The role of ketoconazole in seborrheic dermatitis.

Although the prominent broad-spectrum activity of ketoconazole was reported in the early 1980s, its effect against Malassezia species was most pronounced; thus, it was developed for the treatment of various skin infections in which a link with these fungal species was proposed. Later, a number of ancillary properties were described for ketoconazole that comprised its anti-inflammatory, antiseborrheic, and antiproliferative profile. The incorporation of ketoconazole in an adapted vehicle could further promote its efficacy. Recently, a new formulation-an anhydrous gel containing ketoconazole 2%-was launched in which all of the ancillary properties were optimized.

An expert view on the treatment of acne with systemic antibiotics and/or oral isotretinoin in the light of the new European recommendations.

In 2003 the European Agency for the Evaluation of Medicinal Products amended the summary product characteristics for oral isotretinoin to standardise information provided from the different countries of the European Community. The Committee for Proprietary Medicinal Products recommended that among others, exclusively severe forms of acne (such as nodular or conglobate acne or acne at risk of permanent scarring) resistant to "adequate courses" of standard therapy with systemic antibacterials and local therapy should benefit from oral isotretinoin. However, no indication was provided on what were considered adequate courses or the possibility given to use oral isotretinoin as first line treatment. The aims of the present report were: 1) to provide a specialist view on when it is appropriate to introduce oral isotretinoin as a second line therapy for acne, taking into consideration optimum dosage and duration of systemic antibiotics prior to the start of the oral isotretinoin, and 2) to support the use of oral isotretinoin as first line therapy in specific cases for acne in clinical practice. The recommendations are based on an exhaustive literature review as well as on the personal experience of the members of an European panel of acne specialists. The EEP agreed with the decision made by the CPMP that oral isotretinoin should be administered as 2nd line therapy in those cases of severe acne, which were resistant to or which did not respond successfully to an initial combination regimen with systemic antibiotics and topical treatments (topical retinoids +/- benzoyl peroxide). However, the members emphasized that a number of prognostic factors, as well as psychosocial morbidity should be taken into account when choosing the regimen and that these factors may influence the use of oral isotretinoin as first line therapy.

Skin grafting and wound healing-the "dermato-plastic team approach".

Autologous skin grafts are successfully used to close recalcitrant chronic wounds especially at the lower leg. If wound care is done in a dermato-plastic team approach using the "integrated concept," difficulties associated with harvesting the skin graft as well as the complexities associated with inducing closure at the donor and the recipient site can be minimized. In the context of wound healing, skin transplantation can be regarded as (1) a supportive procedure for epithelialization of the wound surface and (2) mechanical stability of the wound ground. By placing skin grafts on a surface, central parts are covered much faster with keratinocytes. Skin (wound) closure is the ultimate goal, as wound closure means resistance to infection. Depending on the thickness of the skin graft, different amounts of dermis are transplanted with the overlying keratinocytes. The dermal component determines the mechanical (resistance to pressure and shear forces, graft shrinkage), functional (sensibility), and aesthetic properties of the graft. Generally speaking, the thicker the graft the better the mechanical, functional, and aesthetic properties, however, the worse the neo- and revascularization. Skin grafts do depend entirely on the re- and neovascularization coming from the wound bed. If the wound bed is seen as a recipient site for tissue graft, the classification of Lexer (Die freien Transplantationen. Stuttgart: Enke; 1924) turned out to be of extreme value. Three grades can be distinguished: "good wound conditions," "moderate wound conditions," and "insufficient wound conditions." Given good wound conditions, skin grafting is feasible. Nevertheless, skin closure alone might not be sufficient to fulfill the criteria of successful defect reconstruction. In case of moderate or insufficient wound conditions, wound bed preparation is necessary. If wound bed preparation is successful and good wound conditions can be achieved, skin grafting is possible. If, however, this attempt is unsuccessful and moderate or "inadequate wound conditions" are persisting, other methods of defect reconstruction such as local flap transfer, distant flap transfer, free (microvascular) flaps, and ultimately amputation must be considered.

1,25-Dihydroxyvitamin D3 and analogues protect primary human keratinocytes against UVB-induced DNA damage.

