Correction: Physical and compositional analysis of differently cultured 3D human skin equivalents by confocal Raman spectroscopy.

Correction for 'Physical and compositional analysis of differently cultured 3D human skin equivalents by confocal Raman spectroscopy' by Y. Dancik, et al., Analyst, 2018, DOI: .

Physical and compositional analysis of differently cultured 3D human skin equivalents by confocal Raman spectroscopy.

Three-dimensional skin equivalents are increasingly gaining acceptance as non-animal based experimental models of human skin. They are particularly suited to studying differences in physical and compositional properties of normal and diseased skin and their impact on the skin's barrier function. Typically, a culture protocol yielding a model of normal skin is modified to create a model simulating a pathology. Skin layer thicknesses and lipid/protein contents are compared using methods that are invasive, precluding further experiments on the same replicates, and which may be prone to artefacts. We show here that confocal Raman spectroscopy (CRS) is a valuable method for non-invasive discrimination of skin equivalents grown under different culture conditions. Using 3D full-thickness skin equivalents developed in-house, we measure significant differences in stratum corneum and viable epidermis apparent thicknesses resulting from a 7-day difference in the cultures' air-lift phase and from supplementation of the culture medium with interleukin 4. Furthermore, stratum corneum thicknesses obtained by CRS are up to 2.6-fold higher than values measured from histological photomicrographs. Regarding composition, CRS reveals the differential effects of the culture protocol modifications on ceramide, cholesterol and protein composition as a function of depth in the stratum corneum.

Multi-chamber microfluidic platform for high-precision skin permeation testing.

The established in vitro tool used for testing the absorption and penetration of chemicals through skin in pharmacology, toxicology and cosmetic science is the static Franz diffusion cell. While widespread, Franz cells are relatively costly, low-throughput and results may suffer from poor reproducibility. Microfluidics has the potential to overcome these drawbacks. In this paper, we present a novel microfluidic skin permeation platform and validate it rigorously against the Franz cell by comparing the transport of 3 model chemicals of varying lipophilicity: caffeine, salicylic acid and testosterone. Permeation experiments through silicone membranes show that the chip yields higher sensitivity in permeant cumulative amounts and comparable or lower coefficients of variation. Using a skin organotypic culture, we show that the chip decreases the effect of unstirred water layers that can occur in static Franz cells. The validation reported herein sets the stage for efficient skin permeation and toxicity screening and further development of microfluidic skin-on-chip devices.

Activation of the δ-opioid receptor promotes cutaneous wound healing by affecting keratinocyte intercellular adhesion and migration.

BACKGROUND AND PURPOSE: In addition to its analgesic functions, the peripheral opioid receptor system affects skin homeostasis by influencing cell differentiation, migration and adhesion; also, wound healing is altered in δ-opioid receptor knockout mice (DOPr(-/-) ). Hence, we investigated δ-opioid receptor effects on the expression of several proteins of the desmosomal junction complex and on the migratory behaviour of keratinocytes. EXPERIMENTAL APPROACH: Expression levels of desmosomal cadherins in wild-type and DOPr(-/-) mice, and the morphology of intercellular adhesion in human keratinocytes were analysed by immunofluorescence. To investigate the δ-opioid receptor activation pathway, protein expression was studied using Western blot and its effect on cellular migration determined by in vitro live cell migration recordings from human keratinocytes. KEY RESULTS: Expression of the desmosomal cadherins, desmogleins 1 and 4, was up-regulated in skin from DOPr(-/-) mice, and down-regulated in δ-opioid receptor-overexpressing human keratinocytes. The localization of desmoplakin expression was rearranged from linear arrays emanating from cell borders to puncta in cell periphery, resulting in less stable intercellular adhesion. Migration and wound recovery were enhanced in human keratinocyte monolayers overexpressing δ-opioid receptors in vitro. These δ-opioid receptor effects were antagonized by specific PKCα/ß inhibition indicating they were mediated through the PKC signalling pathway. Finally, cells overexpressing δ-opioid receptors developed characteristically long but undirected protrusions containing filamentous actin and δ-opioid receptors, indicating an enhanced migratory phenotype. CONCLUSION AND IMPLICATIONS: Opioid receptors affect intercellular adhesion and wound healing mechanisms, underlining the importance of a cutaneous neuroendocrine system in wound healing and skin homeostasis. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

[Toxic epidermal necrolysis]. / Epidermolyse nécrosante suraiguë de Lyell ou toxic epidermal necrolysis.

