant-inducibility of such defenses in microorganisms. This finding might be important in understanding the role of H(2)O(2) as a key signaling molecule in mammals.

NFY interacts with the promoter region of two genes involved in the rat peroxisomal fatty acid beta-oxidation: the multifunctional protein type 1 and the 3-ketoacyl-CoA B thiolase.

BACKGROUND: Beta-oxidation of long and very long chain fatty acyl-CoA derivatives occurs in peroxisomes, which are ubiquitous subcellular organelles of eukaryotic cells. This pathway releases acetyl-CoA as precursor for several key molecules such as cholesterol. Numerous enzymes participating to cholesterol and fatty acids biosynthesis pathways are co-localized in peroxisomes and some of their encoding genes are known as targets of the NFY transcriptional regulator. However, until now no interaction between NFY transcription factor and genes encoding peroxisomal beta-oxidation has been reported. RESULTS: This work studied the interactions between NFY factor with the rat gene promoters of two enzymes of the fatty acid beta-oxidation, MFP-1 (multifunctional protein type 1) and ThB (thiolase B) and their involvement in the cholesterol dependent-gene regulation. Binding of this nuclear factor to the ATTGG motif of the MFP-1 and of the ThB promoters was demonstrated by EMSA (Electrophoretic Mobility Shift Assay) and super shift assay. In contrast, in spite of the presence of putative Sp1 binding sites in these promoters, competitive EMSA did not reveal any binding. The promoter-dependent luciferase gene expression was downregulated by cholesterol in MFP-1 and ThB promoters harbouring constructs. CONCLUSIONS: This work describes for the first time a NFY interaction with promoter sequences of the peroxisomal beta-oxidation encoding genes. It suggests that cholesterol would negatively regulate the expression of genes involved in beta-oxidation, which generates the initial precursor for its own biosynthesis, via at least the NFY transcription factor.

Isolation and characterization of efficient isoxaben-transforming Microbacterium sp strains from four European soils.

Nutrient-agar plates containing isoxaben (500 mg litre(-1)) were used to isolate isoxaben-metabolising bacteria from four European soils incubated with the herbicide under laboratory conditions. In flask experiments, inoculation of a basal salts medium containing nitrogen and [phenyl-U-14C]isoxaben with an isolate (B2b) resulted in 33% recovery of the initial radioactivity as [14C]carbon dioxide after 2 weeks. A major metabolite identified by GC-MS and NMR analysis as 3-(1-ethyl-1-methylpropyl)isoxazol-5-ylamine accumulated both in basal salts and nutrient broth media. 2,6-Dimethoxybenzoic acid, a suspected metabolite of isoxaben, was not detected in either liquid media. However, the capability of the B2b isolate to use 2,6-dimethoxybenzoic acid as a source of carbon was demonstrated. Soil inoculation with the B2b strain resulted in an increase in the recovery of [14C] carbon dioxide from both [phenyl-U-14C] and [isoxazole-5-14C]isoxaben. The metabolite identified as 3-(1-ethyl-1-methylpropyl)isoxazole-5-ylamine only accumulated if the soil was autoclaved before inoculation. This metabolite was rapidly mineralized by the microflora of a natural soil without history of isoxaben treatment. Homology patterns of sequenced 16S rDNA between isoxaben-transforming isolates and reference strains showed that the four isolates identified belonged to the genus Microbacterium.