Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Impact of the Human Bacterial Environment on Mycobacteriosis and Allergy

My work describes two sectors of the human bacterial environment: 1. The sources of exposure to infectious non-tuberculous mycobacteria. 2. Bacteria in dust, reflecting the airborne bacterial exposure in environments protecting from or predisposing to allergic disorders. Non-tuberculous mycobacteria (NTM) transmit to humans and animals from the environment. Infection by NTM in Finland has increased during the past decade beyond that by Mycobacterium tuberculosis. Among the farm animals, porcine mycobacteriosis is the predominant NTM disease in Finland. Symptoms of mycobacteriosis are found in 0.34 % of slaughtered pigs. Soil and drinking water are suspected as sources for humans and bedding materials for pigs. To achieve quantitative data on the sources of human and porcine NTM exposure, methods for quantitation of environmental NTM are needed. We developed a quantitative real-time PCR method, utilizing primers targeted at the 16S rRNA gene of the genus of Mycobacterium. With this method, I found in Finnish sphagnum peat, sandy soils and mud high contents of mycobacterial DNA, 106 to 107 genome equivalents per gram. A similar result was obtained by a method based on the Mycobacterium-specific hybridization of 16S rRNA. Since rRNA is found mainly in live cells, this result shows that the DNA detected by qPCR mainly represented live mycobacteria. Next, I investigated the occurrence of environmental mycobacteria in the bedding materials obtained from 5 pig farms with high prevalence (>4 %) of mycobacteriosis. When I used for quantification the same qPCR methods as for the soils, I found that piggery samples contained non-mycobacterial DNA that was amplified in spite of several mismatches with the primers. I therefore improved the qPCR assay by designing Mycobacterium-specific detection probes. Using the probe qPCR assay, I found 105 to 107 genome equivalents of mycobacterial DNA in unused bedding materials and up to 1000 fold more in the bedding collected after use in the piggery. This result shows that there was a source of mycobacteria in the bedding materials purchased by the piggery and that mycobacteria increased in the bedding materials during use in the piggery. Allergic diseases have reached epidemic proportions in urbanized countries. At the same time, childhood in rural environment or simple living conditions appears to protect against allergic disorders. Exposure to immunoreactive microbial components in rural environments seems to prevent allergies. I searched for differences in the bacterial communities of two indoor dusts, an urban house dust shown to possess immunoreactivity of the TH2-type and a farm barn dust with TH1-activity. The immunoreactivities of the dusts were revealed by my collaborators, in vitro in human dendritic cells and in vivo in mouse. The dusts accumulated >10 years in the respiratory zone (>1.5 m above floor), thus reflecting the long-term content of airborne bacteria at the two sites. I investigated these dusts by cloning and sequencing of bacterial 16S rRNA genes from dust contained DNA. From the TH2-active urban house dust, I isolated 139 16S rRNA gene clones. The most prevalent genera among the clones were Corynebacterium (5 species, 34 clones), Streptococcus (8 species, 33 clones), Staphylococcus (5 species, 9 clones) and Finegoldia (1 species, 9 clones). Almost all of these species are known as colonizers of the human skin and oral cavity. Species of Corynebacterium and Streptococcus have been reported to contain anti-inflammatory lipoarabinomannans