Duration of wound fluid secretion from chronic venous leg ulcers is critical for interleukin-1α, interleukin-1ß, interleukin-8 levels and fibroblast activation.

Wound fluid collected from chronic wounds may be used as a simple gauge of the processes taking place in the tissue. There is lack of information on the optimal conditions for wound fluid procurement. We have studied possible diurnal variations and duration of wound fluid accumulation using retentive hydrophobic foam on the levels of prototypic cytokines [interleukin (IL)-1α, IL-1ß], a chemokine (IL-8) and proteinases [matrix metalloproteinase (MMP)-9] in 23 chronic venous leg ulcer patients. Bioactivity of 1 and 24 h wound fluids, and serum was also compared. There were no significant temporal changes in the levels of the above-mentioned four proteins, when comparing three consecutive 8-h intervals starting from 0800 that in turn did not differ significantly with the 24-h collection levels. IL-1α, IL-1ß and IL-8 levels were higher (p < 0.05) in 24 h compared with 1 h wound fluids, whereas MMP-9 levels were insensitive to the length of collection. The 24 h wound fluids did not elicit DNA synthesis in adult human dermal fibroblasts in contrast to the 1 h wound fluids (p = 0.046) and serum (p = 0.036). The polyurethane foam alone had no significant effects on the concentration of the examined analytes. The length of collection is critical when monitoring cytokine/chemokine and bioactivity levels of chronic wound fluid. The removal of accumulating unfavorable factors in chronic wound fluid may be important in wound management.

Investigation of the interaction between modified ISCOMs and stratum corneum lipid model systems.

The modified ISCOMs, so-called Posintro nanoparticles, provide an opportunity for altering the surface charge of the particles, which influences their affinity for the negatively charged antigen sites, cell membranes and lipids in the skin. Hypothetically, this increases the passage of the ISCOMs (or their components) and their load through the stratum corneum. The subsequent increase in the uptake by the antigen-presenting cells results in enhanced transcutaneous immunization. To understand the nature of penetration of Posintro nanoparticles into the intercorneocyte space of the stratum corneum, the interaction between the nanoparticles and lipid model systems in form of liposomes and/or supported lipid bilayer was studied. As a lipid model we used Stratum Corneum Lipid (SCL), a mixture similar in composition to the lipids of the intercorneocyte space. By Förster Resonance Energy Transfer (FRET), Atomic Force Microscopy (AFM), Electrochemical Impedance Spectroscopy (EIS) and cryo-Transmission Electron Microscopy (cryo-TEM) it was shown that application of nanoparticles to the SCL bilayers results in lipid disturbance. Investigation of this interaction by means of Isothermal Titration Calorimetry (ITC) confirmed existence of an enthalpically unfavorable reaction. All these methods demonstrated that the strength of electrostatic repulsion between the negatively charged SCL and the nanoparticles affected their interaction, as decreasing the negative charge of the Posintro nanoparticles leads to enhanced disruption of lipid organization.

In vitro cutaneous application of ISCOMs on human skin enhances delivery of hydrophobic model compounds through the stratum corneum.

This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOMs) on human skin penetration of model compounds in vitro to evaluate their potential as a delivery system, ultimately for transcutaneous vaccination. Special focus was on elucidating the mechanisms of penetration. Preparation of ISCOMs was done by dialysis and subsequent purification in a sucrose density gradient. The penetration pathways of acridine-labeled ISCOMs were visualized using confocal laser scanning microscopy (CLSM). Transmission electron microscopy (TEM) was used to evaluate the ultrastructural changes in the skin after application of the ISCOMs with or without hydration. Transcutaneous permeation of the model compound, methyl nicotinate, was evaluated in diffusion cells. The prepared ISCOMs were 42-52 nm in diameter as evaluated by dynamic light scattering with zeta potentials of -33 to -26.1 mV. TEM investigations verified the presence of ISCOM structures. Penetration of acridine into skin was greatly increased by incorporation into ISCOMs as visualized by CLSM. Permeation of methyl nicotinate was enhanced in the presence of ISCOMs. Ultrastructural changes of the intercellular space in the stratum corneum after exposure of ISCOMs were observed on micrographs, especially for hydrated skin. In conclusion, cutaneous application of ISCOMs leads to increased penetration of hydrophobic model compounds through human stratum corneum and thus shows potential as a transcutaneous delivery system. The increased penetration seems to be reflected by a change in the intercellular space between the corneocytes, and the effect is most likely caused by the components of the ISCOMs rather than intact ISCOMs.