The toxic epidermal necrolysis (TEN) is an immunologic reaction in the skin with apoptosis of kératinocytes: most cases are reactions against drugs. The main features of this reaction are a separation of the epidermis and erosions of the mucus membranes, associated with a reduction of the general condition with kidney and liver failure. This leads to problems similar to those observed in severely burned patients. The mortality high of patients with TEN can be reduced, if the responsible drug is immediately withdrawn and if the patient is treated in a specialized burns intensive care unit. The medical treatment to stop the chain reaction of apoptosis is still under discussion. Through a case report, this article will focus on the complexity of the treatment of patients with TEN and will point out the importance of a multidisciplinary approach.

[Pruritus--causes, diagnostics and treatment]. / Prurit: causes, diagnostic et traitement.

Diagnosis and treatment of pruritus is still a great problem. The pathophysiologic principles are mostly unknown, however the progress in neurosciences also added several new neurophysiologic explanations for pruritus. There exist several different types of pruritus and also the treatment of the different forms varies. The treatment of chronic pruritus is not the same as the treatment of acute pruritus. Usually more than one pathological disorder leads to severe pruritus and therefore the diagnostic of the different forms of pruritus and the treatment needs an interdisciplinary approach. One of the major causes of itch is dry skin, this is true for elderly persons and also for patients with atopic dermatitis. Therefore, the treatment of the dry skin is one of the most important measures against pruritus.

Changes of epidermal mu-opiate receptor expression and nerve endings in chronic atopic dermatitis.

There is increasing evidence that neuropeptides such as a substance P, neurotrophins or beta-endorphin, an endogenous agonist for mu-opioid receptor, are involved in the pathogenesis of atopic dermatitis in which mental stress and scratching deteriorate the disease. mu-Opioid receptor, a G-protein-coupled receptor, can be downregulated and internalized by agonists and other factors in vitro. In this study, we investigated the regulation of mu-opioid receptor and nerve endings in atopic dermatitis patients. Skin biopsies from atopic dermatitis patients revealed a significant downregulation of mu-opiate receptor expression in epidermis of atopic dermatitis. Permeabilization of the skin showed that the receptor in keratinocytes from atopic dermatitis is internalized. The mRNA expression pattern of the mu-opiate receptor is different in epidermis taken from patients with chronic atopic dermatitis compared to normal skin. In atopic dermatitis, the mRNA is concentrated in the subcorneal layers of the epidermis and in normal skin in the suprabasal layers. Staining of the nerve endings using protein gene product 9.5 shows a different pattern of epidermal nerve endings in normal skin compared to atopic dermatitis. In normal skin, the epidermal nerve endings are rather thick. However, in atopic dermatitis, the epidermal nerve endings are thin and run straight through the epidermis. Based on these observations and combining the 'intensity' and 'pattern' hypothesis, we propose a new theory especially for histamine-unrelated, peripheral induction of chronic pruritus. We suggest that 'itch' is elicited in the epidermal unmyelinated nerve C-fibers and 'pain' in the dermal unmyelinated nerve fibers. The downregulation of the opioid receptor in the epidermis contributes to the chronic itching. We call this new hypothesis the 'layer hypothesis'.

Mu-opiate receptor and Beta-endorphin expression in nerve endings and keratinocytes in human skin.

We have previously shown that human epidermal keratinocytes express a functionally active micro-opiate receptor, which adds a new dimension to the recently developed research in neuroimmunodermatology and neurogenic inflammation in skin diseases. Human keratinocytes specifically bind and also produce beta-endorphin, the endogenous micro-opiate receptor ligand. Using confocal imaging microscopy, we could now demonstrate that micro-opiate receptors are not only expressed in keratinocytes, but also on unmyelinated peripheral nerve fibers in the dermis and epidermis. Some of the peripheral nerve fibers also express the ligand beta-endorphin. The keratinocytes positive for beta-endorphin staining are clustered around the terminal ends of the unmyelinated nerve fibers. Therefore the opiate receptor system seems to be crucial in the direct communication between nerves and skin. The keratinocytes can influence the unmyelinated nerve fibers in the epidermis directly via secreting beta-endorphin. On the other hand, nerve fibers can also secrete beta-endorphin and influence the migration, differentiation and probably also the cytokine production pattern of keratinocytes.

Anaphylaxis to the carbohydrate carboxymethylcellulose in parenteral corticosteroid preparations.