and immunmoreactive beta-glucans respectively. Streptococcus mitis, found in the urban house dust is known as an inducer of TH2 polarized immunity, characteristic of allergic disorders. I isolated 152 DNA clones from the TH1-active farm barn dust and found species quite different from those found from the urban house dust. Among others, I found DNA clones representing Bacillus licheniformis, Acinetobacter lwoffii and Lactobacillus each of which was recently reported to possess anti-allergy immunoreactivity. Moreover, the farm barn dust contained dramatically higher bacterial diversity than the urban house dust. Exposure to this dust thus stimulated the human dendritic cells by multiple microbial components. Such stimulation was reported to promote TH1 immunity. The biodiversity in dust may thus be connected to its immunoreactivity. Furthermore, the bacterial biomass in the farm barn dust consisted of live intact bacteria mainly. In the urban house dust only ~1 % of the biomass appeared as intact bacteria, as judged by microscoping. Fragmented microbes may possess bioactivity different from that of intact cells. This was recently shown for moulds. If this is also valid for bacteria, the different immunoreactivities of the two dusts may be explained by the intactness of dustborne bacteria. Based on these results, we offer three factors potentially contributing to the polarized immunoreactivities of the two dusts: (i) the species-composition, (ii) the biodiversity and (iii) the intactness of the dustborne bacterial biomass. The risk of childhood atopic diseases is 4-fold lower in the Russian compared with the Finnish Karelia. This difference across the country border is not explainable by different geo-climatic factors or genetic susceptibilities of the two populations. Instead, the explanation must be lifestyle-related. It has already been reported that the microbiological quality of drinking water differs on the two sides of the borders. In collaboration with allergists, I investigated dusts collected from homes in the Russian Karelia and in the Finnish Karelia. I found that bacterial 16S rRNA genes cloned from the Russian Karelian dusts (10 homes, 234 clones) predominantly represented Gram-positive taxa (the phyla Actinobacteria and Firmicutes, 67%). The Russian Karelian dusts contained nine-fold more of muramic acid (60 to 70 ng mg-1) than the Finnish Karelian dusts (3 to 11 ng mg-1). Among the DNA clones isolated from the Finnish side (n=231), Gram-negative taxa (40%) outnumbered the Gram-positives (34%). Out of the 465 DNA clones isolated from the Karelian dusts, 242 were assigned to cultured validly described bacterial species. In Russian Karelia, animal-associated species e.g. Staphylococcus and Macrococcus were numerous (27 clones, 14 unique species). This finding may connect to the difference in the prevalence of allergy, as childhood contacts with pets and farm animals have been connected with low allergy risk. Plant-associated bacteria and plant-borne 16S rRNA genes (chloroplast) were frequent among the DNA clones isolated from the Finnish Karelia, indicating components originating from plants. In conclusion, my work revealed three major differences between the bacterial communtites in the Russian and in the Finnish Karelian homes: (i) the high prevalence of Gram-positive bacteria on the Russian side and of Gram-negative bacteria on the Finnish side and (ii) the rich presence of animal-associated bacteria on the Russian side whereas (iii) plant-associated bacteria prevailed on the Finnish side. One or several of these factors may connect to the differences in the prevalence of allergy.

