Broad spectrum repairing properties of an extract of Aquaphilus dolomiae on in vitro and ex vivo models of injured skin.

BACKGROUND: A biological concentrate was produced from cultures of an Avène aquatic microflora isolate, namely Aquaphilus dolomiae. Some of the beneficial effects on diseased and damaged skin are thought to be due to the presence of this microorganism. AIMS: An extract of A. dolomiae (A. dolomiae extract-G2, ADE-G2) was evaluated for its wound-healing effects using in vitro and ex vivo models of injured skin. METHODS: The effect of ADE-G2 on the proliferation of fibroblasts, migration of keratinocytes and re-epithelialization of ex vivo wounded skin explants was measured. Antimicrobial protection by ADE-G2 was measured by analysing the gene expression of a panel of antimicrobial proteins (AMPs) in keratinocytes (RNASE7, S100A7, DEFB4A/B and DEFb103B), as well as the protein encoded by DEFB4A-B (hBD2) in the medium. RESULTS: ADE-G2 increased fibroblast proliferation and keratinocyte migration, as well as re-epithelialization of wounded ex vivo skin. ADE-G2 induced the expression of all AMP genes analysed in keratinocytes, as well as stimulated the release in to the medium of hBD2 peptide, encoded by DEFB4A/B. CONCLUSIONS: We have shown the broad spectrum of the repairing properties of the A. dolomiae extract, ADE-G2. These results support the use of ADE-G2 as a promising component for use in formulations aimed at repairing skin, limiting wound superinfection and preventing complicated wounds.

Protective effect of Aquaphilus dolomiae extract-G1, ADE-G1, on tight junction barrier function in a Staphylococcus aureus-infected atopic dermatitis model.

BACKGROUND: Atopic dermatitis (AD) is a common skin disease characterized by recurrent pruritic inflammatory skin lesions and defects of the skin barrier. Bacterial infection with Staphylococcus aureus contributes to increased severity of AD by compromising the barrier further. A microorganism component of Avène Thermal Spring Water, Aquaphilus dolomiae, is thought to contribute to some of its beneficial effects to skin, eg AD alleviation. AIMS: Here, we have investigated the effects of an extract of A. dolomiae, A. dolomiae extract-G1 (ADE-G1), on the structural barrier function of keratinocytes, tight junction (TJ) protein expression and the expression of several genes altered in AD patients. METHODS: An epidermal cell culture model mimicking the AD environment and phenotype was used, in which S. aureus-infected cell cultures of normal human epidermal keratinocytes were exposed to a proinflammatory environment. Endpoints measured included the transepithelial electrical resistance (TER) and immunohistological staining of the epidermal TJ proteins, claudin and occludin. Additional analysis was made of several genes known to be differentially regulated in skin from AD patients (C-C motif chemokine ligand 20 (CCL20), interleukin-8 (IL-8), S100 calcium binding protein A7 (S100A7), defensin beta 4 (DEFB4) and filaggrin). RESULTS: Aquaphilus dolomiae extract-G1 strongly increased TER in non-infected cells and provided protection against infection by overcoming the decrease in TER induced by the infection with S. aureus. In infected cells exposed to a pro-inflammatory environment - depicting AD-like conditions - TER protection by ADE-G1 was still observed. Gene expression analysis of infected and pro-inflammatory stimulated cells indicated that ADE-G1 modulated the inflammatory response (induced IL-8 and attenuated CCL20 expression), increased antimicrobial activities (induced DEFB4 and A100A7) and strengthened barrier function (restored filaggrin expression). CONCLUSIONS: ADE-G1 reinforces barrier function and strongly protects TJ barrier disruption induced by bacterial infection and inflammation.

Assessment of the eye irritation potential of chemicals: A comparison study between two test methods based on human 3D hemi-cornea models.

We have recently developed two hemi-cornea models (Bartok et al., Toxicol in Vitro 29, 72, 2015; Zorn-Kruppa et al. PLoS One 9, e114181, 2014), which allow the correct prediction of eye irritation potential of chemicals according to the United Nations globally harmonized system of classification and labeling of chemicals (UN GHS). Both models comprise a multilayered epithelium and a stroma with embedded keratocytes in a collagenous matrix. These two models were compared, using a set of fourteen test chemicals. Their effects after 10 and 60 minutes (min) exposure were assessed from the quantification of cell viability using the MTT reduction assay. The first approach separately quantifies the damage inflicted to the epithelium and the stroma. The second approach quantifies the depth of injury by recording cell death as a function of depth. The classification obtained by the two models was compared to the Draize rabbit eye test and an ex vivo model using rabbit cornea (Jester et al. Toxicol in Vitro. 24, 597-604, 2010). With a 60 min exposure, both of our models are able to clearly differentiate UN GHS Category 1 and UN GHS Category 2 test chemicals.