BACKGROUND: Carboxymethylcellulose is a carbohydrate widely used as additive in tablets, cosmetics, some injectable hormone formulations, food (as E466) and as active principle in hydrocolloid dressings. Anaphylaxis to carboxymethylcellulose in parenteral corticosteroid preparations has previously been reported. Typically, skin tests were positive in such cases, occasionally specific IgE or histamine release have been demonstrated. CASE REPORT: We report on 3 patients who suffered from anaphylactic symptoms after local injection of corticosteroid preparations. Intracutaneous skin tests with carboxymethylcellulose were positive; in 2, sulfidoleukotriene release could be measured in the cellular antigen stimulation test (CAST). Specific IgE could not be identified. Oral provocation tests with typical doses of carboxymethylcellulose as found in food and tablets were negative. CONCLUSION: In patients with anaphylaxis to parenteral administration of carboxymethylcellulose, small amounts are tolerated by the oral route. Skin tests and CAST are useful diagnostic tools.

Different expression of mu-opiate receptor in chronic and acute wounds and the effect of beta-endorphin on transforming growth factor beta type II receptor and cytokeratin 16 expression.

There is evidence that neuropeptides, especially the opiate receptor agonists, are involved in wound healing. We have previously observed that beta-endorphin, the endogenous ligand for the mu-opiate receptor, stimulates the expression of cytokeratin 16 in a dose-dependent manner in human skin organ cultures. Cytokeratin 16 is expressed in hyperproliferative epidermis such as psoriasis and wound healing. Therefore we were interested to study whether epidermal mu-opiate receptor expression is changed at the wound margins in acute and chronic wounds. Using classical and confocal microscopy, we were able to compare the expression level of mu-opiate receptors and the influence of beta-endorphin on transforming growth factor beta type II receptor in organ culture. Our results show indeed a significantly decreased expression of mu-opiate receptors on keratinocytes close to the wound margin of chronic wounds compared to acute wounds. Additionally beta-endorphin upregulates the expression of transforming growth factor beta type II receptor in human skin organ cultures. These results suggest a crucial role of opioid peptides not only in pain control but also in wound healing. Opioid peptides have already been used in animal models in treatment of wounds; they induce fibroblast proliferation and growth of capillaries, and accelerate the maturation of granulation tissue and the epithelization of the defect. Furthermore opioid peptides may fine-tune pain and the inflammatory response while healing takes place. This new knowledge could potentially be used to design new locally applied drugs to improve the healing of painful chronic wounds.

beta-endorphin stimulates cytokeratin 16 expression and downregulates mu-opiate receptor expression in human epidermis.

It has been reported that opioid peptides modulate the differentiation of normal human keratinocytes and that mu-opiate receptors are expressed in human epidermis. The regulation of keratinocyte differentiation is particularly important in psoriasis, and one of the markers for hyperproliferative and differentiating skin diseases is cytokeratin 16. The finding that the endogenous mu-opiate receptor ligand beta-endorphin is increased in serum of patients with psoriasis indicates that the mu-opiate system may play an important role in the pathophysiology of the skin. In this study, we addressed the question whether there is a link between mu-opiate receptor regulation and cytokeratin 16 expression in normal and psoriatic skin. Firstly, we demonstrate that beta-endorphin concentrations between 16 and 1000 nM significantly downregulate mu-opiate receptor expression in epidermis of cultured human skin after 48 h. Secondly, we show that beta-endorphin regulates cytokeratin 16 expression in the epidermis of skin organ cultures exposed to 41-125 nM beta-endorphin for 48 h, leading to elevated cytokeratin 16 production. As expected, the expression of cytokeratin 16 was detected primarily in the suprabasal layer. The same pattern was observed in psoriatic lesional skin, i.e., mu-opiate receptor expression was significantly downregulated and cytokeratin 16 expression upregulated. These results suggest that the mu-opiate receptor system and its ligand beta-endorphin are involved in the pathogenesis of psoriasis, especially in terms of differentiation.

Expression of mu-opiate receptor in human epidermis and keratinocytes.

