Hedgehog- and mTOR-targeted therapies for advanced basal cell carcinomas.

Basal cell carcinomas (BCCs) are the most frequent human cancer. Over 90% of all BCCs have a mutation in PTCH1 or smoothened, two conducting proteins of the Hedgehog pathway. They rarely progress deeply and metastasize; however, if they do, these advanced basal cell carcinoma become amenable to treatment by inhibiting the Hedgehog and the P13K-mTOR pathways. Such innovative drugs include vismodegib, cyclopamine, itraconazole, everolimus and a few other agents that are in early clinical development.

Cyanoacrylate skin surface stripping and the 3S-Biokit advent in tropical dermatology: a look from Liège.

In the dermatopathology field, some simple available laboratory tests require minimum equipment for establishing a diagnosis. Among them, the cyanoacrylate skin surface stripping (CSSS), formerly named skin surface biopsy or follicular biopsy, represents a convenient low cost procedure. It is a minimally invasive method collecting a continuous sheet of stratum corneum and horny follicular casts. In the vast majority of cases, it is painless and is unassociated with adverse events. CSSS can be performed in subjects of any age. The method has a number of applications in diagnostic dermatopathology and cosmetology, as well as in experimental dermatology settings. A series of derived analytic procedures include xerosis grading, comedometry, corneofungimetry, corneodynamics of stratum corneum renewal, corneomelametry, corneosurfametry, and corneoxenometry.

Photoaging under recreational sunbeds.

BACKGROUND: Artificial sources of restricted light wavelength, particularly tanning beds, are progressively gaining importance in photoaging. OBJECTIVE: To assess the kinetics and the long-term evolution of skin pigmentation and tensile functions in sunbed worshippers over a period of 8 years. METHODS: Photoaging was explored in women who were both sunshine and sunbed worshippers. A series of 65 phototype III women aged 31-46 years completed a 100-month survey. Quarterly assessments were performed on the forearms to measure (a) the skin color individual typology angle (ITA°), (b) the extent in mottled subclinical melanoderma (MSM) using the ultraviolet light-enhanced visualization method and (c) the rheological properties of skin. RESULTS: A progressive increase in both skin extensibility and hysteresis was observed, contrasting with a decrease in biologic elasticity. These rheological changes were correlated with the ITA° changes, but not with the MSM extent. The kinetics of evolution of each test variable were distinct over time. DISCUSSION: This work is the first attempt at evaluating the kinetics of changes in physical parameters during a long period of frequent exposures to tanning sunbeds and sunshine for lifestyle purposes. The alterations were quite important in the color, MSM and rheological functions of the skin.

Functionally active macrophage-derived myeloperoxidase in the skin of drug-induced toxic epidermal necrolysis.

BACKGROUND: Drug-induced toxic epidermal necrolysis (TEN) probably results from a complex and specific immune cell reaction involving lymphocytes and macrophages. OBJECTIVE: To assess the functional role of macrophages in TEN. METHODS: Immunohistochemistry was performed on biopsies from early blisters developed in 9 TEN patients. The amount of extracellular myeloperoxidase (MPO) was measured by ELISA in TEN blister fluid and serum. Controls were blister fluids taken from 9 second-degree burns. In addition, 3-chlorotyrosine (a specific marker of MPO activity) was searched for using liquid mass chromatography both in TEN and burn blister fluids. RESULTS: Immunohistochemistry revealed numerous CD68+ macrophages in 8/9 TEN patients; 5-20% of these cells and rare CD15+ neutrophils exhibited MPO immunoreactivity, while keratinocytes were negative. The amount of MPO was significantly higher in TEN blister fluid than in TEN serum, suggesting macrophage production of MPO in the skin. In addition, MPO was significantly more abundant in TEN blister fluid than in burn blister fluid. 3-Chlorotyrosine was detected in 7/9 TEN blister fluids, but in only 2/9 burn blister fluids. DISCUSSION: MPO produced by macrophages was functionally active in most TEN patients, leading to the production of hypochlorous acid, a potent oxidative compound that alters keratinocytes.

Angiogenic fast-growing melanomas and their micrometastases.

