Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study

Abstract Background Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is a skin cancer therapy that still has limitations due to the low penetration of this drug into the skin. We have proposed in this work a delivery system for 5-ALA based on liposomes having lipid composition similar to the mammalian stratum corneum (SCLLs) in order to optimize its skin delivery in Photodynamic Therapy (PDT) of skin cancers. Methods SCLLs were obtained by reverse phase evaporation technique and size distribution of the vesicles was determinated by photon correlation spectroscopy. In vitro permeation profile was characterized using hairless mouse skin mounted in modified Franz diffusion cell. Results Size exclusion chromatography on gel filtration confirmed vesicle formation. SCLLs obtained by presented a degree of encapsulation of 5-ALA around 5.7%. A distribution of vesicle size centering at around 500 nm and 400 nm respectively for SCLLs and SCLLs containing 5-ALA was found. In vitro 5-ALA permeation study showed that SCLLs preparations presented higher skin retention significantly (p < 0.05) on the epidermis without SC + dermis, with a decreasing of skin permeation compared to aqueous solution. Conclusions The in vitro delivery performance provided by SCLLs lead to consider this systems adequate for the 5-ALA-PDT of skin cancer, since SCLLs have delivered 5-ALA to the target skin layers (viable epidermis + dermis) to be treated by topical PDT of skin cancer.

Low temperatures reduce skin healing in the Jacaré do Pantanal (Caiman yacare, Daudin 1802)

Studies of skin wound healing in crocodilians are necessary given the frequent occurrence of cannibalism in intensive farming systems. Air temperature affects tissue recovery because crocodilians are ectothermic. Therefore, the kinetics of skin wound healing in Caiman yacare were examined at temperatures of 33°C and 23°C. Sixteen caiman were selected and divided into two groups of eight maintained at 23°C or 33°C. The studied individuals' scars were photographed after 1, 2, 3, 7, 15 and 30 days of the experimental conditions, and samples were collected for histological processing after 3, 7, 15 and 30 days. Macroscopically, the blood clot (heterophilic granuloma) noticeably remained in place covering the wound longer for the caiman kept at 23°C. Microscopically, the temperature of 23°C slowed epidermal migration and skin repair. Comparatively, new blood vessels, labeled using von Willebrand factor (vWF) antibody staining, were more frequently found in the scars of the 33°C group. The collagen fibers in the dermis were denser in the 33°C treatment. Considering the delayed healing at 23°C, producers are recommended to keep wounded animals at 33°C, especially when tanks are cold, to enable rapid wound closure and better repair of collagen fibers because such lesions tend to compromise the use of their skin as leather.

Evaluation von Nukleotidpolymorphismen für die Analyse des hämatopoetischen Spenderchimärismus auf Ebene der cDNA

