Effect of two shampoo formulations on the prokaryotic and eukaryotic microbiota composition of the human scalp.

OBJECTIVE: The human scalp is characterized by a moderately diverse microbial community, comprising prokaryotic (bacteria) and eukaryotic (fungi) members. Although the details are far from being fully understood, the human scalp microbiota is implicated in several scalp disorders, in particular dandruff formation. Hence, the protection of an intact and diverse scalp microbiota can be regarded as a quality criterion for hair and scalp care formulations. In this study, we investigated the influence of two commercially available, non-antimicrobial shampoo formulations on the structure of the scalp microbiota. METHODS: Scalp microbiota samples, obtained by swab sampling from two cohorts of probands (n = 25, each), were analysed before and after daily use of two different shampoo formulations for 2 weeks, respectively. A polyphasic approach was used, comprising quantitative cultivation of bacteria and fungi on selective media as well as sequencing of PCR-amplified 16S rRNA and 18S rRNA genes, respectively. RESULTS: All analyses revealed a microbiota composition typical for the human scalp. While in particular fungal germ numbers increased significantly during the treatments, overall bacterial and fungal community composition was not affected, based on alpha- and beta-diversity measures. However, we observed an increase in structural bacterial diversity with the age of the probands. CONCLUSIONS: Over an application period of 2 weeks, the investigated shampoo induced quantitative but no qualitative changes in the scalp microbial community structure of the investigated probands, suggesting no adverse but rather preserving or even stimulating effects of the underlying formulations on the scalp microbiota. Further investigation will have to clarify if this is also true for longer application periods and if the formulations might affect community functionality, for example microbial gene expression, rather than community composition.

OBJECTIF: Le cuir chevelu humain se caractérise par une communauté microbienne modérément diversifiée, comprenant des membres procaryotes (bactéries) et eucaryotes (champignons). Bien que l'on soit loin de comprendre totalement les détails, le microbiote du cuir chevelu humain est impliqué dans différents troubles du cuir chevelu, en particulier la formation de pellicules. La protection du microbiote du cuir chevelu intact et diversifié peut être considérée comme un critère de qualité pour les formulations de soins pour les cheveux et le cuir chevelu. Dans cette étude, nous avons examiné l'influence de deux formulations de shampooing non antimicrobien disponibles dans le commerce sur la structure du microbiote du cuir chevelu. MÉTHODES: Des échantillons de microbiote du cuir chevelu, obtenus par écouvillonnage dans deux cohortes de proposants (n = 25 dans chaque cohorte), ont été analysés respectivement avant et après l'utilisation quotidienne de deux formulations de shampooing pendant deux semaines. Une approche en plusieurs phases a été utilisée, dont une culture quantitative de bactéries et de champignons sur des milieux sélectifs et un séquençage respectivement des gènes de l'ARN ribosomique 16S et de l'ARN ribosomique 18S amplifiés par PCR. RÉSULTATS: Toutes les analyses ont révélé une composition du microbiote typique pour le cuir chevelu humain. Bien que le nombre de germes fongiques en particulier ait augmenté significativement pendant les traitements, la composition globale des communautés bactériennes et fongiques n'a pas été affectée, d'après les mesures de diversité alpha et bêta. Cependant, nous avons observé une augmentation de la diversité bactérienne structurelle avec l'âge des proposants. CONCLUSIONS: Sur une période d'utilisation de deux semaines, le shampooing étudié a induit des modifications quantitatives, mais pas qualitatives, de la structure des communautés microbiennes du cuir chevelu des proposants étudiés, ce qui suggère qu'il n'y a pas d'effets indésirables, mais qu'il y a des effets de préservation, voire de stimulation, des formulations sous-jacentes sur le microbiote du cuir chevelu. Des recherches supplémentaires devront clarifier si cela s'avère également pour des périodes d'utilisation plus longues et si les formulations peuvent affecter la fonctionnalité des communautés, par exemple, l'expression des gènes microbiens, plutôt que la composition des communautés.

A novel cell line from human eccrine sweat gland duct cells for investigating sweating physiology.

