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.

Synergistic impacts by an invasive amphipod and an invasive fish explain native gammarid extinction.

BACKGROUND: Worldwide freshwater ecosystems are increasingly affected by invasive alien species. In particular, Ponto-Caspian gobiid fishes and amphipods are suspected to have pronounced effects on aquatic food webs. However, there is a lack of systematic studies mechanistically testing the potential synergistic effects of invasive species on native fauna. In this study we investigated the interrelations between the invasive amphipod Dikerogammarus villosus and the invasive fish species Neogobius melanostomus in their effects on the native amphipod Gammarus pulex. We hypothesized selective predation by the fish as a driver for displacement of native species resulting in potential extinction of G. pulex. The survival of G. pulex in the presence of N. melanostomus in relation to the presence of D. villosus and availability of shelter was analyzed in the context of behavioural differences between the amphipod species. RESULTS: Gammarus pulex had a significantly higher susceptibility to predation by N. melanostomus compared to D. villosus in all experiments, suggesting preferential predation by this fish on native gammarids. Furthermore, the presence of D. villosus significantly increased the vulnerability of G. pulex to fish predation. Habitat structure was an important factor for swimming activity of amphipods and their mortality, resulting in a threefold decrease in amphipods consumed with shelter habitat structures provided. Behavioral differences in swimming activity were additionally responsible for higher predation rates on G. pulex. Intraguild predation could be neglected within short experimental durations. CONCLUSIONS: The results of this study provide evidence for synergistic effects of the two invasive Ponto-Caspian species on the native amphipod as an underlying process of species displacements during invasion processes. Prey behaviour and monotonous habitat structures additionally contribute to the decline of the native gammarid fauna in the upper Danube River and elsewhere.

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.

Effects of sampling techniques on population assessment of invasive round goby Neogobius melanostomus.

In this study, a comparison of point abundance sampling (PAS) electrofishing, angling with two different hook sizes and trap-based fishing was performed in a non-wadeable river to analyse their effects on catch per unit effort (CPUE) and population characteristics of invasive round goby Neogobius melanostomus. PAS electrofishing was identified as the most effective (mean ± s.e. CPUE = 57 ± 4 N. melanostomus min(-1) ) and least selective method in terms of size, feeding status and species composition. Angling had the second highest CPUE, but was more size selective and resulted in a higher proportion of males compared to electrofishing [overall sex ratio angling (female:male) = 1:0.92, electrofishing 1:0.65]. Owing to low CPUE (0.012 ± 0.004) and low frequency of occurrence, minnow traps were least suitable for N. melanostomus population assessment. The results of this study suggest that a higher degree of standardization and inter-calibration is useful to achieve better comparability of population data of invasive N. melanostomus and other benthic fish species.

First evidence for interspecific hybridization between invasive goby species Neogobius fluviatilis and Neogobius melanostomus (Teleostei: Gobiidae: Benthophilinae).

Two hybrids between the monkey goby Neogobius fluviatilis and the round goby Neogobius melanostomus from the Rhine River were identified by genotyping and morphological comparison. These are the first records of goby-hybrids outside the parent species' native ranges worldwide.

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.

Interference of cytokeratin-20 and mammaglobin-reverse-transcriptase polymerase chain assays designed for the detection of disseminated cancer cells.

Several reverse-transcriptase polymerase chain reaction (rtPCR) assays have been designed for the detection of disseminated cancer cells. The specificity of these discussed molecular approaches is controversial. Biological interference of the cytokeratin-20 and mammaglobin rtPCR assays has been investigated. Cell lines of different lineages and bone marrow and peripheral stem cells from patients without epithelial cancer have been examined for the transcription of the cytokeratin-20 (CK20) and mammaglobin messages prior to and after stimulation with different cytokines in a total of 370 liquid cultures. Amplification of both messages from clinical samples prior to stimulation does not support the high specificity for the detection of disseminated epithelial cancer cells as reported. Cytokeratin-20 was amplified from the chronic myeloic leukemia (CML)-derived line K562. Transcription was not influenced by cytokines, either in cell-line experiments or in clinical samples. The thesis of a low-level background transcription in granulocytes is supported. Mammaglobin was induced in cell lines significantly by GM-CSF and in clinical samples additionally by several more cytokines. These results indicate that under certain conditions involving cytokine production, the use of mammaglobin rtPCR for the detection of epithelial cancer cells could be limited. In conclusion, the mechanism of interference of both rtPCR assays are completely different and further research is necessary before the cytokeratin-20 or mammaglobin rtPCR could become standard methods for the detection of disseminated epithelial cancer cells. These factors leading to so-called false-positive results have to be considered in future applications of rtPCR for the detection of minimal residual disease.