Nach einer hämatopoetischen Stammzelltransplantation spielt die Zuordnung hämatopoetischer Zellen zum Spender oder Empfänger für viele transplantationsbezogene Fragestellungen eine wichtige Rolle. Unter anderem ist das Persistieren von dendritischen Zellen des Empfängers, welche allogene T-Zellen des Spenders stimulieren, ein wichtiger Schritt bei der Entstehung der akuten GVHD. Aus diesem Grund wurde in dieser Arbeit die Weiterentwicklung einer Methode angestrebt, die es uns erlaubt, die Zugehörigkeit isolierter hämatopoetischer Zellen dem Spender oder dem Empfänger zuzuordnen (Chimärismusbestimmung) und gleichzeitig Aussagen über das Ursprungsgewebe und den Aktivierungszustand der Zellen machen zu können. Hierfür nutzten wir Einzelbasenpolymorphismen (SNPs). Ziel dieser Arbeit war es, einen Pool von cDNA-kodierten SNPs zu definieren, mit dem auch HLA-identische Geschwister eindeutig unteschieden werden können. rnHierfür wurden zunächst aus publizierten Datenbanken solche SNPs ausgewählt, die in kodierenden Genabschnitten konstitutiv und gewebeunabhängig auf expremierten Genen lagen und zugleich eine hohe Heterozygotenfrequenz in der europäischen Population aufwiesen. Anhand dieser Kriterien wurden mittels der NCBI-Datenbank insgesamt eine Anzahl von 208 Polymorphismen auf 150 Genen identifiziert. Anschließend erfolgte die Gestaltung von Primerpaaren zur Amplifikation der SNP-kodierenden cDNA-Abschnitte. Diese mussten mindestens eine Intron/Exon-Grenze überspannen, um genomische DNA in der PCR ausschließen zu können. Mit Hilfe der etablierten PCR-Reaktion wurden die Gene in unterschiedlichen Geweben auf ihre Expression hin überprüft. Für 45 Gene ließ sich sowohl eine entsprechende PCR etablieren als auch deren konstitutive Expression in verschiedenen hämatopoetischen Zellen nachweisen. Zur Detektion der einzelnen SNPs in der Minisequenzierung wurden Minisequenzierungs-Sonden generiert und geprüft. Im Folgenden wurden für PCR und Minisequezierung Multiplex-Reaktionen aus vier bis sechs Reaktionen zusammengestellt. Zu diesem Zweck wurden die jeweiligen Primerinteraktionen und die unterschiedlichen Basenlängen des PCR-Produktes berücksichtigt.rnVon den 45 etablierten Einzelreaktionen waren 30 für den Multiplexansatz geeignet. Unter Anwendung dieser Multiplex-Reaktionen wurden 24 HLA-identische Geschwisterpaare (Spender und Empfänger) getestet. Zur Kontrolle erfolgte zusätzlich eine konventionelle Sequenzierung der SNP-kodierenden Bereiche auf der cDNA der jeweiligen Proben. Mit Hilfe der SNP-Kombinationen und der etablierten Methodik waren wir in der Lage alle 24 untersuchten Geschwisterpaare in zwischen sechs und 18 SNP-Systemen zu unterscheiden. rnDie Möglichkeiten, die die Analysen des Chimärismus mittels SNPs auf kodierenden Bereichen der DNA mit sich bringen, liegen nicht nur in der gleichzeitigen Bestimmung der Gewebszugehörigkeit und der Detektion des bestehenden Chimärismus sowie dessen Quantifizierung unter Anwendung einer Real-time-PCR. Vielmehr ermöglicht sie auch eine Aussage über die Genexpression der untersuchten Zelle zu machen. Dies ist insbesondere dann von Interesse, wenn geringe Zellzahlen von aus Gewebe isolierten Zellen zur Verfügung stehen. Die in dieser Arbeit etablierten Ansätze werden derzeit für eine Quantifizierung mittels real-time RCR weiterentwickelt und sollen mittelfristig insbesondere für Untersuchungen des Chimärismus von dermalen und epidermalen dendritischen Zellen der Haut und anderer Zielgewebe der GvHD verwendet werden.rn

Percutaneous collagen induction-regeneration in place of cicatrisation?

Ablative procedures that are used for the improvement of a degenerative process that leads to a loss of skin elasticity and integrity, injure or destroy the epidermis and its basement membrane and lead to fibrosis of the papillary dermis. It was recently shown in clinical and laboratory trials that percutaneous collagen induction (PCI) by multiple needle application is a method for safely treating wrinkles and scars and smoothening the skin without the risk of dyspigmentation. In our study, we describe the effect of PCI on epidermal thickness and the induction of genes relevant for regenerative processes in the skin in a small animal model.

MMP19 is upregulated during melanoma progression and increases invasion of melanoma cells

During the progression of cutaneous melanomas, matrix metalloproteinases (MMPs) facilitate the tumour cells to traverse the basement membrane and invade the dermis. In this study, we analysed the expression of MMP19 in the course of melanoma progression. Although MMP19 was absent in melanocytes and melanoma cells of early stages of melanoma development, its expression was strongly upregulated in the neighbouring keratinocytes that may facilitate the vertical outgrowth of melanoma cells. In contrast to early stages, MMP19 was upregulated during the vertical growth phase of melanoma and in metastases. The upregulation of MMP19 in melanoma of Clark levels IV and V correlates with that of MMP2 and also simultaneously with ceased expression of E-cadherin. To reveal whether MMP19 facilitates the invasion of melanomas, we examined adhesion and migratory capacity of selected melanoma cell lines. Melanoma cell lines with low expression of MMP19 exhibited increased adhesion to various substrates and lower migration in comparison with the cell line with higher expression of MMP19. Moreover, ectopic expression of MMP19 could restore the migratory capacity of melanoma cells with low endogenous level of MMP19. These results suggest that the increase of MMP19 expression hallmarks the progression of cutaneous melanoma and might augment melanoma growth by promoting the invasion of tumour cells.

