Cryosurgery during topical imiquimod: a successful combination modality for lentigo maligna

Background Either cryosurgery or topical imiquimod have been used to treat patients with lentigo maligna in cases where surgery is not feasible. Methods We report a patient with lentigo maligna, who was treated with the combination of topical imiquimod and cryosurgery, and review the rationale, which led us to design the present combined cryo‐immunological treatment modality. Results Sustained clearance of lentigo maligna to date (26 months after treatment). The successful treatment of this patient was based on the following rationale: A cryosurgery session during continuing imiquimod application may: (i) reinforce apoptosis of tumor cells; (ii) strengthen antiangiogenesis in the treated tumor; and (iii) build‐up a potent tumor‐destructive immune response by a cascade of events starting with imiquimod‐promoted attraction of immature dendritic antigen‐presenting cells (DCs) into the tumor. DCs further mature within the tumor‐antigen‐rich environment of subsequently cryo‐destructed tumor and upon imiquimod‐driven migration into the peripheral lymph nodes can stimulate a specific antineoplastic cell‐mediated immunity. Finally, continuing imiquimod application after cryosurgery may increase recruitment of activated effector cells into the tumor tissue leading to its destruction. Conclusion Cryosurgery during continued topical imiquimod seems to be a promising treatment for lentigo maligna.     

Cationic membrane‐active peptides – anticancer and antifungal activity as well as penetration into human skin

Cationic antimicrobial peptides are ancient natural broad‐spectrum antibiotics, and several compounds also exhibit anticancer activity. However, most applications pertain to bacterial infections, and treatment for skin cancer is less frequently considered. The cytotoxicity of melittin, cecropin A, protegrin‐1 and histatin 5 against squamous skin cancer cell lines and normal human keratinocytes was evaluated and compared to established drugs. The results show that melittin clearly outperforms 5‐fluorouracil regarding antitumor activity. Importantly, combined melittin and 5‐fluorouracil enhanced cytotoxic effects on cancer cells and reduced toxicity on normal keratinocytes. Additionally, minimum inhibitory concentrations indicate that melittin also shows superior activity against clinical and laboratory strains of Candida albicans compared to amphotericin B. To evaluate its potential for topical applications, human skin penetration of melittin was investigated ex vivo and compared to two non‐toxic cell‐penetrating peptides (CPPs), low molecular weight protamine (LMWP) and penetratin. The stratum corneum prevents penetration into viable epidermis over 6 h; however, the peptides gain access to the viable skin after 24 h. Inhibition of digestive enzymes during skin penetration significantly enhances the availability of intact peptide. In conclusion, melittin may represent an innovative agent for non‐melanoma skin cancer and infectious skin diseases. In order to develop a drug candidate, skin absorption and proteolytic digestion by skin enzymes need to be addressed.     

Topical ceramides neither enhance UVB‐induced apoptosis in normal human keratinocytes nor affect viability in UVB‐irradiated reconstructed human epidermis

Ceramides are the major lipid of lamellar sheets present in intercellular spaces of the stratum corneum contributing to epidermal barrier properties. Therefore, ceramides and their analogues have been studied for barrier enhancing and water‐holding properties for decades. In vitro studies have indicated cytotoxic potential for cell‐permeable ceramides thereby raising the question whether topical ceramide application might contribute to UVB‐induced apoptosis. Phytosphingosine, N‐hexanoyl‐phytosphingosine and N‐stearoylphytosphingosine (ceramide III) in concentrations ≤5 μm have been used for co‐stimulation with low (160 J/m²) or high (600 J/m²) UVB doses in subconfluent basal and confluent differentiating keratinocytes. Significantly, increased caspase‐3 activity was observed in basal keratinocytes irradiated with 600 J/m² UVB and in differentiating keratinocytes with both UVB doses. Co‐stimulation with the named ceramides did not further increase (i) caspase‐3 activity and (ii) nucleosomal fragmentation in differentiating keratinocytes. Moreover, co‐stimulation with 1‐mm ceramides did not further affect viability/lactate dehydrogenase release in UVB‐irradiated reconstructed human epidermis corroborating the safety of these ceramides.     