Exposure to UVB irradiation is a major risk factor for the development of skin cancer. Therefore, it is important to identify agents that can offer protection against UVB-caused damage. Photocarcinogenesis is caused largely by mutations at sites of incorrectly repaired DNA photoproducts, of which the most common are the cyclobutane pyrimidine dimers (CPDs). In this study, we demonstrated that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] protects primary human keratinocytes against the induction of CPDs by UVB. This protection required pharmacologic doses 1,25(OH)2D3 and an incubation period of at least 8 h before irradiation. Furthermore, we provided arguments indicating that the anti-proliferative capacity of 1,25(OH)2D3 underlies its protective effect against UVB-induced DNA damage. Finally, we showed that 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527), two low-calcemic analogues of 1,25(OH)2D3, were even 100 times more potent than the parent molecule in inhibiting UVB-caused DNA damage. These molecules are therefore promising candidates for the chemoprevention of UVB-induced skin cancer.

AKT status controls susceptibility of malignant keratinocytes to the early-activated and UVB-induced apoptotic pathway.

In previous work, we have described an early-activated and ultraviolet B (UVB)-induced apoptotic pathway in human keratinocytes, which can be completely inhibited by AKT activation. We now compared this response of primary human keratinocytes with the response of two p53-mutated squamous cell carcinoma (SCC)-derived cell lines (A431 and A253) to an apoptotic UVB dose. In these cell lines, both the basal AKT phosphorylation status and the apoptotic response to UVB diverged strongly from the response of healthy primary keratinocytes. Even more, a remarkable correlation was found between the two. Although a constitutive dual phosphorylation of AKT rendered the A253 SCC cell line completely resistant to the early-activated and UVB-induced apoptotic pathway, deficient T308 phosphorylation of AKT in the SCC cell line A431 led to a greatly augmented sensitivity to the early-activated, UVB-induced apoptotic pathway. These results indicate that the preservation of a healthy AKT pathway is essential for a wild-type UVB-induced apoptotic response in skin, and suggest that AKT-mediated dysregulation of the early-activated apoptotic response to UVB is an important event in the oncogenic transformation of keratinocytes.

Eicosapentaenoic acid, a n-3 polyunsaturated fatty acid differentially modulates TNF-alpha, IL-1alpha, IL-6 and PGE2 expression in UVB-irradiated human keratinocytes.

In response to UVB-irradiation keratinocytes release a variety of cytokines and prostaglandins, including tumor necrosis factor alpha (TNF-alpha), interleukin 1alpha (IL-1alpha), interleukin 6 (IL-6), and prostaglandin E2 (PGE2). n-3 Polyunsaturated fatty acids (PUFA), mainly present in fish oil, can modulate cytokine synthesis, as predominantly studied in macrophages. In order to investigate the immune modulating actions of n-3 PUFA on the UVB response in human skin, we investigated the effect of eicosapentaenoic acid (EPA), a n-3 PUFA and a precursor of eicosanoid biosynthesis, on UVB-modulated TNF-alpha, IL-1alpha, IL-6, and PGE2 expression in normal human keratinocytes (NHK). We show that cultured NHK can efficiently take up EPA. Basal TNF-alpha expression is very low in NHK. IL-1alpha on the contrary is significantly present in untreated cultured NHK. Upon UVB-irradiation (32 mJ per cm2) TNF-alpha mRNA expression and secretion is induced and IL-1alpha mRNA expression is reduced, although IL-1alpha secretion is induced. EPA treatment results in higher TNF-alpha and IL-1alpha expression, both in nonirradiated and UVB-irradiated keratinocytes. Moreover EPA and UVB appear to act synergistically to superinduce TNF-alpha expression. EPA treatment results also in lipid peroxidation and in decreased PGE2 and IL-6 secretion after UVB-irradiation. In contrast to EPA, oleic acid (monounsaturated fatty acid) and linoleic acid (n-6 PUFA) treatment did not result in higher TNF-alpha or IL-1alpha levels in nonirradiated or UVB-irradiated NHK, indicating that the observed effects are specific for EPA. In conclusion, these results show that EPA can differentially modulate UVB-induced cytokine and prostaglandin synthesis in NHK.

Induction of tumor necrosis factor-alpha by UVB: a role for reactive oxygen intermediates and eicosanoids.