Malignant melanoma (MM), particularly its fast-growing type, is prone to interstitial, intravascular and extravascular migratory metastases. There is no information linking their growth fraction, the configuration of the MM advancing edge, the extent in vascularity and the propensity to metastatic progression. The objective of this study was to determine the growth fraction, the size of vascularity and the contours of the progression border of 32 fast-growing MM with regard to the presence or absence of a micrometastatic spread inside the skin and overt metastases in the sentinel lymph nodes. In vivo high resolution colorimetry was performed as a clinical estimate of MM vascularity. Euclidean geometry and fractal analysis were used on immunohistochemical sections. The relative microvasculature profile area beneath MM, and the fractal dimension D of the MM frontline were assessed. The MIB/Ki-67 index was determined in MM cells. Value a* of colorimetry was similarly increased in the presence or absence of micro-metastases. No difference in growth fraction was revealed between these neoplasms. Correlations were found between vascularity and angiotropism, and between the micrometastatic process and the sentinel lymph node involvement. By contrast, no correlation was shown between vascularity and the fractal D dimension of the MM advancing edge. In sum, this study establishes a link between the extent of MM growth fraction, vascularity and the presence of dermal and nodal micrometastases in fast-growing MM.

Molecular mapping of Factor XIIIa-enriched dendrocytes in the skin (Review).

The human dermis contains a series of dendritic cells which express different phenotypes including Factor XIIIa immunoreactivity. This compound is related to a blood coagulation factor participating in angiogenesis, in the final stages of the clotting cascade and in wound healing. In normal skin, Factor XIIIa is expressed in specific dermal dendrocytes (DD) derived from the monocyte/macrophage lineage or from a mesenchymal origin. DD are located predominantly around the microvasculature in the adventitial dermis, at the dermo-epidermal junction, and around skin appendages, but normally not within the epidermis. Increased numbers of Factor XIIIa+ DD are present in a host of specific cutaneous inflammatory and fibrotic conditions. In tumor pathology, immunophenotypic differences are found between dermatofibromas and other fibrohistiocytic entities, most notably dermatofibrosarcoma protuberans. In addition, Factor XIIIa+ DD are likely to be involved in the progression and regression of some malignancies including cutaneous melanoma and basal cell carcinoma.

Dew point effect of cooled hydrogel pads on human stratum corneum biosurface.

BACKGROUND: Cooled hydrogel pads are used to prevent overheating effects of laser therapy. They do not induce cold injuries to the skin, but their more subtle physiological effects have not been thoroughly studied. PURPOSE: To describe the changes in transepidermal water loss and electrometric properties of the skin surface following application of cooled hydrogel pads. Measurements were performed on normal forearm skin of 27 healthy volunteers and on freshly excised skin from abdominoplasty. METHODS: LaserAid hydrogel pads cooled to 4 degrees C were placed for 15 min on the forearm skin. Measurements of transepidermal water loss (TEWL) and electrometric properties (Corneometer, Nova DPM 900) were performed before application and after removal of the cooled pads. RESULTS: A consistent increase in corneometer units, dermal phase meter (DPM) values and TEWL were recorded at removal of the cooled hydrogel pads. Both the in vivo and in vitro assessments brought similar information. DISCUSSION: The similar changes disclosed in vitro and in vivo suggest that a common physical process is operating in these conditions. The observed phenomenon is opposite to the predicted events given by the Arrhenius law probably because of the combination of cooling and occlusion by the pads. A dew point effect (air temperature at which relative humidity is maximal) is likely involved in the moisture content of the stratum corneum. Thus, the biological impact of using cooling hydrogel pads during laser therapy is different from the effect of a cryogenic spray cooling procedure. The better preservation of the water balance in the stratum corneum by the cooled hydrogel pads could have a beneficial esthetic effect on laser treated areas.

Crossroads between actinic keratosis and squamous cell carcinoma, and novel pharmacological issues.

Actinic keratoses (AKs) and their derived squamous cell carcinomas are distinctive lesions forming a continuum in a multi-step carcinogenesis process. They are typically found on chronically sun exposed skin. AKs merit to be recognized as such and to be distinguished from squamous cell carcinomas both conceptually and for therapeutic implications. The histological differences between these lesions are well defined and should not be blurred. A brief review is presented about the biological features responsible for AKs and the clinicopathologically distinctive aspects of these lesions. In addition, recent findings are presented about pharmacotherapy using anti-epidermal growth factor receptors, imidazoquinolines, diclofenac-hyaluronan, and methyl aminolevulinate photodynamic therapy.

