Hypothermia reduces sulphur mustard toxicity

The effect of temperature on the development of sulphur mustard (HD)-induced toxicity was investigated in first passage cultures of human skin keratinocytes and on hairless guinea pig skin. When cells exposed to HD were incubated at 37 degrees C, a concentration-dependent decline in viability was observed that was maximal by 2 days. In contrast, no significant HD-induced toxicity was evident up to 4 days posttreatment when the cells were incubated at 25 degrees C. However, these protective effects were lost by 24 h when the cells were switched back to 37 degrees C. The protective effects of hypothermia were also demonstrated when apoptotic endpoints were examined. The HD concentration-dependent induction of fragmented DNA (as quantitated using soluble DNA and the TUNEL reaction), morphology, and p53 expression were all significantly depressed when cell cultures were incubated at 25 degrees C compared to 37 degrees C. When animals were exposed to HD vapour for 2, 4, and 6 min and left at room temperature, lesions were produced whose severity was dependent on exposure time and that were maximal by 72 h posttreatment. Moderate cooling (5-10 degrees C) of HD exposure sites posttreatment (4-6 h) significantly reduced the severity of the resultant lesions. However, in contrast to the in vitro results, these effects were permanent. It appears that the early and noninvasive act of cooling HD-exposed skin may provide a facile means of reducing the severity of HD-induced cutaneous lesions.     

Enhanced constitutive invasion activity in human nontumorigenic keratinocytes exposed to a low level of barium for a long time

We have recently demonstrated that exposure to barium for a short time (≤4 days) and at a low level (5 µM = 687 µg/L) promotes invasion of human nontumorigenic HaCaT cells, which have characteristics similar to those of normal keratinocytes, suggesting that exposure to barium for a short time enhances malignant characteristics. Here we examined the effect of exposure to low level of barium for a long time, a condition mimicking the exposure to barium through well water, on malignant characteristics of HaCaT keratinocytes. Constitutive invasion activity, focal adhesion kinase (FAK) protein expression and activity, and matrix metalloproteinase 14 (MMP14) protein expression in primary cultured normal human epidermal keratinocytes, HaCaT keratinocytes, and HSC5 and A431 human squamous cell carcinoma cells were augmented following an increase in malignancy grade of the cells. Constitutive invasion activity, FAK phosphorylation, and MMP14 expression levels of HaCaT keratinocytes after treatment with 5 µM barium for 4 months were significantly higher than those of control untreated HaCaT keratinocytes. Taken together, our results suggest that exposure to a low level of barium for a long time enhances constitutive malignant characteristics of HaCaT keratinocytes via regulatory molecules (FAK and MMP14) for invasion. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 161–167, 2015.     

Decellularized dermis in combination with cultivated keratinocytes in a short- and long-term animal experimental investigation

Decellularized human dermis as a potentially ideal scaffold for dermal substitution in severe burns was examined in a two‐staged animal experiment. In an initial step, an in vitro generated composite graft consisting of human keratinocytes and decellularized dermis (AlloDerm®) was transplanted onto nude mice in a short‐term trial (n = 20, 14 days). Subsequently, a combined one‐step grafting of full thickness wounds with both decellularized dermis (in part preincubated with fibroblasts) and cultivated autologous keratinocytes as a cell suspension in fibrin glue was done in a long‐term porcine animal model (n = 10, 6 months). In both series, macroscopic wound healing was evaluated by planimetry. Histological investigations included morphological as well as immunohistochemical parameters. The short‐term study showed both successful integration of the composite grafts and reduction of wound contraction compared with the control group (epithelial grafts). The long‐term porcine study displayed reduced myofibroblast formation and contraction in the wounds that had been treated with fibroblast‐preincubated dermis. After 4 weeks, a decline of the structural integrity of the dermal matrix could be noticed. The utility of decellularized dermis as template for both dermal reconstitution and keratinocyte delivery vehicle was shown. The closure of full thickness wounds by a single‐step combination of an autologous keratinocyte fibrin sealant suspension and acellular dermis in a pig animal model could be shown. Incorporation of fibroblasts led to reduced wound contraction but could not prevent the loss of dermal integrity. The engineered ‘skin’ remained viable and stable over a period of 6 months.     

Immunohistochemical localization of ornithine decarboxylase in human skin

The localization of ornithine decarboxylase (ODC) in human skin and cultured keratinocytes was studied with an immuno-histochemical method. ODC was found in the epidermis, hair follicles, sweat glands and errector muscles. Irritation induced by stripping or UV-B irradiation did not change the staining pattern in the epidermis. In psoriasis, the staining was most marked at the tip of the epidermal rete ridges. In basal cell carcinoma, there was a homogeneous labelling of the tumor cells and, in squamous cell carcinoma, the labelling was strong but less homogeneous. Melanoma and dermal naevus also positively stained for ODC. Cultured human keratinocytes also showed ODC positive immunofluorescence. This technique detects the ODC antigen present rather than levels of ODC activity.     