UVB irradiation induces nuclear factor-kappaB (NF-kappaB) activation, tumor necrosis factor-alpha (TNF-alpha) expression and reactive oxygen intermediates (ROI) in keratinocytes. We investigated whether ROI play a role in UVB-induced TNF-alpha mRNA expression. The antioxidants N-acetyl cysteine, NAC, epigallocathin gallate, EGCG, butylated hydroxyanisole (BHA) and vitamin C could reduce UVB-induced TNF-alpha mRNA levels to various degrees; vitamin E (alpha-tocopherol) had no effect. BHA was the most potent inhibitor. The oxidant tertiary butylated hydroperoxide could effectively induce TNF-alpha mRNA expression. Nordihydroguaiaretic acid (NDGA) and MK-886, inhibitors of lipoxygenase (LOX), and indometacin and quinacrine, inhibitors of cyclooxygenase (COX) and phospholipase A2, respectively, could also reduce UVB-induced TNF-alpha mRNA expression. Inhibition by NDGA was in concordance with the results for BHA. NDGA, indometacin, quinacrine and BHA could also effectively inhibit the inhibitor of NF-kappaB degradation, thereby maintaining NF-kappaB inactivity. In conclusion, we show that ROI are implicated in the induction of TNF-alpha mRNA by UVB and that not all antioxidants are equally effective inhibitors. COX products and more importantly LOX products, which themselves are products of an oxidative metabolism, are the main ROI implicated in this induction of TNF-alpha expression by UVB probably via activation of NF-kappaB.

Two 14-epi analogues of 1,25-dihydroxyvitamin D3 protect human keratinocytes against the effects of UVB.

In search of photoprotective agents, we recently demonstrated a protective effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] against different events mediated by ultraviolet B (UVB) in human keratinocytes. Pharmacological doses of 1,25(OH)(2)D(3) were required to obtain significant UVB protection; however, these doses cannot be used in vivo due to the calcemic properties of 1,25(OH)(2)D(3). Therefore, we evaluated the photoprotective capacities of two low-calcemic 14-epi analogues of 1,25(OH)(2)D(3), 19-nor-14-epi-23-yne-1,25(OH)(2)D(3) (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3) (TX 527). Using cultured human keratinocytes, we investigated the influence of TX 522 and TX 527 on two hallmark events in UVB-irradiated keratinocytes: the induction of apoptosis and the production of interleukin-6 (IL-6). Treatment of the keratinocytes with TX 522 or TX 527, 24 h before irradiation, resulted in a significant and dose-dependent reduction of both UVB-induced apoptosis and IL-6 production. Both analogues were equally efficient in their anti-UVB effects and at least 100 times more potent than 1,25(OH)(2)D(3). We further demonstrated that metallothionein (MT) mRNA expression was clearly induced by 1,25(OH)(2)D(3) and both analogues. MT acts as a radical scavenger in oxygen-mediated UVB injury and its induction may therefore be relevant for the anti-UVB effects of 1,25(OH)(2)D(3) and both analogues. Taken together, these findings create new perspectives for the use of active vitamin D analogues as photoprotective agents.

European recommendations on the use of oral antibiotics for acne.

Non-rational prescribing of oral antibiotics in acne is common, and there is currently an unmet need for up-to-date guidelines that specifically address these issues. Presented here is a set of recommendations on the use of oral antibiotics in acne, developed by a group of European acne specialists, designed to be considered by dermatologists and general practitioners in their daily practice throughout Europe. Recommendations cover optimal choice of antibiotic, drug doses, duration of treatment, combination treatment, and maintenance therapy.

Contact allergic reactions of the vulva: a 14-year review.

BACKGROUND: In women with vulval complaints, irritant contact dermatitis is more common than allergic contact dermatitis, but secondary sensitization has to be taken into consideration since these patients often use several topical medications. OBJECTIVE: The aims of this retrospective study were to establish the prevalence of allergic contact dermatitis in patients with vulval complaints and to verify how many patients with allergic contact dermatitis suffered from a previous pathology. METHODS: We reviewed patch- and prick-test results from 92 women. The women had all been administered the European Standard series, and most had also been tested for other allergens such as the ingredients of topical pharmaceutical products and cosmetics. RESULTS: Thirty-five patients (38%) presented with one or more positive allergic reactions. For 15 of the patients, these reactions were considered to be relevant to their clinical condition and were most often due to contact dermatitis from topical pharmaceutical products. Three patients presented with positive and relevant contact urticaria syndrome from latex, and two patients presented with protein contact dermatitis from human seminal plasma. CONCLUSION: Patients with vulval dermatitis are at risk of developing contact sensitivities, particularly to topical pharmaceutical products; therefore, attention should be paid to this problem when such products are prescribed.