Development of a mass spectrometry method for the determination of a melanoma biomarker, 5-S-cysteinyldopa, in human plasma using solid phase extraction for sample clean-up.

5-S-cysteinyldopa is a well-known pigment intermediate and analysis of its plasma concentration is interesting for the early diagnosis, as well as for evaluation of treatment and follow-up of malignant melanoma. A determination method of 5-SCD in human plasma was developed using solid phase extraction (SPE) on disposable cartridges and liquid chromatography electrospray mass spectrometry (LC-ESI-MS-MS). Compound's sensitivity to light and oxidation requires the addition of anti-oxidative agents (AO), to work in acidic media at 4 degrees C and to avoid light exposure of samples since blood collection. Different solid phases involving covalent binding to phenylboronic groups or dual retention mechanisms were evaluated and extraction cartridges containing both hydrophobic and strong cation exchange functionalities were finally selected. The LC separation of 5-SCD from endogenous catecholamines was achieved by gradient elution on a C18 stationary phase. 5-SCD was detected by multiple reaction monitoring (MRM) performed on ES(+) generated ions. Finally, the method was prevalidated in the lower ng/ml range. Good results with respect to accuracy, trueness and precision were obtained.

Toxic epidermal necrolysis: revisiting the tentative link between early apoptosis and late necrosis (review).

Toxic epidermal necrolysis (TEN) is a dramatic drug-induced reaction that may lead to full destruction of the epidermis and epithelial mucosae. The keratinocytes themselves seem to play a major role in the pathogenic mechanism. Biochemical and morphological studies performed on early epidermal lesions demonstrated that keratinocytes undergo apoptosis, but histological and clinical data show evidence of necrosis of the epidermis later in the disease evolution. Based on the limited information currently available about TEN pathomechanism, we present a 'mitochondrial hypothesis' that may explain both early apoptosis and late necrosis in TEN epidermis. Strong electrophilic drug metabolites are generated in TEN-affected keratinocytes due to an impaired detoxication pathway. These compounds presumably disrupt the electron transfer chain in the mitochondriae resulting in a decline in ATP production, loss of electrochemical gradient across the inner membrane (Deltapsim), and partial reduction in O2 with production of reactive oxygen species (ROS). The latter compounds directly damage cell membranes and act as intracellular chemical messengers stimulating proapoptotic systems such as CD95 and TNF-alpha, which in turn can activate nitric oxide (NO) metabolism. NO interacts with ROS to enhance their toxic effects. These proapoptotic events represent swift processes in the involved cells. The loss of Deltapsim and the opening of permeability transition pores in the mitochondrial membrane lead to osmotic swelling and rupture of these organelles with subsequent necrosis of the cell. The necrotic events follow apoptosis when both phenomena are present.

Treatment of pyogenic granulomas with the Nd-YAG laser.

Pyogenic granuloma is a common vascular tumour that can be treated by various means. However, some large lesions and those located on some difficult-to-treat body sites may represent a difficult challenge to the clinician (residual pain after treatment, difficult to cover with a dressing, or risk of sequelae). We report the successful treatment of such lesions in three patients using the Nd-YAG laser.

Drug-induced toxic epidermal necrolysis and pancytopenia: a puzzling association.