SIRT6 Promotes COX-2 Expression and Acts as an Oncogene in Skin Cancer

SIRT6 is a SIR2 family member that regulates multiple molecular pathways involved in metabolism, genomic stability, and aging. It has been proposed previously that SIRT6 is a tumor suppressor in cancer. Here, we challenge this concept by presenting evidence that skin-specific deletion of SIRT6 in the mouse inhibits skin tumorigenesis. SIRT6 promoted expression of COX-2 by repressing AMPK signaling, thereby increasing cell proliferation and survival in the skin epidermis. SIRT6 expression in skin keratinocytes was increased by exposure to UVB light through activation of the AKT pathway. Clinically, we found that SIRT6 was upregulated in human skin squamous cell carcinoma. Taken together, our results provide evidence that SIRT6 functions as an oncogene in the epidermis and suggest greater complexity to its role in epithelial carcinogenesis. (C) 2014 AACR.

Microbial community profiling shows dysbiosis in the lesional skin of Vitiligo subjects

Healthy human skin harbours a diverse array of microbes that comprise the skin microbiome. Commensal bacteria constitute an important component of resident microbiome and are intricately linked to skin health. Recent studies describe an association between altered skin microbial community and epidemiology of diseases, like psoriasis, atopic dermatitis etc. In this study, we compare the differences in bacterial community of lesional and non-lesional skin of vitiligo subjects. Our study reveals dysbiosis in the diversity of microbial community structure in lesional skin of vitiligo subjects. Although individual specific signature is dominant over the vitiligo-specific microbiota, a clear decrease in taxonomic richness and evenness can be noted in lesional patches. Investigation of community specific correlation networks reveals distinctive pattern of interactions between resident bacterial populations of the two sites (lesional and non-lesional). While Actinobacterial species constitute the central regulatory nodes (w.r.t. degree of interaction) in non-lesional skin, species belonging to Firmicutes dominate on lesional sites. We propose that the changes in taxonomic characteristics of vitiligo lesions, as revealed by our study, could play a crucial role in altering the maintenance and severity of disease. Future studies would elucidate mechanistic relevance of these microbial dynamics that can provide new avenues for therapeutic interventions.

Skin Color-Based Video Segmentation under Time-Varying Illumination

A novel approach for real-time skin segmentation in video sequences is described. The approach enables reliable skin segmentation despite wide variation in illumination during tracking. An explicit second order Markov model is used to predict evolution of the skin-color (HSV) histogram over time. Histograms are dynamically updated based on feedback from the current segmentation and predictions of the Markov model. The evolution of the skin-color distribution at each frame is parameterized by translation, scaling and rotation in color space. Consequent changes in geometric parameterization of the distribution are propagated by warping and resampling the histogram. The parameters of the discrete-time dynamic Markov model are estimated using Maximum Likelihood Estimation, and also evolve over time. The accuracy of the new dynamic skin color segmentation algorithm is compared to that obtained via a static color model. Segmentation accuracy is evaluated using labeled ground-truth video sequences taken from staged experiments and popular movies. An overall increase in segmentation accuracy of up to 24% is observed in 17 out of 21 test sequences. In all but one case the skin-color classification rates for our system were higher, with background classification rates comparable to those of the static segmentation.

Substance P induces histamine release from human pulmonary mast cells.

Substance P elicits histamine release from human skin and rodent mast cells. Since neuropeptide-mediated reflexes may be important in asthma, we examined the ability of substance P to stimulate human mast cells obtained at bronchoalveolar lavage (BAL). BAL samples were obtained at routine bronchoscopy from 35 non-preselected patients. Histamine release experiments were performed in a standard manner using substance P and the calcium ionophore A23187. Both substance P (50 μM) and A23187 caused histamine release (median 26.7%, range 6.2–62.8% and 32.1%, 7.7–56.8% respectively) which was significantly greater (P < 0.0001) than the spontaneous release (median 15.6%, range 4.1–33.4%), i.e. that in the absence of any stimulus. Substance P induced histamine release was via an energy dependent process and was blocked by preincubation with antimycin A. A significant correlation was observed between substance P induced release and spontaneous release but was not observed with A23187 induced release. Mast cell counts correlated significantly with substance P induced release but not with spontaneous or A23187 induced release. The substance P induced histamine secretion was elicited at similar concentrations to those used with rodent and human skin mast cells. Asthma is associated with increased numbers of mast cells which have both increased spontaneous and stimulated secretory responses. Thus, in vivo, the bronchoconstrictor action of substance P may in part result from activation of mast cells in the bronchial lumen.

Skin permeation and metabolism of di(2-ethylhexyl) phthalate (DEHP).

Phthalates are suspected to be endocrine disruptors. Di(2-ethylhexyl) phthalate (DEHP) is assumed to have low dermal absorption; however, previous in vitro skin permeation studies have shown large permeation differences. Our aims were to determine DEHP permeation parameters and assess extent of skin DEHP metabolism among workers highly exposed to these lipophilic, low volatile substances. Surgically removed skin from patients undergoing abdominoplasty was immediately dermatomed (800 μm) and mounted on flow-through diffusion cells (1.77 cm(2)) operating at 32°C with cell culture media (aqueous solution) as the reservoir liquid. The cells were dosed either with neat DEHP or emulsified in aqueous solution (166 μg/ml). Samples were analysed by HPLC-MS/MS. DEHP permeated human viable skin only as the metabolite MEHP (100%) after 8h of exposure. Human skin was able to further oxidize MEHP to 5-oxo-MEHP. Neat DEHP applied to the skin hardly permeated skin while the aqueous solution readily permeated skin measured in both cases as concentration of MEHP in the receptor liquid. DEHP pass through human skin, detected as MEHP only when emulsified in aqueous solution, and to a far lesser degree when applied neat to the skin. Using results from older in vitro skin permeation studies with non-viable skin may underestimate skin exposures. Our results are in overall agreement with newer phthalate skin permeation studies.