Processing of procollagen III by meprins: new players in extracellular matrix assembly?

Meprins ? and ?, a subgroup of zinc metalloproteinases belonging to the astacin family, are known to cleave components of the extracellular matrix, either during physiological remodeling or in pathological situations. In this study we present a new role for meprins in matrix assembly, namely the proteolytic processing of procollagens. Both meprins ? and ? release the N- and C-propeptides from procollagen III, with such processing events being critical steps in collagen fibril formation. In addition, both meprins cleave procollagen III at exactly the same site as the procollagen C-proteinases, including bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family. Indeed, cleavage of procollagen III by meprins is more efficient than by BMP-1. In addition, unlike BMP-1, whose activity is stimulated by procollagen C-proteinase enhancer proteins (PCPEs), the activity of meprins on procollagen III is diminished by PCPE-1. Finally, following our earlier observations of meprin expression by human epidermal keratinocytes, meprin ? is also shown to be expressed by human dermal fibroblasts. In the dermis of fibrotic skin (keloids), expression of meprin ? increases and meprin ? begins to be detected. Our study suggests that meprins could be important players in several remodeling processes involving collagen fiber deposition.

The adhesion modulating properties of tenascin-w

Tenascins are extracellular matrix glycoproteins associated with cell motility, proliferation and differentiation. Tenascin-C inhibits cell spreading by binding to fibronectin; tenascin-R and tenascin-X also have anti-adhesive properties in vitro. Here we have studied the adhesion modulating properties of the most recently characterized tenascin, tenascin-W. C2C12 cells, a murine myoblast cell line, will form broad lamellipodia with stress fibers and focal adhesion complexes after culture on fibronectin. In contrast, C2C12 cells cultured on tenascin-W fail to spread and form stress fibers or focal adhesion complexes, and instead acquire a multipolar shape with short, actin-tipped pseudopodia. The same stellate morphology is observed when C2C12 cells are cultured on a mixture of fibronectin and tenascin-W, or on fibronectin in the presence of soluble tenascin-W. Tenascin-W combined with fibronectin also inhibits the spreading of mouse embryo fibroblasts when compared with cells cultured on fibronectin alone. The similarity between the adhesion modulating effects of tenascin-W and tenascin-C in vitro led us to study the possibility of tenascin-W compensating for tenascin-C in tenascin-C knockout mice, especially during epidermal wound healing. Dermal fibroblasts harvested from a tenascin-C knockout mouse express tenascin-W, but dermal fibroblasts taken from a wild type mouse do not. However, there is no upregulation of tenascin-W in the dermis of tenascin-C knockout mice, or in the granulation tissue of skin wounds in tenascin-C knockout animals. Similarly, tenascin-X is not upregulated in early wound granulation tissue in the tenascin-C knockout mice. Thus, tenascin-W is able to inhibit cell spreading in vitro and it is upregulated in dermal fibroblasts taken from the tenascin-C knockout mouse, but neither it nor tenascin-X are likely to compensate for missing tenascin-C during wound healing.

Oral mucosal morphea: a new variant

Morphea is a cutaneous disorder characterized by an excessive collagen deposition. While in almost all cases the sclerosing process exclusively affects the skin, there are anecdotal cases in which associated mucosal involvement has been described. We here report the case of a woman developing a whitish indurated plaque over the left upper vestibular mucosa and hard palate leading to dental mobility and exposure of the roots of several teeth. Cone beam computed tomography of the left maxilla showed bone resorption involving the upper cuspid to the second molar region with widened periodontal ligament spaces, while light microscopy studies demonstrated epithelial atrophy and fibrosis of the dermis extending into the submucosa with hyalinization of subepithelial collagen. Our observation expands the spectrum of clinical presentations of morphea and provides the first example of isolated oral morphea. Its recognition is important to avoid significant local complications.