The molecular mechanisms involved in the pathogenesis of toxic epidermal necrolysis (TEN) remain not fully understood. We report a unique case of antibiotic-induced TEN developed in a patient who also suffered from prolonged severe methotrexate-induced pancytopenia. The objective of the study was to explore the nature of the cutaneous inflammatory infiltrate and the density in dermal dendrocytes (DD). Immunohistochemistry was used to identify activated T lymphocytes (CD45R0), monocyte-macrophages (Mac 387, CD68), DD (Factor XIIIa), and Langerhans cells (CD1a). The proliferation marker (Ki67) and the antibody to Fas receptor (CD95R) were also used to assess the distribution of the germinative pool of keratinocytes and the FAS-related apoptotic process, respectively. Numerous Factor XIIIa+ DD were present in the papillary dermis with only sparce perivascular CD45RO+ T lymphocytes and scattered CD68+ or Mac 387+ macrophages. Double immunostainings revealed that a minority of Factor XIIIa+ DD co-expressed the CD68 glycoprotein (a marker of phagocytic activity). No cells co-expressed factor XIIIa and Mac 387 immunoreactivities. CD45RO+ T lymphocytes, CD68+ and Mac 387+ macrophages were absent in the epidermis. The expression of CD95R was present although restricted to the basal keratinocytes, while the L1-protein (Mac 387+) was diffusely present in the epidermis. Langerhans cells (CD1a+) were sparce, but normal in distribution. The presence of a great number of Factor XIIIa+ DD without any possible recent recruitment from bone marrow suggests that these cells differentiated from resident cells of the skin. Indeed, there was no co-expression of Factor XIIIa and L1-protein, thus showing the absence of recruitment from monocytes. The simultaneous over-expression of Factor XIIIa and CD68 in some DD indicates some phagocytic activity. In view of the absence of inflammatory cells in the epidermis, keratinocytes appeared responsible for their own destruction through CD95-mediated and/or calcium-dependent apoptotic pathways. This finding entails that TEN treatments should target the keratinocyte metabolism rather than the circulating inflammatory cells which presumably play a limited role, if any, in the epidermal destructive process.

Analytical quantification of the inflammatory cell infiltrate and CD95R expression during treatment of drug-induced toxic epidermal necrolysis.

The treatment of drug-induced toxic epidermal necrolysis (TEN) remains unsatisfactory. Intravenous immunoglobulins (IVIg) and intravenous cyclosporin A (CsA) have shown some efficacy in short series of patients. We assessed the effects of IVIg and CsA on TEN lesional and apparently uninvolved skin using standard histology and immunohistochemistry. Cutaneous biopsies were taken from necrotic and clinically uninvolved TEN skin at admission (D1) before any treatment, and after a 5-day treatment (D5). Two IVIg-treated patients (0.75 g/kg/day), two CsA-treated patients (5 mg/kg/day) and two control patients only receiving supportive care were compared. Biopsies were examined by standard histology and immunohistochemistry using antibodies directed to CD68 antigen (macrophages), CD45R0 antigen (activated T lymphocytes), Factor XIIIa (dermal dendrocytes) and the CD95 receptor (apoptosis marker). The different cell densities were evaluated by computerized image analysis. The clinical outcomes with the different treatments were also recorded. There was no obvious difference in the duration of hospitalization in intensive care unit between the three groups but one patient passed away in each of the IVIg- and CsA-groups. At D5, no differences were found between the three groups in the histological and clinical rate of re-epithelialization, and in the evolution of T lymphocyte, macrophage and dendrocyte densities in the epidermis and dermis. However, the expression of the CD95 receptor was similarly and strongly abated at D5 in the epidermis of IVIg- and CsA-treated patients, while it was conversely increased in the two patients under supportive care only. Such a difference was found both in necrotic and uninvolved sites. IVIg and CsA treatments thus appeared to exert no obvious effect on the inflammatory infiltrate, but both abated the expression of the CD95 receptor in the skin of TEN patients. This effect did not seem sufficient to fully reverse the clinical evolution of the disease. It is inferred that IVIg and CsA do not completely abate the TEN process.

Keratinocyte injury in drug-induced toxic epidermal necrolysis: Simultaneous but distinct topographic expression of CD95R and calprotectin.

Keratinocyte injury in drug-induced toxic epidermal necrolysis (TEN) is attributed to a dysregulation in the complex apoptotic machinery. This study was performed to investigate some aspects of the apoptotic pathomechanism involving calprotectin and the CD95 receptor (CD95R, FasR) in TEN. The expression of these two molecules corresponds to calcium-dependent and calcium-independent processes, respectively. Biopsies were collected from blistering skin in 21 TEN patients and from clinically uninvolved skin in 16 of these patients. Immunohistochemistry was performed using specific antibodies directed to CD95R and calprotectin. Half (8/16) of the biopsy specimens taken from clinically uninvolved sites showed the expression of calprotectin throughout the epidermis or restricted to the suprabasal layers. In these samples, a strong CD95R immunoreactivity was often restricted to the basal layer (8/16). Calprotectin was present in all biopsies of bullous skin, especially in suprabasal layers (15/21) or throughout the epidermis (6/21). By contrast, the prominent expression of CD95R was confined almost exclusively to the basal layer (15/21), and more rarely throughout the epidermis (2/21), and it remained sometimes unexpressed (4/21). A clear-cut boundary was often present between the areas labeled by the two antibodies with exceptional overlap between them. The simultaneous expression of calprotectin and CD95R in TEN at distinct levels of the epidermis indicates that the pathomechanism leading to keratinocyte death does not belong to a single process in TEN. Their expression in clinically uninvolved skin suggests that these processes are early and widespread events in TEN.