Netting neutrophils in skin wounds recruit and activate plasmacytoid dentritic cells

Les mécanismes qui régulent le processus de guérison de la peau lésée ne sont pas entièrement compris. Nous avons précédemment montré que les cellules dendritiques plasmocytoïdes (pDCs) sont normalement absentes de la peau saine mais infiltrent rapidement la peau humaine ainsi que celle des souris après une blessure cutanée. Après avoir infiltré la peau, ces pDCs sont capables de détecter les acides nucléiques par l'expression des récepteurs de type Toll 7 et 9 ce qui les active à produire de 1' interféron (IFN) de type I. Ce processus est primordial pour la re- épithélisation des blessures cutanées. Cependant, les mécanismes conduisant à l'infiltration et à 1'activation des pDCs restent inconnus. Dans notre projet, nous montrons que la chimiokine CxcllO est responsable de l'infiltration des pDCs. De façon importante, nous démontrons que les neutrophiles qui infiltrent également la peau lésée sont la source majeure de cette chimiokine. La déplétion des neutrophiles abolit d'ailleurs le recrutement des pDCs confirmant ainsi que CxcllO produit par les neutrophiles est responsable de l'infiltration des pDCs dans la peau endommagée. De façon intéressante, nous avons trouvé que CxcllO en plus de son activité chimiotactique, est capable de former des complexes avec l'ADN et d'activer ainsi les pDCs à produire de l'IFN de type I. De plus, nous avons observé que les neutrophiles qui infiltrent la peau forment des Neutrophil Extracellular Traps (NETs). Ces NETs sont constitués de filaments extracellulaires d'ADN recouverts par de nombreuses protéines principalement d'origine granulaire. D'une manière frappante, le blocage de la NETose ou l'utilisation de souris déficientes pour la formation de NETs altère le recrutement et l'activation des pDCs ainsi que la réponse inflammatoire qui en découle ainsi que le processus de re-epithélisation qui s'ensuit. En prenant en compte toutes ces données, nos résultats démontrent que suite à une blessure de la peau, les neutrophiles par la production de CxcllO contrôlent l'infiltration des pDCs dans la peau lésée et par la formation de NETs, promeuvent l'activation des pDCs. Notre étude fournit donc de nouvelles informations sur les mécanismes de guérison de la peau et ouvre de nouvelles perspectives thérapeutiques quant à la réparation tissulaire de la peau soit dans le but de l'amplifier ou de l'inhiber. -- The mechanisms that regulate healing of the injured skin are not well understood. We have previously shown that plasmacytoid dendritic cells (pDCs) are normally absent from the healthy skin, but rapidly infiltrate both murine and human skin upon injury. Upon skin infiltration, pDCs sense nucleic acids via TLR7/TLR9 and are activated to produce type I interferon (IFN), a process that is crucial for re-epithelialisation of skin wounds. However, the mechanisms that drive pDCs recruitment and activation in injured skin remain unclear. We show that CxcllO is responsible for pDCs infiltration. Importantly, we demonstrate that skin infiltrating neutrophils are the major source of this chemokine. Neutrophils depletion completely abrogated pDCs recruitment confirming that CxcllO- driven pDCs recruitment is controlled by neutrophils. Interestingly, CxcllO was also found to form complexes with DNA and to activate pDCs to produce Type I IFN in addition to its chemotactic activity. Moreover, we observed that infiltrating neutrophils release Neutrophils Extracellular Traps (NETs) composed of DNA filaments decorated with neutrophils-derived proteins. Strikingly, blocking NETosis or using mice deficient for NETs production impaired pDCs recruitment and activation as well as the subsequent inflammatory response and the re-epithelialisation process. Altogether, these data demonstrate that upon skin injury, neutrophils control pDCs infiltration into the injured skin by the release of CxcllO and via the production of NETs, they allow complex formation between CxcllO and NET-DNA leading to pDCs activation. Our findings provide new insights into the mechanisms of wound healing and open new avenues for potential therapeutic interventions to boost or inhibit wound repair in the skin.