OBJECTIVE: Human eccrine sweat glands (eSG) represent vital components of the skin involved in regulating body temperature. Especially the eccrine duct, which opens directly into the skin surface and releases the aqueous sweat, constitutes the first contact point with topically applied substances. For scientific investigations and to understand the underlying sweating mechanism on a cellular level defined cellular material is beneficial. We, therefore, strived to generate a cell line derived from human eSG duct cells for identifying new mechanisms in sweating control, as such a standardized cell line is currently lacking. MATERIAL AND METHODS: Isolated primary human eSG duct cells were transduced with simian virus 40 large T antigen (SV40T) by lentiviral transduction. Successfully SV40T-transduced clones were selected by single-cell cloning with one clone, named 1D10, being particularly described in this work. RESULTS: In performed cellular investigations, SV40T-transduced duct-derived cells exhibited an extended lifespan with stable population doubling times suggesting its immortality. Besides, 1D10 clonal cell culture demonstrated similarities with parental, primary duct cells regarding gene expression of selected sweat gland-related markers. When combined with primary coil cells in a hanging drop co-culture, those transduced duct-derived cells showed some duct cell-like features. Further, a certain degree of cellular communication and a specific reaction towards substance application was observed. CONCLUSION: Generated and herein described cell line derived from isolated human eSG duct cells is, based on the presented scientific findings, considered as immortal. Besides, this cell line shows some similarity with primary duct cells, although alterations from native glands were detected, among which is loss of expression of cystic fibrosis transmembrane conductance regulator. Provided some further investigations, presented SV40T-transduced duct-cell derived cell line seems a suited surrogate of primary eccrine duct cells.

OBJECTIF: les glandes sudoripares eccrines (GSe) humaines représentent des composants vitaux de la peau impliqués dans la régulation de la température corporelle, en particulier le canal eccrine, qui s'ouvre directement à la surface de la peau et libère la sueur aqueuse, et constitue le premier point de contact avec les substances appliquées par voie topique. Un matériel cellulaire défini est bénéfique pour les recherches scientifiques et pour comprendre le mécanisme de sudation sous-jacent au niveau cellulaire. Nous nous sommes donc appliqués à générer une lignée cellulaire dérivée de cellules de canaux de GSe humaines pour identifier de nouveaux mécanismes dans le contrôle de la transpiration, car ce type de lignée cellulaire standardisée n'est actuellement pas disponible. MATÉRIELS ET MÉTHODES: des cellules primaires isolées de canaux de GSe humaines ont été transduites avec le grand antigène T du virus simien 40 (SV40T) par transduction lentivirale. Les clones SV40T transduits avec succès ont été sélectionnés par clonage unicellulaire, et un clone, nommé 1D10, est décrit plus particulièrement dans les présents travaux. RÉSULTATS: dans les investigations cellulaires réalisées, les cellules transduites avec le SV40T présentaient une durée de vie plus longue, avec des temps de doublement de la population stables suggérant leur immortalité. En outre, la culture cellulaire clonale 1D10 présentait des similitudes avec les cellules de canaux primaires parentaux concernant l'expression génique de certains marqueurs liés aux glandes sudoripares. Lorsqu'elles ont été associées à des cellules spirale primaires dans une co-culture suspendue, ces cellules transduites dérivées des canaux présentaient certaines caractéristiques des cellules de canaux. De plus, un certain degré de communication cellulaire et une réaction spécifique à l'application de substances ont été observés. CONCLUSION: la lignée cellulaire produite et décrite ici, dérivée de cellules isolées de canaux de GSe humaines, est, d'après les résultats scientifiques présentés, considérée comme immortelle. En outre, cette lignée cellulaire présente une certaine similarité avec les cellules de canaux primaires, bien que des modifications des glandes natives aient été détectées, dont la perte d'expression du régulateur de la conductance transmembranaire de la mucoviscidose. Sous réserve de recherches supplémentaires, la lignée cellulaire dérivée de cellules de canaux primaires transduites avec le SV40T présentée ici semble être un substitut approprié des cellules de canaux eccrines primaires.

A novel organotypic 3D sweat gland model with physiological functionality.