Clinical forms of dermatophytosis (ringworm infection).

Dermatophytic infections of the skin, hairs and nails are very common and are very variable in aspect. In skin, inflammatory symptoms are often absent, but in other cases they may be very pronounced, in particular when caused by zoophilic dermatophytes. In onychomycosis, it is very difficult to make the differential diagnosis with other causes of onychodystrophy on purely clinical grounds; indeed, even in case of fungal infection, the causative agent can be suspected on clinical grounds only in a minority of cases. The clinical presentation of skin infections, infections of the scalp and beard, and the nails are presented more in detail.

Detection of basophil-activating IgG autoantibodies in chronic idiopathic urticaria by induction of CD 63.

BACKGROUND: Approximately 40% to 50% of patients with chronic idiopathic urticaria (CIU) have functional IgG autoantibodies against FcepsilonRIalpha or IgE, which induce histamine release from basophils and cutaneous mast cells. A positive autologous serum skin test response is believed to reflect the presence of these autoantibodies. OBJECTIVE: We sought to further define the functional properties of and develop a sensitive functional assay for detection of autoantibodies in patients with CIU. METHODS: Sera from patients with CIU (n=61) and sera from healthy control subjects (n=23) were incubated with donor basophils. Activation of basophils was determined on the basis of CD 63 surface expression, as analyzed on a FACScan flow cytometer. RESULTS: A positive basophil activation test result was found in 51% of patients with CIU, and basophil-activating properties were present in the IgG fractions of sera. When both the in vitro test and the autologous serum skin test were considered, basophil/mast cell-activating autoantibodies were present in 62% of the patients. Patients with a positive basophil activation test result had a significantly higher prevalence of other autoantibodies, had more severe urticaria, and were more likely to have angioedema. CONCLUSION: The results demonstrate the presence of basophil-activating autoantibodies in about 50% of patients with CIU. The data support the autoimmune cause of the disease and provide a simple test for detection of these autoantibodies.

Insulin-like growth factor-1-mediated AKT activation postpones the onset of ultraviolet B-induced apoptosis, providing more time for cyclobutane thymine dimer removal in primary human keratinocytes.

Insulin-like growth factor-1 (IGF-1) acts as a potent survival factor in numerous cell lines, primarily through activation of the AKT signaling pathway. Although some targets of this pathway have known anti-apoptotic functions, its relationship with the improved survival of cells after exposure to environmental stresses, including UVB, remains largely unclear. We report that in growth factor-deprived keratinocytes, IGF-1 significantly and consistently delayed the onset of UVB-induced apoptosis by >7 h. This delay allowed IGF-1-supplemented keratinocytes to repair significantly more cyclobutane thymine dimers than their growth factor-deprived counterparts. This increase in cyclobutane thymine removal resulted in enhanced survival if the amount of DNA damage was not too high. To increase cell survival after UVB irradiation, IGF-1 supplementation was required only during this initial time period in which extra repair was executed. Finally, we show that IGF-1 mediated this delay in the onset of UVB-induced apoptosis through activation of the AKT signaling pathway. We therefore believe that the AKT signaling pathway increases cell survival after a genotoxic insult such as UVB irradiation not by inhibiting the apoptotic stimulus, but only by postponing the induction of apoptosis, giving the DNA repair mechanism more time to work.

1,25-Dihydroxyvitamin D3 inhibits ultraviolet B-induced apoptosis, Jun kinase activation, and interleukin-6 production in primary human keratinocytes.

We investigated the capacity of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to protect human keratinocytes against the hazardous effects of ultraviolet B (UVB)-irradiation, recognized as the most important etiological factor in the development of skin cancer. Cytoprotective effects of 1,25(OH)(2)D(3) on UVB-irradiated keratinocytes were seen morphologically and quantified using a colorimetric survival assay. Moreover, 1,25(OH)(2)D(3) suppressed UVB-induced apoptotic cell death. An ELISA, detecting DNA-fragmentation, demonstrated that pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM for 24 h reduced UVB-stimulated apoptosis by 55-70%. This suppression required pharmacological concentrations 1,25(OH)(2)D(3) and a preincubation period of several hours. In addition, 1,25(OH)(2)D(3) also inhibited mitochondrial cytochrome c release (90%), a hallmark event of UVB-induced apoptosis. Furthermore, we demonstrated that 1,25(OH)(2)D(3) reduced two important mediators of the UV-response, namely, c-Jun-NH(2)-terminal kinase (JNK) activation and interleukin-6 (IL-6) production. As shown by Western blotting, pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM diminished UVB-stimulated JNK activation with more than 30%. 1,25(OH)(2)D(3) treatment (1 microM) reduced UVB-induced IL-6 mRNA expression and secretion with 75-90%. Taken together, these findings suggest the existence of a photoprotective effect of active vitamin D(3) and create new perspectives for the pharmacological use of active vitamin D compounds in the prevention of UVB-induced skin damage and carcinogenesis.