Characterization of canine dendritic cells in healthy, atopic, and non-allergic inflamed skin

Atopic dermatitis in humans and dogs is a chronic relapsing allergic skin disease. Dogs show a spontaneous disease similar to the human counterpart and represent a model to improve our understanding of the immunological mechanisms, the pathogenesis of the disease, and new therapy development. The aim of the study was to determine the frequency and phenotype of dendritic cells (DC) in the epidermis and dermis of healthy, canine atopic dermatitis lesional, and non-allergic inflammatory skin to further validate the model and to obtain insights into the contribution of DC to the pathogenesis of skin diseases in dogs. We first characterized canine skin DC using flow-cytometric analysis of isolated skin DC combined with an immunohistochemical approach. A major population of canine skin dendritic cells was identified as CD1c(+)CD11c(+)CD14(-)CD80(+)MHCII(+)MAC387(-) cells, with dermal DC but not Langerhans cells expressing CD11b. In the epidermis of lesional canine atopic dermatitis and non-allergic inflammatory skin, we found significantly more dendritic cells compared with nonlesional and control skin. Only in canine atopic dermatitis skin did we find a subset of dendritic cells positive for IgE, in the epidermis and the dermis. Under all inflammatory conditions, dermal dendritic cells expressed more CD14 and CD206. MAC387(+) putative macrophages were absent in healthy but present in inflamed skin, in particular during non-allergic diseases. This study permits a phenotypic identification and differentiation of canine skin dendritic cells and has identified markers and changes in dendritic cells and macrophage populations related to allergic and non-allergic inflammatory conditions. Our data suggest the participation of dendritic cells in the pathogenesis of canine atopic dermatitis similar to human atopic dermatitis and further validate the only non-murine spontaneous animal model for this disease.

Penetration of ASM 981 in canine skin: a comparative study

ASM 981 has been developed for topical treatment of inflammatory skin diseases. It specifically inhibits the production and release of pro-inflammatory cytokines. We measured the skin penetration of ASM 981 in canine skin and compared penetration in living and frozen skin. To make penetration of ASM 981 visible in dog skin, tritium labelled ASM 981 was applied to a living dog and to defrosted skin of the same dog. Using qualitative autoradiography the radioactive molecules were detected in the lumen of the hair follicles until the infundibulum, around the superficial parts of the hair follicles and into a depth of the dermis of 200 to 500 microm. Activity could not be found in deeper parts of the hair follicles, the dermis or in the sebaceous glands. Penetration of ASM 981 is low in canine skin and is only equally spread in the upper third of the dermis 24 hours after application. Penetration in frozen skin takes even longer than in living canine skin but shows the same distribution.

Facets of lupus erythematosus: panniculitis responding to thalidomide

Lupus erythematosus profundus or lupus panniculitis is a rare clinical variant of lupus erythematosus, which involves the deep dermis and subcutaneous fat. Diagnosis may be difficult in cases with isolated involvement. Further manifestations of lupus erythematosus may thus be essential for diagnosis, which depends on the clinical picture, histopathology and a positive lesional lupus band test. We report a severe, mutilating case of lupus panniculitis, which responded well to thalidomide.

Composite skin grafts for basal cell carcinoma defects of the nose

Basal cell carcinoma is the most frequent cutaneous cancer of the nose and is characterized by its local spreading and exceptionally rare tendency to metastasize. Since a significant advantage has been seen in surgery compared to other treatments, surgical excision ensuring the highest chance of cure is frequently employed. Excision defects of the nose may be covered with either local flap or a full-thickness skin graft. In resurfacing such defects following excision of basal cell carcinomas, we favor the technique of composite-skin grafting which involves the harvesting of composite-skin graft including the epidermis, dermis and superficial layers of subcutaneous tissue to obtain the required thickness in the recipient site. This technique was used for defects remaining after the excision of basal cell carcinomas in a series of 15 patients. The areas involved were lateral nasal region (5 cases), nasal tip (4 cases), dorsum (3 cases), alar lobule (2 cases), and soft triangle (1 case). The mean follow-up was 14.2 months. The color, texture and thickness of the composite-skin graft harvested from the preauricular site and the neck compare favorably with the skin of the nose region. Satisfactory results, both clinically and in patient appreciation, have been obtained in both the reconstruction site and the appearance of the donor site in all patients.