There is increasing evidence that neurotransmitters play a crucial role in skin physiology and pathology. The expression and production of proopiomelanocortin molecules such as beta-endorphin in human epidermis suggest that an opiate receptor is present in keratinocytes. In this paper we show that human epidermal keratinocytes express a mu-opiate receptor on both the mRNA level and the protein level. Performing polymerase chain reaction with cDNA libraries from human epidermal keratinocytes gave the polymerase chain reaction products of the expected length, which were confirmed as mu-opiate receptors by Southern blot analysis. Using in situ hybridization techniques with a specific probe for mu-opiate receptors we detected the receptor in human epidermis. There was a cytoplasmic expression in all layers of the epidermis, which was more distinct in the suprabasal layers. Immunohistochemistry using the mu-opiate receptor-specific antibody indicates that epidermis expresses protein as well, and that the protein level is more elevated in the basal layer. The correlation between the locations of both mRNA and protein expression in skin indicates that the mu-opiate receptor has not only been transcribed but also has a specific function. To prove a function of the receptor we performed a functional assay using skin organ cultures from human skin transplants. After 48 h incubation with Naloxone or beta-endorphin the expression of the mu-opiate receptor in epidermis was significantly downregulated compared with the control. These results show that a functional receptor indeed exists in human epidermis.

Keratinocyte muscarinic acetylcholine receptors: immunolocalization and partial characterization.

We have reported previously that human keratinocytes synthesize and secrete acetylcholine and that muscarinic cholinergic drugs have effects on keratinocyte proliferation, adhesion, and migration. This study defines the location of muscarinic acetylcholine receptors in human epidermis and describes some pharmacologic and molecular properties of these receptors. Confocal microscopy employing the anti-muscarinic receptor monoclonal antibody M35 visualized the receptors in the intercellular areas of normal human epidermis. Using immunoelectron microscopy, the receptors appeared to be attached to the keratinocyte plasma membranes. Functional, high-density (Bmax = 8.3 nmol/2 x 10(6) cells) and high-affinity (Kd = 21.5 nM) muscarinic receptors were demonstrated by saturable binding of the reversible radioligand [3H]quinuclidinyl benzilate to the surfaces of freshly isolated epidermal cells at 0 degrees C. Receptor proteins were separated by gel electrophoresis. An apparent isoelectric point of pH 4.3 was determined in immunoblots of sodium-cholate-solubilized receptors separated on isoelectric-focusing gels. Three protein bands, two at approximately 60 kDa and one at 95 kDa, were visualized in immunoblots of membrane-bound or solubilized receptors separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The covalent, irreversible ligand [3H]propylbenzilylcholine mustard confirmed these results. Thus, human keratinocytes express a heterogeneous population of muscarinic cholinergic receptors. Because human keratinocytes also express nicotinic cholinergic receptors, endogenously secreted acetylcholine may control different biologic processes in these cells by activating different types of their cholinergic receptors.

Effects of detergents on proliferation and metabolism of human keratinocytes.

Effects of low concentrations of detergents on cultured human foreskin keratinocytes were assessed in vitro. The viability and activity of keratinocytes were assessed by measuring reduction of a tetrazolium dye as an indicator of mitochondrial metabolism. The keratinocyte proliferative responses after incubation with detergents were assessed by a spectrophotometric assay employing crystal violet dye and a fluorometric assay determining total DNA content. Both the cationic detergent cetyltrimethylammonium bromide [CTAB] and the anionic detergent sodium lauryl sulfate [SLS] showed toxic effects on keratinocytes at concentrations as low as 3 micrograms/mg, but SLS was less toxic. However, both SLS and CTAB activated keratinocytes at very low concentrations. Proliferative activity and mitochondrial metabolism increased. Serum partially protected keratinocytes against toxic and stimulatory activities of both detergents. We suggest that detergents may directly damage keratinocytes and thereby produce irritant contact dermatitis, but activation of keratinocytes by low concentrations may also produce dermatitis, perhaps by causing keratinocytes to release cytokines.

Computerized microassay of keratinocyte cell-plastic attachment and proliferation for assessing net stimulatory, inhibitory and toxic effects of compounds on nonimmortalized cell lines.

Testing of pharmacological agents that affect growth of epidermal keratinocytes (EK) requires a standardized assay. We have developed an assay measuring net effects of stimulatory (e.g. growth factors), inhibitory (e.g. methotrexate) or toxic (e.g. Triton X-100) compounds. The amount of crystal violet staining viable EK attached to the wells of standard 96-well microplates is measured in situ using an ELISA plate reader. Optical density readings are directly converted into cell counts by computer software. Counts obtained by this method strongly correlate with the results obtained using the [3H]thymidine uptake assay and direct cell counts. The assay standardizes measurements of nonimmortalized EK lines with different innate proliferative properties and allows accurate quantitation of EK numbers in the range of 2,500-500,000 EK/well.