Oestrogenic activity of benzyl salicylate, benzyl benzoate and butylphenylmethylpropional (Lilial) in MCF7 human breast cancer cells in vitro

Benzyl salicylate, benzyl benzoate and butylphenylmethylpropional (Lilial) are added to bodycare cosmetics used around the human breast. We report here that all three compounds possess oestrogenic activity in assays using the oestrogen-responsive MCF7 human breast cancer cell line. At 3 000 000-fold molar excess, they were able to partially displace [H-3]oestradiol from recombinant human oestrogen receptors ER alpha and ER beta, and from cytosolic ER of MCF7 cells. At concentrations in the range of 5 x 10(-5) to 5 x 10(-4) M, they were able to increase the expression of a stably integrated oestrogen-responsive reporter gene (ERE-CAT) and of the endogenous oestrogen-responsive pS2 gene in MCF7 cells, albeit to a lesser extent than with 10(-8) M 17 beta-oestradiol. They increased the proliferation of oestrogen-dependent MCF7 cells over 7 days, which could be inhibited by the antioestrogen fulvestrant, suggesting an ER-mediated mechanism. Although the extent of stimulation of proliferation over 7 days was lower with these compounds than with 10(-8) M 17 beta-oestradiol, given a longer time period of 35 days the extent of proliferation with 10(-4) M benzyl salicylate, benzyl benzoate or butylphenylmethylpropional increased to the same magnitude as observed with 10(-8) M 17 beta-oestradiol over 14 days. This demonstrates that benzyl salicylate, benzyl benzoate and butylphenylmethylpropional are further chemical components of cosmetic products which give oestrogenic responses in a human breast cancer cell line in culture. Further research is now needed to investigate whether oestrogenic responses are detectable using in vivo models and the extent to which these compounds might be absorbed through human skin and might enter human breast tissues. Copyright (C) 2009 John Wiley & Sons, Ltd.

Iron-regulated surface determinant protein a mediates adhesion of staphylococcus aureus to human corneocyte envelope proteins

The ability of Staphylococcus aureus to colonize the human nares is a crucial prerequisite for disease. IsdA is a major S. aureus surface protein that is expressed during human infection and required for nasal colonization and survival on human skin. In this work, we show that IsdA binds to involucrin, loricrin, and cytokeratin K10, proteins that are present in the cornified envelope of human desquamated epithelial cells. To measure the forces and dynamics of the interaction between IsdA and loricrin (the most abundant protein of the cornified envelope), single-molecule force spectroscopy was used, demonstrating high-specificity binding. IsdA acts as a cellular adhesin to the human ligands, promoting whole-cell binding to immobilized proteins, even in the absence of other S. aureus components (as shown by heterologous expression in Lactococcus lactis). Inhibition experiments revealed the binding of the human ligands to the same IsdA region. This region was mapped to the NEAT domain of IsdA. The NEAT domain also was found to be required for S. aureus whole-cell binding to the ligands as well as to human nasal cells. Thus, IsdA is an important adhesin to human ligands, which predominate in its primary ecological niche.