Human beta-defensin-2 and psoriasin are overexpressed in lesions of acne inversa

BACKGROUND: Acne inversa is a chronic inflammatory disorder of apocrine gland-bearing skin. The role of the innate immune system in the pathogenesis of the disease is controversial. OBJECTIVES: We investigated the expression of antimicrobial peptide/proteins in acne inversa. METHODS: Tissue samples were obtained from patients with acne inversa and compared with normal-appearing skin. The expression of psoriasin and human beta-defensin (hBD)-2 on messenger RNA and protein level was analyzed. RESULTS: Both messenger RNA and protein levels of psoriasin and hBD-2 were significantly increased in acne inversa. Macrophages expressing hBD-2 were found in the dermis. LIMITATIONS: Small sample size is a limitation. CONCLUSIONS: Antimicrobial peptide/proteins are overexpressed in acne inversa lesions as compared with normal-appearing skin. The site of the major expression depends on the particular antimicrobial peptide/protein. Psoriasin is overexpressed in epidermal keratinocytes whereas hBD-2 is produced mainly by dermal macrophages, leaving a relative deficiency of hBD-2 in the epidermis of acne inversa lesions.

Potent and broad-spectrum antimicrobial activity of CXCL14 suggests an immediate role in skin infections

The skin is constantly exposed to commensal microflora and pathogenic microbes. The stratum corneum of the outermost skin layer employs distinct tools such as harsh growth conditions and numerous antimicrobial peptides (AMPs) to discriminate between beneficial cutaneous microflora and harmful bacteria. How the skin deals with microbes that have gained access to the live part of the skin as a result of microinjuries is ill defined. In this study, we report that the chemokine CXCL14 is a broad-spectrum AMP with killing activity for cutaneous gram-positive bacteria and Candida albicans as well as the gram-negative enterobacterium Escherichia coli. Based on two separate bacteria-killing assays, CXCL14 compares favorably with other tested AMPs, including human beta-defensin and the chemokine CCL20. Increased salt concentrations and skin-typical pH conditions did not abrogate its AMP function. This novel AMP is highly abundant in the epidermis and dermis of healthy human skin but is down-modulated under conditions of inflammation and disease. We propose that CXCL14 fights bacteria at the earliest stage of infection, well before the establishment of inflammation, and thus fulfills a unique role in antimicrobial immunity.

Melan-a-positive "pseudomelanocytic nests": a pitfall in the histopathologic and immunohistochemical diagnosis of pigmented lesions on sun-damaged skin

We encountered recently 3 cases with a histopathologic diagnosis of melanoma in situ on sun-damaged skin (male = 2, female = 1; median age: 59 years; range: 52-60 years). The diagnosis was based mainly on the finding of actinic elastosis in the dermis and increased number of melanocytes in the epidermis and was confirmed by strong positivity for Melan-A in single cells and in small nests ("pseudomelanocytic nests"), located at the dermoepidermal junction. Indeed, examination of slides stained with hematoxylin and eosin revealed the presence of marked hyperpigmentation and small nests of partially pigmented cells at the dermoepidermal junction, positive for Melan-A. The histologic and especially the immunohistochemical features were indistinguishable from those of melanoma in situ on chronic sun-damaged skin. In addition, a variably dense lichenoid inflammation was present. Clinicopathologic correlation, however, showed, in all patients, the presence of a lichenoid dermatitis (phototoxic reaction, 1 case; lichen planus pigmentosus, 1 case; and pigmented lichenoid keratosis, 1 case). Our cases clearly show the histopathologic pitfalls represented by lichenoid reactions on chronic sun-damaged skin. Immunohistochemical investigations, especially if performed with Melan-A alone, may lead to confusing and potentially disastrous results. The unexpected staining pattern of Melan-A in cases like ours raises concern about the utility of this antibody in the setting of a lichenoid tissue reaction on chronic sun-damaged skin. It should be underlined that pigmented lesions represent a paradigmatic example of how immunohistochemical results should be interpreted carefully and always in conjunction with histologic and clinical features.