Dysregulated human eccrine sweat glands can negatively impact the quality-of-life of people suffering from disorders like hyperhidrosis. Inability of sweating can even result in serious health effects in humans affected by anhidrosis. The underlying mechanisms must be elucidated and a reliable in vitro test system for drug screening must be developed. Here we describe a novel organotypic three-dimensional (3D) sweat gland model made of primary human eccrine sweat gland cells. Initial experiments revealed that eccrine sweat gland cells in a two-dimensional (2D) culture lose typical physiological markers. To resemble the in vivo situation as close as possible, we applied the hanging drop cultivation technology regaining most of the markers when cultured in its natural spherical environment. To compare the organotypic 3D sweat gland model versus human sweat glands in vivo, we compared markers relevant for the eccrine sweat gland using transcriptomic and proteomic analysis. Comparing the marker profile, a high in vitro-in vivo correlation was shown. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), muscarinic acetylcholine receptor M3 (CHRM3), Na+-K+-Cl- cotransporter 1 (NKCC1), calcium-activated chloride channel anoctamin-1 (ANO1/TMEM16A), and aquaporin-5 (AQP5) are found at significant expression levels in the 3D model. Moreover, cholinergic stimulation with acetylcholine or pilocarpine leads to calcium influx monitored in a calcium flux assay. Cholinergic stimulation cannot be achieved with the sweat gland cell line NCL-SG3 used as a sweat gland model system. Our results show clear benefits of the organotypic 3D sweat gland model versus 2D cultures in terms of the expression of essential eccrine sweat gland key regulators and in the physiological response to stimulation. Taken together, this novel organotypic 3D sweat gland model shows a good in vitro-in vivo correlation and is an appropriate alternative for screening of potential bioactives regulating the sweat mechanism.

In vitro skin irritation: facts and future. State of the art review of mechanisms and models.

The skin is the main target tissue for exogenous noxes, protecting us from harmful environmental hazards, UV-irradiation and endogenous water loss. It is composed of three layers, whereas the outermost epidermis is a squamous epithelium that mainly consists of keratinocytes. These cells execute a terminal differentiation, which finally results in the assembly of the stratum corneum. This layer, consisting of cornified keratinocytes, is an effective barrier against a vast number of substances. Apart of this, keratinocytes play crucial roles in the immune surveillance and the initiation, modulation and regulation of inflammation in the epidermis. Regarding cutaneous inflammatory reactions, skin irritation is one of the most common adverse effect in humans. For reasons of human safety assessment new chemicals are still evaluated for irritant potentials by application to animals followed by visible changes such as erythema and oedema. Testing for skin irritation in animals potentially cause them pain and discomfort. Furthermore, the results are not always predictive for those found in humans. In order to replace animal testing and to improve the prediction of irritants, the cosmetic and toiletry industry, in Europe represented by Colipa, develops and uses several alternative in vitro test systems. In this respect, the use of in vitro reconstructed organotypic skin equivalents are mostly favored, because of their increasingly close resemblance to human skin. Due to ethical and scientific questions and on account of the 7th amendment of the European Council Directive 76/768/EEC, the authors see the requirement to drive the development of alternative tests for irritants. Therefore, this article centres on cosmetic ingredients and provides the readership an overview of the state of art of cellular mechanisms of skin irritation and summarizes the results of the commonly used skin equivalents to evaluate irritation in vitro.

Expression of hurpin, a serine proteinase inhibitor, in normal and pathological skin: overexpression and redistribution in psoriasis and cutaneous carcinomas.