Molecular pathways involved in the anti-apoptotic effect of 1,25-dihydroxyvitamin D3 in primary human keratinocytes.

We previously reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] protects primary human keratinocytes against ultraviolet (UV)B-induced apoptosis. Here, we confirmed the anti-apoptotic effect of 1,25(OH)2D3 in keratinocytes, using cisplatin and doxorubicin as apoptotic triggers. We further showed that 1,25(OH)2D3 activates two survival pathways in keratinocytes: the MEK/extracellular signal regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway. Activation of ERK and Akt by 1,25(OH)2D3 was transient, required a minimal dose of 10(-9) M and could be blocked by actinomycin D and cycloheximide. Moreover, inhibition of Akt or ERK activity with respectively a PI-3K inhibitor (LY294002) or MEK inhibitors (PD98059, UO126), partially or totally suppressed the anti-apoptotic capacity of 1,25(OH)2D3. Finally, 1,25(OH)2D3 changed the expression of different apoptosis regulators belonging to the Bcl-2 family. Indeed, 1,25(OH)2D3 treatment increased levels of the anti-apoptotic protein Bcl-2 and decreased levels of the pro-apoptotic proteins Bax and Bad in a time- and dose-dependent way. Induction of Bcl-2 by 1,25(OH)2D3 was further shown to be mediated by ERK and, to a lesser extent, by Akt. In conclusion, 1,25(OH)2D3 clearly protects keratinocytes against apoptosis (1) by activating the MEK/ERK and the PI-3K/Akt survival pathways and (2) by increasing the Bcl-2 to Bax and Bad ratio.

Loss of membranous expression of beta-catenin is associated with tumor progression in cutaneous melanoma and rarely caused by exon 3 mutations.

beta-Catenin plays a fundamental role in the regulation of the E-cadherin-catenin cell adhesion complex. It also plays a role in the Wnt signaling pathway by activating T-cell factor- and lymphoid enhancer factor-regulated gene transcription. The level of beta-catenin in cells is tightly controlled in a multiprotein complex, and mutations in the glycogen synthase kinase 3beta (GSK-3beta) phosphorylation sites of the beta-catenin gene (CTNNB1) result in nuclear and/or cytoplasmic accumulation of beta-catenin and constitutive transactivation of T-cell factor and lymphoid enhancer factor target genes, a mechanism occurring in many cancers. Melanoma cell lines may harbor beta-catenin mutations; in vivo, however, cellular accumulation of beta-catenin is rarely caused by CTNNB1 mutations. In our study, 43 primary cutaneous melanoma and 30 metastases were screened for CTNNB1 exon 3 mutations by using a denaturing gradient gel electrophoresis technique and sequencing. beta-Catenin mutations were found in 2 primary melanomas and 1 metastatic melanoma and were not correlated with nuclear accumulation of beta-catenin in these cases. Cellular expression of beta-catenin was evaluated by immunohistochemistry and by reverse polymerase chain reaction (RT-PCR) in 80 and 70 cases, respectively. Immunohistochemistry revealed a significant loss of membranous beta-catenin staining between the primary and metastatic melanomas as well as between radial and vertical growth phase. RT-PCR showed a significant inverse correlation between the amount of RNA and the proportion of cells with membranous expression of beta-catenin (P =.0015); no correlation existed between the amount of RNA and the number of cells with nuclear or cytoplasmic expression of beta-catenin. In conclusion, nuclear expression of beta-catenin is seen in cutaneous melanoma but, in contrast to the case of many other cancers, does not correlate with tumor stage or mutation status. A combination of immunohistochemistry and RT-PCR showed that down-regulation of membranous beta-catenin was associated with an increased amount of beta-catenin RNA in primary or metastatic melanoma. Our results suggest that posttranslational events, rather than CTNNB1 mutations, are responsible for the altered distribution of beta-catenin in cutaneous melanoma.