Paraben esters: review of recent studies of endocrine toxicity, absorption, esterase and human exposure, and discussion of potential human health risks

This toxicology update reviews research over the past four years since publication in 2004 of the first measurement of intact esters of p-hydroxybenzoic acid (parabens) in human breast cancer tissues, and the suggestion that their presence in the human body might originate from topical application of bodycare cosmetics. The presence of intact paraben esters in human body tissues has now been confirmed by independent measurements in human urine, and the ability of parabens to penetrate human skin intact without breakdown by esterases and to be absorbed systemically has been demonstrated through studies not only in vitro but also in vivo using healthy human subjects. Using a wide variety of assay systems in vitro and in vivo, the oestrogen agonist properties of parabens together with their common metabolite (p-hydroxybenzoic acid) have been extensively documented, and, in addition, the parabens have now also been shown to possess androgen antagonist activity, to act as inhibitors of sulfotransferase enzymes and to possess genotoxic activity. With the continued use of parabens in the majority of bodycare cosmetics, there is a need to carry out detailed evaluation of the potential for parabens, together with other oestrogenic and genotoxic co-formulants of bodycare cosmetics, to increase female breast cancer incidence, to interfere with male reproductive functions and to influence development of malignant melanoma which has also recently been shown to be influenced by oestrogenic stimulation. Copyright (C) 2008 John Wiley & Sons, Ltd.

Cannabinoids inhibit human keratinocyte proliferation through a non-CB1/CB2 mechanism and have a potential therapeutic value in the treatment of psoriasis

Background: Cannabinoids from cannabis (Cannabis sativa) are anti-inflammatory and have inhibitory effects on the proliferation of a number of tumorigenic cell lines, some of which are mediated via cannabinoid receptors. Cannabinoid (CB) receptors are present in human skin and anandamide, an endogenous CB receptor ligand, inhibits epidermal keratinocyte differentiation. Psoriasis is an inflammatory disease also characterised in part by epidermal keratinocyte hyper-proliferation. Objective: We investigated the plant cannabinoids Delta-9 tetrahydrocannabinol, cannabidiol, cannabinol and cannabigerol for their ability to inhibit the proliferation of a hyper-proliferating human keratinocyte cell line and for any involvement of cannabinoid receptors. Methods: A keratinocyte proliferation assay was used to assess the effect of treatment with cannabinoids. Cell integrity and metabolic competence confirmed using lactate-dehydrogenase and adenosine tri-phosphate assays. To determine the involvement of the receptors, specific agonist and antagonist were used in conjunction with some phytocannabinoids. Western blot and RT-PCR analysis confirmed presence of CB1 and CB2 receptors. Results: The cannabinoids tested all inhibited keratinocyte proliferation in a concentration-dependent manner. The selective CB2 receptor agonists JWH015 and BML190 elicited only partial inhibition, the non-selective CB agonist HU210 produced a concentration-dependent response, the activity of theses agonists were not blocked by either C81 /C82 antagonists. Conclusion: The results indicate that while CB receptors may have a circumstantial role in keratinocyte proliferation, they do not contribute significantly to this process. Our results show that cannabinoids inhibit keratinocyte proliferation, and therefore support a potential role for cannabinoids in the treatment of psoriasis. (c) 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Liposomes and skin: From drug delivery to model membranes

The early eighties saw the introduction of liposomes as skin drug delivery systems, initially promoted primarily for localised effects with minimal systemic delivery. Subsequently, a novel ultradeformable vesicular system (termed "Transfersomes" by the inventors) was reported for transdermal delivery with an efficiency similar to subcutaneous injection. Further research illustrated that the mechanisms of liposome action depended on the application regime and the vesicle composition and morphology. Ethical, health and supply problems with human skin have encouraged researchers to use skin models. 'IYaditional models involved polymer membranes and animal tissue, but whilst of value for release studies, such models are not always good mimics for the complex human skin barrier, particularly with respect to the stratum corneal intercellular lipid domains. These lipids have a multiply bilayered organization, a composition and organization somewhat similar to liposomes, Consequently researchers have used vesicles as skin model membranes. Early work first employed phospholipid liposomes and tested their interactions with skin penetration enhancers, typically using thermal analysis and spectroscopic analyses. Another approach probed how incorporation of compounds into liposomes led to the loss of entrapped markers, analogous to "fluidization" of stratum corneum lipids on treatment with a penetration enhancer. Subsequently scientists employed liposomes formulated with skin lipids in these types of studies. Following a brief description of the nature of the skin barrier to transdermal drug delivery and the use of liposomes in drug delivery through skin, this article critically reviews the relevance of using different types of vesicles as a model for human skin in permeation enhancement studies, concentrating primarily on liposomes after briefly surveying older models. The validity of different types of liposome is considered and traditional skin models are compared to vesicular model membranes for their precision and accuracy as skin membrane mimics. (c) 2008 Elsevier B.V. All rights reserved.