Hurpin was identified by differential display analysis studying UV-repressible genes in the keratinocyte cell line HaCaT. We have previously reported that hurpin mRNA is overexpressed in psoriatic skin compared to non-lesional or normal skin; hurpin inhibits cathepsin L and that, after overexpression in keratinocytes, hurpin decreases UV-induced apoptosis. To further study the expression of hurpin, we have isolated monoclonal antibodies against hurpin and analyzed its expression in normal and diseased skin by immunohistochemistry (IHC). In the epidermis of normal skin, we found hurpin to be mainly expressed in the stratum basale. In contrast, we found an enhanced expression of hurpin in the stratum spinosum and stratum granulosum in the majority of diseased skin samples. Within the dermis of normal and diseased skin, hurpin was detected in sebaceous and sweat glands, hair follicles, and endothelial cells of blood vessels. Hurpin was localized in the cytoplasm in normal and diseased skin. Additionally to IHC, we analyzed hurpin expression in selected skin diseases by semiquantitative reverse-transcription polymerase chain reaction. We found overexpression of hurpin mRNA in psoriasis, squamous cell carcinoma (SCC), and actinic keratosis. In contrast, expression of hurpin in melanoma and basal cell carcinoma was comparable to that in normal skin. Overall, the strongest overexpression was observed in SCC and psoriasis. Individual differences in hurpin expression between patients were observed. The increased expression and redistribution of hurpin in diseased skin suggests its possible involvement in inflammatory processes or the regulation of endogenous or pathogen-derived proteinase activity. Additional studies will elucidate the physiological role of hurpin.

Molecular basis of basal cell carcinoma: analysis of differential gene expression by differential display PCR and expression array.

Basal cell carcinoma (BCC) is the most common tumor in the Caucasian population. Although BCC rarely metastasize and cause death, they are problematic due to their destructive growth and the frequent localization on the face. Until now the knowledge of genes differentially expressed in BCC has been incomplete. To elucidate the complex alterations in BCC-associated gene expression, we took advantage of 2 techniques: the differential display RT-PCR (DD-PCR) and the differential hybridization of cDNA arrays. Using DD-PCR, we showed differential expression of genes known from other biological contexts (e.g., rac, ubiquitin hydrolase), which could now be associated with BCC. In addition, we detected unknown genes possibly contributing to the carcinogenesis of BCC. Of the 588 genes screened by differential hybridization of the Atlas human cDNA array, differences in the expression levels of BCC were observed for 10 genes. These data were obtained with RNA probes pooled from several BCC of different donors and were subsequently confirmed by semiquantitative RT-PCR for Janus protein tyrosine kinase 3 (Jak3), microsomal glutathione S-transferase 1 (GST 12), teratocarcinoma-derived growth factor cripto, glutaredoxin and the monocyte chemoattractant protein 1 (MCP-1) in 10 individual BCC specimens, 2 squamous cell carcinoma (SCC), the cell line HaCaT and cultured normal human keratinocytes (NHK) in comparison to normal skin. These genes are candidates from gene families with known association to tumors, but they have not been reported in the carcinogenesis of BCC yet. In summary, both approaches allow the detection of differentially expressed genes possibly involved in the carcinogenesis of BCC.

Hurpin is a selective inhibitor of lysosomal cathepsin L and protects keratinocytes from ultraviolet-induced apoptosis.

Hurpin (headpin/PI13/serpinB13) is an intracellular, differentially spliced member of the serpin superfamily that has been linked to differentiation and apoptosis of human keratinocytes. It is transiently downregulated by UV light and overexpressed in psoriatic skin lesions. Although it has all of the features of an inhibitory serpin, a productive interaction between hurpin and a proteinase has not yet been reported. Here we demonstrate that hurpin is a potent and selective inhibitor of the archetypal lysosomal cysteine proteinase cathepsin L (catL). Recombinant hurpin inhibits human catL with a stoichiometry of inhibition (SI) of 1.7 and a rate constant k(assoc) of (4.6 +/- 0.14) x 10(5) M(-1) s(-1). It inefficiently inhibits catV and does not inhibit papain, catB, or catK. To investigate the inhibitory mechanism, we determined the P1-P1' bond in the reactive center loop cleaved by catL ((356)Thr-(357)Ser) and expressed variants in which the proximal hinge, P1 residue, or differentially spliced CD loop was mutated. The results of assays using these proteins suggest that inhibition of catL by hurpin occurs via the conventional serpin inhibitory mechanism and that the CD loop plays no role in the process. Finally, it was found that the majority of hurpin is cytosolic and that its overexpression in human keratinocytes confers resistance to UV-induced apoptosis. Given that lysosomal disruption, release of catL, and catL-mediated caspase activation are known to occur in response to cellular stress, we propose that a physiological role of hurpin is to protect epithelial cells from ectopic catL.