Endoplasmic reticulum stress is involved in hydrogen peroxide induced apoptosis in immortalized and malignant human oral keratinocytes

Background: Although hydrogen peroxide may play an important role in the development of cancer, it can be an efficient inducer of apoptosis in cancer cells; the exact mechanism by which this action occurs is not completely understood in oral cancer cells. Method: In this study, the mechanisms by which H₂O₂ inhibited growth and induced apoptosis were differentially investigated using HPV‐immortalized human oral keratinocytes (IHOK) and oral cancer cells (HN4). Results: H₂O₂ treatment sensitively and dose‐dependently induced growth inhibition and typical apoptosis in IHOK and HN4 cells, as demonstrated by a decreased level of cell viability, an increased population of cells in the sub‐G₀/G₁ phase, ladder formation of the genomic DNA, chromatin condensation and accumulation of Annexin V⁺/PI⁺ cells. Furthermore, the expression of Bax, p53 and p21^WAF1/CIP1 increased, whereas the expression of Bcl‐2 decreased in immortalized and malignant keratinocytes that were treated with H₂O₂. In addition, cytochrome‐c from the mitochondria was observed in H₂O₂‐treated IHOK and oral cancer cells, and this was accompanied by the activation of caspase‐3 and ‐9. Additionally, H₂O₂ treatment induced upregulation of CHOP, GRP78 and several representative endoplasmic reticulum (ER) stress‐responsive proteins, including heme oxygenase‐1. Conclusion: Overall, these results suggest that H₂O₂ triggers apoptosis via the mitochondrial and ER stress pathway in IHOK and HN4 cells, and that increasing the cellular levels of H₂O₂ sufficiently may lead to selective killing of oral cancer cells and therefore be therapeutically useful.     

Wound healing properties of jojoba liquid wax: an in vitro study

Aim of the study

The wound healing properties of jojoba (Simmondsia chinensis) liquid wax (JLW) were studied in vitro on HaCaT keratinocytes and human dermal fibroblasts, which are involved in wounded skin repair.

Materials and methods

JLW cytotoxicity was evaluated by the crystal violet staining and the neutral red uptake endpoint. Induction of wound healing by JLW was assessed by scratch wound assay on cell monolayers. The involvement of signaling pathways was evaluated by the use of the Ca²⁺ chelator BAPTA and of kinase inhibitors, and by Western blot analysis of cell lysates using anti-phospho antibodies. Collagen and gelatinase secretion by cells were assayed by in-cell ELISA and zymography analysis, respectively.

Results

Cytotoxicity assays showed that the toxic effects of JLW to these cells are extremely low. Scratch wound experiments showed that JLW notably accelerates the wound closure of both keratinocytes and fibroblasts. The use of inhibitors and Western blot revealed that the mechanism of action of JLW is strictly Ca²⁺ dependent and requires the involvement of the PI3K-Akt-mTOR pathway and of the p38 and ERK1/2 MAPKs. In addition, JLW was found to stimulate collagen I synthesis in fibroblasts, while no effect was detected on the secretion of MMP-2 and MMP-9 gelatinases by HaCaT or fibroblasts.

Conclusions

Taken together, data provide a pharmacological characterization of JLW properties on skin cells and suggest that it could be used in the treatment of wounds in clinical settings.     

Mathematical model for wound healing following autologous keratinocyte transplantation

In times of increasing economical pressure on the health care systems, it is important to optimise the outpatient treatment of chronic wounds. Another aim of wound healing research is to discover agents to accelerate healing. Wound healing trajectories or healing velocities can provide information to demonstrate the endpoints for wound healing. A great problem in clinical trials is to specify these parameters. Therefore, we developed a mathematical model for more transparency. In this initial project, we observed 19 wounds to construct the wound healing trajectories after transplantation of autologous keratinocytes, and the results are so encouraging that investigation in this area will continue. The developed mathematical model describes the clinical observed healing process. It was possible to find parameters to distinguish between old and young patients, retrospectively or prospectively calculate the healing rates and to determine exactly the endpoint of healing. Therefore, our model might be very useful in practices or for studies.     

Regulation of PDGF and PDGF receptor in cultured dermal papilla cells and follicular keratinocytes of the human hair follicle

Platelet-derived growth factor (PDGF) is a potent mitogenic factor for many cell types and has been shown to be important in follicular development and vasculogenesis. In this study, we examined the expression pattern of both PDGF factors and their corresponding receptors in mesenchyme-derived dermal papilla cells (DPCs) and epithelial follicular keratinocytes (FKs). Both types of PDGF receptors are expressed in FKs, whereas DPCs only express PDGF receptor beta on the protein level, a finding also seen in whole organ cultures. By examining the expression of PDGF ligands, we were able to show that cultured FKs synthesize both PDGF-A and PDGF-B, whereas, DPCs only express PDGF-A. As immunomodulatory cytokines were shown to affect hair growth, we investigated the effects of IL-1beta, IL-4, TNF-alpha, TGF-beta and IFN-gamma on the expression levels of PDGF factors in cultured DPCs and FKs. Interestingly, we could show a significant down-regulatory effect by catagen-inducing cytokines like IL-1beta or IFN-gamma, suggesting a possible involvement of PDGF signaling in the induction of catagen. The question concerning the latter hypothesis remains to be elucidated in further studies on whole organ cultures.