Skin blood flow in necrobiosis lipoidica diabeticorum

Background Necrobiosis lipoidica diabeticorum (NLD) is a granulomatous skin reaction found in < 1% of diabetic patients. Our purpose was to determine if NLD represented areas of cutaneous ischemia. Methods Using laser Doppler flowmetry, we measured cutaneous blood flow in nine diabetic patients at NLD lesions and at contiguous uninvolved sites. Flow values were also determined at several reference sites noncontiguous with the NLD lesions and compared to age‐ and sex‐matched controls: 24 diabetic subjects without skin abnormalities, 18 diabetic patients with dermopathy, and 40 nondiabetic subjects. Results NLD lesions exhibited significantly higher blood flow (4.8 ± 0.7 ml/min/100 g) than areas of unaffected skin close to the lesions (1.2 ± 0.1 ml/min/100 g) (P < 0.01 for both comparisons). There were no significant differences in flow between normal skin sites in NLD patients and normal sites in diabetic patients without skin lesions. Conclusions Our findings refute the hypothesis that NLD is a manifestation of microvascular ischemic disease of the skin. The increased blood flow seen in NLD lesions suggests an ongoing inflammatory process.     

Spleen tyrosine kinase (SYK) is a potential target for the treatment of cutaneous lupus erythematosus patients

Spleen tyrosine kinase (SYK) is a protein kinase involved in cell proliferation and the regulation of inflammatory pathways. Due to the increasing evidence that kinase inhibitors have potential as specific anti‐inflammatory drugs, we have investigated the potential for SYK inhibition as a therapeutic target in autoimmune diseases, particularly cutaneous lupus erythematosus (CLE). Skin samples of patients with different CLE subtypes and appropriate controls were analysed for the expression of SYK and SYK‐associated pro‐inflammatory mediators via gene expression analysis and immunohistochemistry. The functional role of SYK in keratinocytes was investigated in vitro, using LE‐typical pro‐inflammatory stimuli and a selective inhibitor of SYK. SYK‐associated genes are strongly upregulated in CLE skin lesions. Importantly, phosphorylated SYK (pSYK) is strongly expressed by several immune cell types and also keratinocytes in CLE skin. In vitro, immunostimulatory nucleic acids are capable of inducing SYK phosphorylation in keratinocytes leading to the induction of pro‐inflammatory cytokines, while small‐molecule SYK inhibition decreases the expression of these proteins. The results demonstrate that pSYK is expressed by immune cells and keratinocytes in skin lesions of CLE patients. LE‐typical stimuli induce the expression of pSYK in vitro. Small‐molecule SYK inhibition leads to a reduction of pSYK expression and downregulation of pro‐inflammatory cytokines in keratinocytes. We therefore believe that pSYK provides a potential future drug target for the treatment of patients who suffer from CLE and related skin disorders. Specifically, our study reveals evidence supporting the use of topical SYK inhibitors in treating lupus.     

In vitro cytotoxicity of antimicrobial agents to human keratinocytes

Backround The degree of bacterial contamination in the wound bed plays a key role in determining the level of “take” of keratinoeyte grafts in burn patients. This study was done to evaluate the best suited antimicrobial agent for topical application in relation to antimicrobial activity and cytotoxic effect on graft cells. Objective The cytotoxicity of 7 antibacterial agents (gentamicin sulphate, ciprofloxacin hydrochioride, bacitracin, polymyxin B sulphate, diethanolamin salt of fusidic acid, teicoplanin, vancomycin hydrochtoride), one anlifungal substance (amphotcricin B sodium deoxycholate) and one antiseptic agent (povidone iodine) on cultured human keratinocytes, HaCaT keratinocytes and fibroblasts was investigated. Material and methods The effects of these agents were tested using two separate assays - the neutral red assay and the Bradford protein assay. In order to assess toxicity of the agent, the obtained midpoint cytotoxicity (MC50) was compared to the respective minimal inhibitory concentration (MIC) for receiving the cytotoxic potential in microbiologically effective doses. Results Antibiotics effective against Staphylococcus aureus with a small potential of toxicity are Bacitracin, Fusidic acid, Vancomycin and Tcicoplanin. Gentamicin and Ciprofloxacin are effective against both Staphylococcus aureus and Pseudomonas aeruginosa with a low potential of toxicity. Conclusion The use of antimicrobial agents with a midpoint cytotoxicity (MC50) higher than the minimal inhibitory concentration (MIC) by at least a factor of 100 might reduce the risk of toxic injury to graft cells.     