The Pseudomonas aeruginosa autoinducer dodecanoyl-homoserine lactone inhibits the putrescine synthesis in human cells.

Pseudomonas aeruginosa uses acyl-homoserine lactones to coordinate gene transcription in a process called quorum sensing (QS). The QS molecules C4-HSL and C12-oxo-HSL are synthesized from the universal precursor S-adenosyl methionine, which is also a precursor of polyamines in human cells. Polyamines are required for mitotic cell division and peak during this phase. The polyamine putrescine is synthesized by ornithine decarboxylase (ODC) as a rate-limiting step. The ODC enzyme concentration also peaks during the mitotic phase. This peak is mediated by translation of ODC mRNA by the ITAF45 protein, which translocates from the nuclear compartment to the cytoplasm in a phosphorylation-dependent manner. We observed that C12-HSL-treated human epidermal cells had a higher cytoplasm-to-nuclear ITAF45 protein concentration and this translocation was dependent on the dephosphorylation of ITAF45. Finally, C12-HSL-treated cells also had a time-course-dependent higher concentration of ODC mRNA. Based on these mitotic markers, more human cells were apparently trapped in the mitotic phase when treated with C12-HSL. This should normally imply higher levels of putrescine. However, C12-HSL-treated human cells had a significantly lower concentration of putrescine and displayed a lower cell proliferation rate. In conclusion, the P. aeruginosa autoinducer C12-oxo-HSL apparently arrests human cells in the mitotic phase by lowering the concentration of putrescine.

Ultrastructural localization and chemical binding of silver ions in human organotypic skin cultures.

Organotypic cultures of human breast skin incubated with silver bandage or treated with silver sulfadiazine accumulated silver in epithelial cells and in macrophages, fibroblasts and collagen fibrils and fibres of underlying connective tissue. Ultrastructurally, the accumulated silver was found in lysosome-like vesicles of the different cells and evenly spread along collagen structures. Apoptotic nuclei were present but few. Autometallographic amplification of 2D-PAGE gels revealed that glutathione S-transferase and glutathion detoxify silver ions in the epidermal cell by binding them in silver-sulphur nanocrystals. Thus, the cytotoxic effect of silver ions seems to be muted by silver ions by being: (1) taken up by undamaged cells, neutralised by glutathione (GSH) and accumulated in lysosomal vesicles, (2) bound extracellularly to SH-groups of the collagen fibres.

Skin changes induced by a zinc oxide dressing compared with a hydrocolloid dressing in healthy individuals.

BACKGROUND/PURPOSE: Incidence of skin complications in ostomy patients constitutes a well-known and well-described problem. The reasons are, however, very difficult to describe because of the many factors contributing to the problem. This article describes the skin changes derived exclusively from the adhesives used in a carefully controlled, long-term study using two fundamentally different types of adhesives: a hydrocolloid adhesive and a zinc oxide adhesive. METHODS: The adhesives were changed daily on the volar forearm of 11 volunteers for a 4-week period. Once a week, transepidermal water-loss (TEWL), water content of the skin, erythema and the peel force applied for removal of the adhesives were measured. On the last day of the study, a replica of the skin surface was obtained to determine changes in the skin topography, and a biopsy was taken to study changes at the cellular level. RESULTS AND CONCLUSION: We found increased TEWL and decreased water content in skin treated with the zinc oxide adhesive, but increased water-loss and water content when the hydrocolloid adhesive was used. In addition, the area treated with zinc oxide adhesive showed significant increase of epidermal thickness, scaly appearance and parakeratosis with similarities to pathological dry skin diseases such as psoriasis and atopic dermatitis, changes that were not found when using the hydrocolloid adhesive. The skin response seems to be the result of the content of zinc oxide and the mechanical interaction of the zinc oxide adhesive. We conclude that the nature of the adhesive plays an important role in the skin response to repeated application of adhesives, as seen in peristomal skin.