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.

Identification of the omega4400 regulatory region, a developmental promoter of Myxococcus xanthus.

Omega4400 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn5 lac omega4400. The DNA upstream of the omega4400 insertion has been cloned, the promoter has been localized, and a partial open reading frame has been identified. From the deduced amino acid sequence of the partial open reading frame, the gene disrupted by Tn5 lac omega4400 may encode a protein with an ATP- or GTP-binding site. Expression of the gene begins 6 to 12 h after starvation initiates development, as measured by beta-galactosidase production in cells containing Tn5 lac omega4400. The putative transcriptional start site was mapped, and deletion analysis has shown that DNA downstream of -101 bp is sufficient for C-signal-dependent, developmental activation of this promoter. A deletion to -76 bp eliminated promoter activity, suggesting the involvement of an upstream activator protein. The promoter may be transcribed by RNA polymerase containing a novel sigma factor, since a mutation in the M. xanthus sigB or sigC gene did not affect Tn5 lac omega4400 expression and the DNA sequence upstream of the transcriptional start site did not match the sequence of any M. xanthus promoter transcribed by a known form of RNA polymerase. However, the omega4400 promoter does contain the sequence 5'-CATCCCT-3' centered at -49 and the C-signal-dependent omega4403 promoter also contains this sequence at the same position. Moreover, the two promoters match at five of six positions in the -10 regions, suggesting that these promoters may share one or more transcription factors. These results begin to define the cis-acting regulatory elements important for cell-cell interaction-dependent gene expression during the development of a multicellular prokaryote.

The composition of NF-defined emulsifiers: sorbitan monolaurate, monopalmitate, monostearate, monooleate, polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

Using the analytical constants for sorbitan monolaurate, monopalmitate, monostearate, and monooleate given in the National Formulary (NF), calculations were carried out that indicated that these emulsifiers are esters of sorbitol mono- and dianhydrides. Contrary to the NF description, no significant amount of sorbitol ester was calculated to be present, in agreement with recent experimental findings. Further calculations were made using the NF-defined analytical constants of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, which indicate that these emulsifiers are esters primarily of polyoxyethylated sorbitol monoanhydride (i.e., sorbitan), with lesser quantities of polyoxyethylated esters of sorbitol dianhydride. Since all hydroxyl groups of the polysorbates are primary, random distribution of acyl groups on the available hydroxyls can be assumed, and the likely composition of these emulsifiers can be calculated. The most abundant compounds appear to be polyoxyethylated sorbitan mono-, di-, and triesters. Although the polysorbates are stated to contain 20 moles of ethylene oxide per mole of ester, the oxyethylene contents stated in the Food Chemicals Codex reveal that somewhat more than 20 moles of ethylene oxide are combined.

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.

Phloem Loading by the PmSUC2 Sucrose Carrier from Plantago major Occurs into Companion Cells.

High levels of mRNA for the sucrose-H+ symporter PmSUC2 have been found in the vascular bundles of petioles from Plantago major. The possible role of PmSUC2 in phloem loading was studied with antiserum raised against the recombinant PmSUC2 protein. This antiserum labeled a single 35-kD protein band in detergent extracts of P. major vascular bundles. It showed no cross-reaction with the P. major sucrose carrier PmSUC1, which was tested with detergent extracts from plasma membranes of transgenic yeast strains containing either the P. major sucrose transporter PmSUC1 or PmSUC2. The antiserum was used to determine the site of PmSUC2 expression in leaves, petioles, and roots of P. major. In cross-sections and longitudinal sections, the PmSUC2 protein was found in only one single cell type. These cells were identified as companion cells because they are nucleated, contain a dense cytoplasm, and are always adjacent to a sieve element. The labeled cells had the same longitudinal extension as did their sister sieve elements and always ended next to the sieve plates, which were characterized by specific staining. PmSUC2 mRNA and PmSUC2 protein were also detected in P. major roots. The function of PmSUC2 in the different organs and its role in phloem loading are discussed.