Keratins 2 and 4/13 in reconstituted human skin are reciprocally regulated by retinoids binding to nuclear receptor RARα

Please cite this paper as: Keratins 2 and 4/13 in reconstituted human skin are reciprocally regulated by retinoids binding to nuclear receptor RARα. Experimental Dermatology 2010; 19: 674–681. Abstract: Disorders of keratinization are often treated with vitamin A derivatives (retinoids) which affect keratinocyte differentiation, including keratin (KRT) gene expression. In vivo, suprabasal keratinocytes normally express only keratin (K) 1, K2 and K10, but after topical application of all‐trans retinoic acid (ATRA), the granular cells will additionally express K4 and K13, i.e. keratins normally present in oral mucosa and in cultured epidermal keratinocytes. To learn more about the retinoid regulation of keratin expression under in vivo‐like conditions, we cultured keratinocytes on de‐epidermized dermis in only 0.5% serum. These cells produce a normal‐looking epidermis that expresses high mRNA levels of KRT1, KRT2 and KRT10, but minimal amounts of KRT4 and KRT13. Addition of ATRA to the medium for 48 h caused a dose‐dependent increase in KRT4/KRT13 and a down‐regulation of KRT2 mRNA. An increase in K4 protein was also found. The response was greater than the up‐regulation of another retinoid‐regulated gene, CRABPII. By studying 10 retinoids with different affinities for the retinoic acid receptors (RAR) and retinoid X receptors (RXR) isoforms, the reciprocal expression of KRT2 and KRT4/KRT13 could be connected with agonists for RARα. Two of these agonists, CD336/Am580 and CD2081, altered the expression profile with similar potency as the pan‐RAR agonists ATRA and CD367. Co‐addition of a pan‐RAR antagonist (CD3106/AGN193109) markedly inhibited the induction of KRT4/KRT13 expression, whereas the down‐regulation of KRT2 was less affected. In conclusion, RARα agonists elicit a reciprocal modulation of KRT2 and KRT4/KRT13 expression in human epidermis, but whether or not the keratin genes also possess RARα‐specific regulatory elements is still unclear.     

NF‐κB is involved in inhibition of lipoxin A4 on dermal inflammation and hyperplasia induced by mezerein

Please cite this paper as: NF‐κB is involved in inhibition of lipoxin A₄ on dermal inflammation and hyperplasia induced by mezerein. Experimental Dermatology 2010; 19 : e286–e288. Abstract: The mechanisms by which lipoxin A₄ (LXA₄) inhibit skin inflammation remain unclear. In the present studies, the ear inflammatory model was induced by topical application of mezerein. Treatment of the mouse ear with LXA₄ exhibited the inhibitory effects on oedema, neutrophil infiltration, vascular permeability, expressions of interleukin (IL)‐1, IL‐6 and IL‐8 mRNA, DNA‐binding activity of nuclear factor‐κB (NF‐κB), and on dermal hyperplasia. NF‐κB reporter activities and nuclear translocations of NF‐κB p65 in cultured keratinocytes stimulated by mezerein were inhibited by pretreatment of the cells with LXA₄. LXA₄ reduced degradation, but not phosphorylation of IκBα in cultured keratinocytes stimulated by mezerein, suggesting that LXA₄‐attenuated IκBα degradation may restore the mezerein‐blocked inhibitory effects of IκB on nuclear translocation and DNA‐binding activity of NF‐κB. Our results demonstrated that LXA₄ displays the anti‐inflammatory and anti‐proliferative role on ear inflammatory model induced by mezerein and these effects were related with downregulation of DNA‐binding activity of NF‐κB.     

Topical application of TRPM8 agonists accelerates skin permeability barrier recovery and reduces epidermal proliferation induced by barrier insult: role of cold‐sensitive TRP receptors in epidermal permeability barrier homoeostasis

Please cite this paper as: Topical application of TRPM8 agonists accelerates skin permeability barrier recovery and reduces epidermal proliferation induced by barrier insult: role of cold‐sensitive TRP receptors in epidermal permeability barrier homoeostasis. Experimental Dermatology 2010; 19 : 791–795. Abstract: TRPA1 and TRPM8 receptors are activated at low temperature (A1: below 17°C and M8: below 22°C). Recently, we observed that low temperature (below 22°C) induced elevation of intracellular calcium in keratinocytes. Moreover, we demonstrated that topical application of TRPA1 agonists accelerated the recovery of epidermal permeability barrier function after disruption. In this study, we examined the effect of topical application of TRPM8 modulators on epidermal permeability barrier homoeostasis. Immunohistochemical study and RT‐PCR confirmed the expression of TRPM8 or TRPM8‐like protein in epidermal keratinocytes. Topical application of TRPM8 agonists, menthol and WS 12 accelerated barrier recovery after tape stripping. The effect of WS12 was blocked by a non‐selective TRP antagonist, Ruthenium Red, and a TRPM8‐specific antagonist, BTCT. Topical application of WS12 also reduced epidermal proliferation associated with barrier disruption under low humidity, and this effect was blocked by BTCT. Our results indicate that TRPM8 or a closely related protein in epidermal keratinocytes plays a role in epidermal permeability barrier homoeostasis and epidermal proliferation after barrier insult.     