Epidermal tight junctions in health and disease.

The skin, the largest organ of the body, is an essential barrier that under homeostatic conditions efficiently protects and/or minimizes damage from both environmental (e.g. microorganisms, physical trauma, ultraviolet radiation) and endogenous (e.g., cancers, inflammation) factors. This formidable barrier function resides mainly in the epidermis, a dynamic, highly-stratified epithelium. The epidermis has 2 major barrier structures: stratum corneum, the outmost layer and tight junctions, intercellular junctions that seal adjacent keratinocytes in the stratum granulosum, found below the stratum corneum. In recent years there have been significant advances in our understanding of tight junction function, composition and regulation. Herein we review what is known about tight junctions in healthy skin and keratinocyte culture systems and highlight the dynamic crosstalk observed between tight junctions and the cutaneous immune system. Finally we discuss the preliminary observations suggesting that tight junction function or protein expression may be relevant for the pathogenesis of a number of common cutaneous inflammatory and neoplastic conditions.

Evidence for distinct populations of human Merkel cells.

Merkel cells (MCs) are neuroendocrine cells of unknown origin located in the skin. They are identified at electron microscopic level by electron dense granules, at light microscopic level by the presence of cytokeratins 8, 18, 19 and 20. Contradictory reports concerning the presence of other molecules of epithelial as well as neural origin prompted us to investigate whether there are distinct populations of human MCs. Here, we show the heterogeneous expression of villin, N-CAM, NGF-R, and neurofilaments in MCs. Synaptophysin is found in all MCs but with different intensity, nestin is absent. Expression patterns vary between interfollicular epidermis, hair follicles and glabrous epidermis. We conclude that there are distinct populations of MCs, but all populations contain markers for epithelial as well as neural cells. Putative functions of the distinct populations are discussed.

An ex-vivo oral mucosa infection model for the evaluation of the topical activity of antifungal agents.

Although Nystatin has been used since 1950s as a non-absorbable antifungal agent, there is still no reliable in-vivo data available stating a dose-effect relationship of Nystatin-suspension in the treatment of oropharyngeal infection with Candida albicans. Here, we studied the efficacy of a commercially available topical Nystatin suspension in a new ex-vivo model of candidiasis using porcine oral mucosa. After 48 and 96 h of C. albicans infection, 230 IU Nystatin (standard dosage), 100 IU and 20 IU proved to be equally efficacious. Multiple applications of Nystatin were not superior compared with single application. In dosages of 10 and 0.1 IU the activity of Nystatin suspension against C. albicans was no longer confirmed. In an agar diffusion model, the minimal biocidal concentration of Nystatin proved to be 0.25 IU. Our results suggest that the proposed porcine ex-vivo model is much closer to the in-vivo situation compared with other established in-vitro models of the treatment of muco-cutaneous candidiasis and may provide a substitute for animal models in the investigation of antifungal agents. Additionally, it seems to be a valuable tool for further investigations of the pathogenesis of C. albicans infections.

Pores in the epidermis: aquaporins and tight junctions.

Water homeostasis of the epidermis is important for the appearance and physical properties of the skin, as well as for water balance in the body. It depends on several factors, e.g. barrier quality, uptake of water into the epidermis, concentration of water-retaining humectants, and external humidity. Aquaporins (AQPs) are pores in the plasmamembranes of cells. Monomeric AQPs form barrel-like structures that are primarily water selective, some AQPs also transport glycerol and possibly other small solutes. In the epidermis, AQP3 is the predominant AQP. It is localized mainly in basal but also in suprabasal layers of the epidermis and is permeable for water as well as for glycerol, a humectant. Mice deficient in AQP3 exhibit reduced stratum corneum (SC) hydration and impaired SC barrier recovery after SC removal. In skin diseases associated with elevated transepidermal water loss (TEWL) and reduced SC hydration, altered expression of AQP3 was shown. Tight junctions (TJ) are cell-cell junctions, which play a central role in sealing the intercellular space of cell sheets and thereby establishing a paracellular barrier. Within the TJ, pores are postulated to exist, which allow the controlled diffusion of water and solutes via the paracellular pathway. In the epidermis, TJ structures were demonstrated in the stratum granulosum whereas TJ proteins were found in all viable layers. Mice which overexpress or are deficient of key-proteins of TJ die soon after birth because of a tremendous TEWL. In various skin diseases that are accompanied by elevated TEWL and reduced skin hydration, staining patterns of TJ proteins are altered. This review will summarize our current knowledge of the involvement of AQPs and TJ in the water homeostasis of the epidermis.