Differential pathomechanisms of epidermal necrolytic blistering diseases.

Staphylococcal scalded skin syndrome (SSSS) results from the effect of exfoliative-toxins produced by staphylococcal strains. The disease affects predominantly children, and is rare in adults. We report two cases of the adult type of SSSS. Corticotherapy, chronic alcohol abuse and epilepsy-related immune changes might have been predisposing factors in these patients. The immunopathological characteristics of the inflammatory cell infiltrate in adults SSSS have not been thoroughly explored so far in the literature. Biopsies from 2 patients with bullous SSSS skin were studied by means of immunochemistry using a panel of 10 antibodies directed to FXIIIa, CD15, CD31, CD45R0, CD50, CD54, CD62E, CD95, CD106, and L1-protein, respectively. Cutaneous biopsies from related blistering diseases were compared. They included drug-induced toxic epidermal necrolysis (TEN), bullous impetigo and superficial pemphigus. A dense cell infiltrate composed of granulocytes (CD15+), macrophages (L1 protein+) and memory T cells (CD45R0+) and a strong expression of ICAM-3 (CD50) were present in the epidermis. CD95+ keratinocytes were lining the intraepidermal blisters. Type I dermal dendrocytes (Factor XIIIa+) were numerous and plump in the dermis. Bullous impetigo exhibited the same pattern of inflammatory cells, but with a lower density in type I dermal dendrocytes. TEN differed from SSSS by both the absence of CD15+ granulocytes and a stronger expression of the pro-apoptotic CD95 antigen in the epidermis. In superficial pemphigus, CD95 antigen was not expressed, and CD15+ granulocytes, CD45R0+ lymphocytes and L1 protein+ monocytes were much less numerous. It is concluded that the specific binding of SSSS-induced exotoxins to the desmosomes alters the keratinocyte metabolism leading to an inflammatory reaction followed by focal apoptosis. Our findings are in line with the concept that SSSS exotoxins might be superantigens. A common pathomechanism leading to epidermal destruction is likely operative in SSSS and bullous impetigo. The inflammatory cell composition in TEN and superficial pemphigus markedly differs from that in SSSS.

Deciphering the impaired cytokine cascades in chronic leg ulcers (review).

Chronic leg ulcers are typically wounds that do not heal at a normal rate. Impaired healing appears to be due to primary microvascular changes and it is aggravated by ongoing bacteria-driven vasculitis. The various cytokines identified in experimental wounds are also present in leg ulcers. VEGF is strongly implicated as a promoter of blood vessel growth in patients with venous disease. In addition, there is good evidence of increased expression of bFGF, TGF-beta1, and PDGF in lipodermatosclerosis. All of these growth factors are involved in wound healing. Upregulated TGF-beta1 is probably one of the main causes of the fibrosis observed in lipodermatosclerosis. In leg ulcers, cytokines appear to be trapped in the perivascular fibrinoid deposits. It is not the nature and amount of cytokines that are inadequate in leg ulcers, but rather their spatial distribution. Dermal dendrocytes (DD) are resident factor XIIIa-enriched macrophages. They likely play a role in tissue repair when boosted adequately. New therapies aiming at helping the release of cytokines by DD apparently promote and improve the healing phase.

Skin viscoelasticity in incipient gravitational syndrome.