Can drug-bearing liposomes penetrate intact skin?

Using liposomes to deliver drugs to and through human skin is controversial, as their function varies with type and composition. Thus they may act as drug carriers controlling release of the medicinal agent. Alternatively, they may provide a localized depot in the skin so minimizing systemic effects or can be used for targeting delivery to skin appendages (hair follicles and sweat glands). Liposomes may also enhance transdermal drug delivery, increasing systemic drug concentrations. With such a multiplicity of functions, it is not surprising that mechanisms of liposomal delivery of therapeutic agents to and through the skin are unclear. Accordingly, this article provides an overview of the modes and mechanisms of action of different vesicles as drug delivery vectors in human skin. Our conclusion is that vesicles, depending on the composition and method of preparation, can vary with respect to size, lamellarity, charge, membrane fluidity or elasticity and drug entrapment. This variability allows for multiple functions ranging from local to transdermal effects. Application to dissimilar skins (animal or human) via diverse protocols may reveal different mechanisms of action with possible vesicle skin penetration reaching different depths, from surface assimilation to (rarely) the viable tissue and subsequent systemic absorption.

Human microbiota in health and disease

Each human body plays host to a microbial population which is both numerically vast (at around 1014 microbial cells) and phenomenally diverse (over 1,000 species). The majority of the microbial species in the gut have not been cultured but the application of culture-independent approaches for high throughput diversity and functionality analysis has allowed characterisation of the diverse microbial phylotypes present in health and disease. Studies in monozygotic twins, showing that these retain highly similar microbiota decades after birth and initial colonisation, are strongly indicative that diversity of the microbiome is host-specific and affected by the genotype. Microbial diversity in the human body is reflected in both richness and evenness. Diversity increases steeply from birth reaching its highest point in early adulthood, before declining in older age. However, in healthy subjects there appears to be a core of microbial phylotypes which remains relatively stable over time. Studies of individuals from diverse geopraphies suggest that clusters of intestinal bacterial groups tend to occur together, constituting ‘enterotypes’. So variation in intestinal microbiota is stratified rather than continuous and there may be a limited number of host/microbial states which respond differently to environmental influences. Exploration of enterotypes and functional groups may provide biomarkers for disease and insights into the potential for new treatments based on manipulation of the microbiome. In health, the microbiota interact with host defences and exist in harmonious homeostasis which can then be disturbed by invading organisms or when ‘carpet bombing’ by antibiotics occurs. In a portion of individuals with infections, the disease will resolve itself without the need for antibiotics and microbial homeostasis with the host’s defences is restored. The administration of probiotics (live microorganisms which when administered in adequate amounts confer a health benefit on the host) represents an artificial way to enhance or stimulate these natural processes. The study of innate mechanisms of antimicrobial defence on the skin, including the production of numerous antimicrobial peptides (AMPs), has shown an important role for skin commensal organisms. These organisms may produce AMPs, and also amplify the innate immune responses to pathogens by activating signalling pathways and processing host produced AMPs. Research continues into how to enhance and manipulate the role of commensal organisms on the skin. The challenges of skin infection (including diseases caused by multiply resistant organisms) and infestations remain considerable. The potential to re-colonise the skin to replace or reduce pathogens, and exploring the relationship between microbiota elsewhere and skin diseases are among a growing list of research targets. Lactobacillus species are among the best known ‘beneficial’ bacterial members of the human microbiota. Of the approximately 120 species known, about 15 are known to occur in the human vagina. These organisms have multiple properties, including the production of lactic acid, hydrogen peroxide and bacteriocins, which render the vagina inhospitable to potential pathogens. Depletion of the of the normal Lactobacillus population and overgrowth of vaginal