Calcipotriol induces apoptosis in psoriatic keratinocytes

In recent years, vitamin D3 analogues have become one of the most widely prescribed topical treatments for mild or moderate chronic plaque psoriasis. These molecules are effective and safe, but their exact mechanism of action is not completely understood. In vitro studies have shown that D3 analogues decrease proliferation and induce differentiation of keratinocytes, and have strong immunomodulating effects, but there are no conclusive data about apoptosis. The aim of this study was to evaluate differences in apoptotic response between lesional and perilesional keratinocytes of patients with psoriasis before and after treatment with calcipotriol, a synthetic vitamin D3 analogue. Keratinocytes were isolated from psoriatic plaques including lesional and perilesional skin, and cultured. Cells were treated with calcipotriol for 20 h and examined under confocal microscopy after staining with propidium iodide. The number of apoptotic cells after incubation with calcipotriol was significantly higher in lesional than in perilesional keratinocytes (P < 0.05) or non‐treated psoriatic keratinocytes (P < 0.05). In conclusion, calcipotriol seems to induce apoptosis in psoriatic keratinocytes.     

Immunostimulatory activity of murine keratinocyte‐derived exosomes

It has long been known that keratinocytes influence cutaneous immunity through secretion of soluble factors. Exosomes, small membrane vesicles of endocytotic origin, have been implicated in intercellular communication processes such as the transfer of tumor cell antigens and the activation of recipient dendritic cells (DC). However, little is known about immunomodulatory functions of keratinocyte‐derived exosomes. To address this question, we analysed exosome secretion of the murine keratinocyte cell line MPEK under steady state as well as inflammatory conditions (+/? IFNγ). These exosomes were readily taken up by bone marrow‐derived DC (BMDC) in vitro resulting in a matured phenotype, as evidenced by increased CD40 expression as well as by the production of large amounts of IL‐6, IL‐10 and IL‐12. When the transfer of antigen‐specific information through exosomes was investigated, it was found that keratinocytes took up antigen (ovalbumin) and transferred it to their exosomes. However, these antigen‐harbouring exosomes failed to induce antigen‐specific T cell responses via BMDC. Together, this novel biological function suggests that keratinocytes are able to direct unspecific immune processes but do not elicit specific immune responses.     

Effects of a 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitor and low‐density lipoprotein on proliferation and migration of keratinocytes

Background Keratinocytes can obtain cholesterol either by de novo synthesis or by extraction, primarily from low‐density lipoprotein (LDL). LDL is internalized following binding to the LDL receptor (LDLR). Because LDLR is expressed at a higher level in the cells of the basal layer of the epidermis, it might be assumed that LDLR upregulation is associated with keratinocyte proliferation. However, the effect of LDLR stimulation on keratinocyte function remains unclear. Objectives To investigate the effects and mechanism of action of pitavastatin and effects of LDL on proliferation and migration of keratinocytes. Methods Pitavastatin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, was used to induce upregulation of LDLR. LDLR expression was evaluated by immunofluorescence staining, fluorescence‐activated cell sorting, immunohistochemical staining and real‐time polymerase chain reaction (PCR). HaCaT cells and normal human keratinocytes (NHKs) were used for evaluation of migration. 5‐Bromo‐2′‐deoxyuridine incorporation was used to evaluate keratinocyte proliferation and differentiation. C57BL6 mice were used for in vivo evaluation of the effect of topical pitavastatin or lovastatin. Results Pitavastatin was most effective in LDLR induction at a concentration of 1 μmol L^?1 in NHKs. Real‐time PCR showed that pitavastatin significantly increased LDLR and liver X receptor (LXR) β mRNA expression in these cells. Similar results were obtained in vivo. However, pitavastatin had no effect on the migration of NHKs. After the addition of LDL and/or mevalonate concomitantly with pitavastatin to NHK cultures, or topical application of pitavastatin on mouse skin, keratinocyte proliferation was significantly increased. Conclusions Pitavastatin significantly upregulates LDLR in both NHKs and C57BL6 mouse skin, resulting in increased keratinocyte proliferation. LXRβ may be involved in the pitavastatin‐induced keratinocyte proliferation.     