Tight junction proteins in the skin.

It has long been accepted that tight junctions (TJ) are crucial for the formation and maintenance of the paracellular barrier and for cell polarity in simple epithelia and endothelia. Moreover, it is long known that they play a role in barrier function of amphibian skin. However, only in recent years were TJ and TJ proteins identified in the epidermis of men and mice. Their involvement in the barrier function of mammalian skin has been shown. This review summarizes our current knowledge about TJ and TJ proteins in mammalian skin.

Caffeine improves barrier function in male skin.

The influence of androgens, especially testosterone and its effector dihydrotestosterone, results in a constitutive disadvantage for male skin, e.g. reduced viability of hair at the scalp and reduced epidermal permeability barrier repair capacity. Dihydrotestosterone can act, among others, as an adenyl cyclase inhibitor. Caffeine on the other hand is an inexpensive and (in regular doses) harmless substance used in various cosmetic products, which can act as a phosphodiesterase inhibitor. To prove the hypothesis that caffeine as a phosphodiesterase inhibitor is able to override testosterone-induced effects on barrier function, we performed a double-blind placebo controlled study with healthy volunteers. In this study, 0.5% caffeine in a hydroxyethylcellulose gel preparation (HEC) was applied on one forearm, HEC without caffeine on the other forearm of male and female volunteers for 7 days and transepidermal water loss (TEWL) was measured before and at the end of the treatment period. Basal TEWL did not differ significantly between male and female subjects but the application of caffeine significantly reduced TEWL in male skin compared with female skin. We conclude that caffeine is beneficial for barrier function in male skin.

Identification and characterization of a novel kind of nuclear protein occurring free in the nucleoplasm and in ribonucleoprotein structures of the "speckle" type.

We have identified, by cDNA cloning and immunodetection, a novel type of constitutive nuclear protein which occurs in diverse vertebrate species, from Xenopus laevis to man, in the form of two different gene products (79.1 kDa and 82.1 kDa in Xenopus, 81.6 kDa and 84.6 kDa in man), remarkably differing in pI (5.4-7.2). This type of protein is characterized by a carboxyterminal domain extremely rich in hydroxyamino acid residues, notably Ser (S), and tetrapeptide repeats of the type XSRS, and hence is termed "domain rich in serines" (DRS) protein. It has been immunolocalized exclusively in the cell nucleus such as in blood cell smears, cultured cells of very different origins and tissue sections, and has also been identified in Xenopus oocyte nuclei, both in sections and by biochemical methods in manually isolated nuclei. In many cell types the protein appears in two different physical states: (i) nuclear granules, identified as ribonucleoprotein (RNP) structures of the "speckle" category by colocalization and cofractionation with certain splicing factors and Sm-proteins, and (ii) in molecules diffusible throughout the nucleoplasm. During mitosis and also in meiosis (Xenopus eggs) the protein is transiently dispersed throughout the cytoplasm but rapidly reaccumulates into the reforming daughter-nuclei. In agreement with this, biochemical experiments have shown that during meiosis (eggs) the protein is recovered in a approximately 11-13S complex of the fraction of soluble cell components. We discuss general constitutive nuclear functions of this apparently ubiquitous and evolutionarily conserved protein.

Evidence that "pinin", reportedly a differentiation-specific desmosomal protein, is actually a widespread nuclear protein.

A protein recently described as a desmosome-specific molecule involved in anchoring intermediate-sized filaments (IFs) to the desmosomal plaque, and hence named "pinin" [43], has been known in our laboratory for several years as a strictly nuclear protein occurring in a wide range of cell types, including many that are totally devoid of desmosomes. Using a series of specific antibodies we have localized the protein in the nucleoplasm of cultured cells, blood cells and solid tissues of diverse vertebrate species, from oocytes to erythrocytes of amphibia and from liver to connective tissue and fibroblasts in mammals. Desmosomes have consistently been negative, and the nuclear specificity of the immunolocalization reactions has also been directly demonstrated by double-label immunofluorescence microscopy. From our results we conclude that this nuclear protein, characterized by a domain exceptionally rich in serine residues and hence termed DRS-protein, occurs in at least two genetically different forms in a diffusible state as well as in special ribonucleoprotein-particles, "speckles" [6], and is a widespread if not ubiquitous nuclear protein. Consequently it must serve nuclear functions rather than "pinning" IFs to plasma membranes and does not provide a new reliable marker for desmosomes and epithelial or myocardial differentiation.