BACKGROUND: The gravitational syndrome resulting from venous pressure elevation occasionally develops on the legs during pregnancy. The limb tends to enlarge and become stiffer. The body contours are altered. AIMS: To assess incipient gravitational edema due to chronic venous insufficiency using measurements of the skin tensile strength. METHOD: A total of 21 women aged 28-37 years were enrolled in the study. Evaluations were made twice in each subject following an alternate use and avoidance of daytime elastic contention. Skin viscoelasticity was measured on the mid portion of the calves using a computerized suction device. RESULTS: The discretely increased consistency of skin showing abnormal rheological characteristics at the site of incipient gravitational edema was significantly improved by contention therapy. Under progressive suction measurements, both skin distensibility, biologic elasticity and hysteresis were increased after wearing tight stockings. The biologic elasticity appears to be the most sensitive parameter pointing to the diagnosis of gravitational syndrome. CONCLUSION: Noninvasive measurements of the skin viscoelasticity, particularly the biologic elasticity, represent an objective assessment of both early gravitational edema and its control by contention therapy.

Effect of N-acetylcysteine combined with infliximab on toxic epidermal necrolysis. A proof-of-concept study.

INTRODUCTION: The pathophysiology of toxic epidermal necrolysis (TEN) is thought to be related to a drug-induced oxidative stress combined with TNFα overexpression by keratinocytes. None of the current treatments for TEN including systemic corticosteroids, cyclosporine and intravenous administration of immunoglobulins has proven superior over supportive care only. METHODS: A total of 10 TEN patients were enrolled to be treated at admission in burn units with the antioxidant N-acetylcysteine [NAC, 150mg/kg in a 20-h intravenous (IV) administration], or the combination of the same IV NAC perfusion with the anti-TNFα antibody infliximab (Remicade(®)), administered at a 5mg/kg dosage as a single 2-h IV administration. TEN was confirmed by a skin biopsy taken from a bullous lesion. At entry in the trial and 48h later, the illness auxiliary score (IAS) of clinical severity was determined and the extent in altered skin area (erythema and blisters) was assessed as a relative body area. Skin biopsies of both clinically uninvolved and erythematous areas were collected and immunohistochemistry was performed for assessing the density of inflammatory cells (CD8+ T cells, CD68+ macrophages) and keratinocytes enriched in intracellular calcium (Ca(++)) identified by the Mac387 anti-calprotectin antibody. RESULTS: No unexpected drug-induced adverse event was noticed. After 48h of both treatment modalities, improvements were not observed in the extent of skin involvement and in IAS. Immunohistopathology showed the absence of reduction in the amount of intraepidermal inflammatory cells. An increased intracellular Ca(++) load in clinically uninvolved keratinocytes and in erythematous epidermis was noticed. This latter finding suggested the progression in the way of the apoptotic process. On burn unit discharge, the survival in each modality of treatment was not improved compared to the expected outcomes determined from the IAS at admission. CONCLUSIONS: In this proof-to-concept attempt, NAC treatment or its combination with infliximab did not appear to reverse the evolving TEN process.

Deciphering supportive treatment strategies for toxic epidermal necrolysis.

Toxic epidermal necrolysis (TEN) is a dreadful life-threatening syndrome typically induced by an adverse drug reaction. This condition is characterized by the sudden and extensive destruction of the epidermis. The patient should be promptly addressed to a burn unit where three types of treatment should be administered, namely, (a) specific topical care of the bullous/eroded skin areas, (b) systemic anti-apoptotic/necrotic treatments, and (c) supportive care preventing secondary internal organ failures. This latter aspect is covered by the present review and focuses on (a) early withdrawal of the causative drug, (b) airway management, (c) hydro-electrolytic control, (d) nutritional support, (e) antibiotherapy, (f) prevention of venous thrombosis and gastroduodenal ulcers, and (g) analgesia and anesthesia.

Drug interactions with normal and TEN epidermal keratinocytes.

Human epidermal keratinocytes (EKs) are metabolically involved in various drug transport mechanisms, as well as in detoxification or activation processes. The overall cell mechanisms of drug metabolization, and more specifically drug processing are reviewed in normal EKs. The overall drug metabolism involves different phases corresponding to the uptake, biotransformation and anti-transport steps. In EKs, both the enzymes and transportassociated proteins are different from those involved in the hepatocyte metabolism. Some cytochrome P450 enzymes and the flavin-containing mono-oxygenases are particularly involved in EKs. Basically, EKs represent key cells likely involved during the initial stage of drug-induced toxic epidermal necrolysis (TEN). Only limited advances have been made so far in this field. Nevertheless, mitigating EKs metabolic disturbances in TEN probably represent a promising specific treatment of the disease.