Obesity exacerbates imiquimod‐induced psoriasis‐like epidermal hyperplasia and interleukin‐17 and interleukin‐22 production in mice

Psoriasis is a chronic inflammatory skin disorder that is accompanied by an imbalance between the proliferation and differentiation of keratinocytes. A number of studies have suggested an association between obesity and severe psoriasis; however, it remains to be clarified whether obesity exacerbates psoriasis. To address this unsolved question, we induced psoriasiform dermatitis in mouse models for obesity. We found that obesity exaggerated the severity of psoriasiform dermatitis induced by topical application of the Toll‐like receptor (TLR) 7 agonist, imiquimod. Ear swelling and epidermal hyperplasia were more prominent in the obese mice than in the control mice. When compared to imiquimod‐treated control mice, imiquimod‐treated obese mice expressed higher levels of psoriasis mediators, interleukin‐17A (IL‐17A) and IL‐22 in the skin. Food intake restriction partially abrogated enhanced ear swelling and cytokine overproduction in obese mice. Furthermore, the obesity environment and imiquimod treatment synergistically induced an IL‐17A downstream molecule, regenerating islet‐derived 3γ (Reg3γ), which is a critical molecule for psoriatic epidermal hyperplasia. Palmitic acid, one of the fatty acids released by subcutaneous adipocytes, increased the expression of REG3A (a human homologue of mouse Reg3γ) in both the HaCaT keratinocyte cell line and normal human keratinocytes. Taken together, these results strongly suggest that obesity exacerbates psoriasiform dermatitis in mice by upregulating IL‐17A, IL‐22 and Reg3γ.     

Increased cutaneous absorption reflects impaired barrier function of reconstructed skin models mimicking keratinisation disorders

The aim of this study was to assess a recently established 3D model of congenital ichthyosis, representing severe epidermal barrier function defects, for skin penetration and permeation. We have generated disease models by knock‐down of either TGM1 or ALOXE3 in primary human keratinocytes, and using keratinocytes and fibroblasts from patients with congenital ichthyosis. The results indicate disturbed barrier function as demonstrated by increased permeation of testosterone and caffeine particularly in TGM1 knock‐down models compared to control models. In addition, enhanced penetration of the model dye nile red incorporated into solid lipid nanoparticles and core‐multishell nanotransporters, respectively, was evident in disease models. Thus, in vitro skin disease models reproduce differences in barrier permeability and function seen in congenital ichthyosis and pave the way to personalised disease models. Furthermore, our findings indicate that nanocarriers may be useful in new, topical therapeutic approaches for the currently very limited treatment of congenital ichthyosis.     

Curcumin inhibits UVB‐induced matrix metalloproteinase‐1/3 expression by suppressing the MAPK‐p38/JNK pathways in human dermal fibroblasts

Curcumin (diferuloylmethane) is a polyphenol derived from turmeric (Curcuma longa), which is commonly used as a spice. Recent studies have shown that curcumin has a wide range of pharmacological activities, including anticarcinogenic, antioxidant, anti‐inflammatory and antiangiogenic activities. However, the antiphotoageing effects of curcumin have yet to be characterized. In this study, we investigated the inhibitory effects of curcumin on matrix metalloproteinase (MMP)‐1 and MMP‐3 expression in human dermal fibroblast cells. Western blot analysis revealed that curcumin inhibited ultraviolet (UV) B‐induced MMP‐1 and MMP‐3 expression. Furthermore, curcumin significantly blocked UVB‐induced reactive oxygen species generation in fibroblasts. Curcumin treatment significantly blocked the UVB‐induced activation of nuclear factor (NF)‐κB and activator protein (AP)‐1. Additionally, curcumin strongly repressed the UVB‐induced phosphorylation of p38 and c‐Jun N‐terminal kinase. Curcumin prevented UVB‐induced MMP expression through mitogen‐activated protein kinase/NF‐κB inhibition and AP‐1 activation. In conclusion, curcumin may be useful for preventing and treating skin photoageing.     

IL‐26 in allergic contact dermatitis: Resource in a state of readiness

In this study, we investigated the role of IL‐26 in allergic contact dermatitis (ACD), highlighting its’ contribute in the cytotoxic mechanism responsible for the tissue injury. IL‐26 is a signature Th17 cytokine, and immune cells are its predominant sources. Recently, it has shown that Th17 cell‐derived‐IL‐26 functions like an antimicrobial peptide. Here, we hypothesized that IL‐26 could be involved in cytotoxicity mechanism that underlies ACD. Indeed, we have attributed a role to IL‐26 in this context, through PBMC cytotoxicity assays vs HaCat. To demonstrate that IL‐26 was effectively involved in this activity, we performed the assay using transfected ACD PBMCs by siRNA for IL‐26. Indeed, we demonstrated that these cells were less able to kill keratinocytes compared with ACD PBMCs (P < .01). In conclusion, our findings support the idea that this emergent cytokine, IL‐26, is implicated in the killing mechanisms of KC observed during ACD.