anaerobes, accompanied by the loss of normal vaginal acidity can lead to bacterial vaginosis – the commonest cause of abnormal vaginal discharge in women. Some vaginal anaerobes are associated with the formation of vaginal biofilms which serve to act as a reservoir of organisms which persists after standard antibiotic therapy of bacterial vaginosis and may help to account for the characteristically high relapse rate in the condition. Administration of Lactobacillus species both vaginally and orally have shown beneficial effects in the treatment of bacterial vaginosis and such treatments have an excellent overall safety record. Candida albicans is a frequent coloniser of human skin and mucosal membranes, and is a normal part of the microbiota in the mouth, gut and vagina. Nevertheless Candida albicans is the most common fungal pathogen worldwide and is a leading cause of serious and often fatal nosocomial infections. What turns this organism from a commensal to a pathogen is a combination of increasing virulence in the organism and predisposing host factors that compromise immunity. There has been considerable research into the use of probiotic Lactobacillus spp. in vaginal candidiasis. Studies in reconstituted human epithelium and monolayer cell cultures have shown that L. rhamnosus GG can protect mucosa from damage caused by Candida albicans, and enhance the immune responses of mucosal surfaces. Such findings offer the promise that the use of such probiotic bacteria could provide new options for antifungal therapy. Studies of changes of the human intestinal microbiota in health and disease are complicated by its size and diversity. The Alimentary Pharmabiotic Centre in Cork (Republic of Ireland) has the mission to ‘mine microbes for mankind’ and its work illustrates the potential benefits of understanding the gut microbiota. Work undertaken at the centre includes: mapping changes in the microbiota with age; studies of the interaction between the microbiota and the gut; potential interactions between the gut microbiota and the central nervous system; the potential for probiotics to act as anti-infectives including through the production of bacteriocins; and the characterisation of interactions between gut microbiota and bile acids which have important roles as signalling molecules and in immunity. The important disease entity where the role of the gut microbiota appears to be central is the Irritable Bowel Syndrome (IBS). IBS patients show evidence of immune activation, impaired gut barrier function and abnormal gut microbiota. Studies with probiotics have shown that these organisms can exert anti-inflammatory effects in inflammatory bowel disease and may strengthen the gut barrier in IBS of the diarrhoea-predominant type. Formal randomised trials of probiotics in IBS show mixed results with limited benefit for some but not all. Studies confirm that administered probiotics can survive and temporarily colonise the gut. They can also stimulate the numbers of other lactic acid bacilli in the gut, and reduce the numbers of pathogens. However consuming live organisms is not the only way to influence gut microbiota. Dietary prebiotics are selectively fermented ingredients that can change the composition and/or activity of the gastrointestinal microbiota in beneficial ways. Dietary components that reach the colon, and are available to influence the microbiota include poorly digestible carbohydrates, such as non-starch polysaccharides, resistant starch, non-digestible oligosaccharides (NDOs) and polyphenols. Mixtures of probiotic and prebiotic ingredients that can selectively stimulate growth or activity of health promoting bacteria have been termed ‘synbiotics’. All of these approaches can influence gut microbial ecology, mainly to increase bifidobacteria and lactobacilli, but metagenomic approaches may reveal wider effects. Characterising how these changes produce physiological benefits may enable broader use of these tactics in health and disease in the future. The current status of probiotic products commercially available worldwide is less than ideal. Prevalent problems include misidentification of ingredient organisms and poor viability of probiotic microorganisms leading to inadequate shelf life. On occasions these problems mean that some commercially available products cannot be considered to meet the definition of a probiotic product. Given the potential benefits of manipulating the human microbiota for beneficial effects, there is a clear need for improved regulation of probiotics. The potential importance of the human microbiota cannot be overstated. ‘We feed our microbes, they talk to us and we benefit. We just have to understand and then exploit this.’ (Willem de Vos).