Long-term exposure of immortalized keratinocytes to arsenic induces EMT, impairs differentiation in organotypic skin models and mimics aspects of human skin derangements.

Arsenic is one of the most important human carcinogens and environmental pollutants. However, the evaluation of the underlying carcinogenic mechanisms is challenging due to the lack of suitable in vivo and in vitro models, as distinct interspecies differences in arsenic metabolism exist. Thus, it is of high interest to develop new experimental models of arsenic-induced skin tumorigenesis in humans. Consequently, aim of this study was to establish an advanced 3D model for the investigation of arsenic-induced skin derangements, namely skin equivalents, built from immortalized human keratinocytes (NHEK/SVTERT3-5). In contrast to spontaneously immortalized HACAT cells, NHEK/SVTERT3-5 cells more closely resembled the differentiation pattern of primary keratinocytes. With regard to arsenic, our results showed that while our new cell model was widely unaffected by short-time treatment (72 h) with low, non-toxic doses of ATO (0.05-0.25 µM), chronic exposure (6 months) resulted in distinct changes of several cell characteristics. Thus, we observed an increase in the G2 fraction of the cell cycle accompanied by increased nucleus size and uneven tubulin distribution. Moreover, cells showed strong signs of de-differentiation and upregulation of several epithelial-to-mesenchymal transition markers. In line with these effects, chronic contact to arsenic resulted in impaired skin-forming capacities as well as localization of ki67-positive (proliferating) cells at the upper layers of the epidermis; a condition termed Bowen's disease. Finally, chronically arsenic-exposed cells were characterized by an increased tumorigenicity in SCID mice. Taken together, our study presents a new model system for the investigation of mechanisms underlying the tumor-promoting effects of chronic arsenic exposure.

Proteome analysis identifies L1CAM/CD171 and DPP4/CD26 as novel markers of human skin mast cells.

BACKGROUND: The function of skin mast cells has been well documented in IgE-mediated allergic reactions, whereas other mast cell functions are poorly defined. This study aimed at identifying novel mast cell proteins by proteome analysis of primary human skin mast cells. METHODS: The proteome of skin mast cells was compared to other cell types and analyzed using bioinformatics. The expression and function of two proteins hitherto not described in skin mast cells was investigated in isolated mast cells as well as in mast cells in situ. RESULTS: Within the mast cell proteome, we identified 49 highly expressed proteins previously not described in mast cells; 21 of these proteins were found to be selectively expressed in mast cells. Two proteins, the neural cell adhesion molecule L1 and dipeptidyl peptidase 4, were further studied. L1 was found to be highly expressed in mast cells in normal, psoriasis, and mastocytosis skin. Dipeptidyl peptidase 4 was found to be expressed in mast cells in normal, psoriasis, and mastocytosis skin as well as in bone marrow mast cells in patients with systemic mastocytosis. In normal skin, mast cells were identified as a major source of dipeptidyl peptidase 4 and we also found that skin mast cells and fibroblasts secrete an active form of this enzyme. CONCLUSIONS: In a systematic proteomics approach we identified two novel mast cell proteins potentially relevant to skin homeostasis: neural cell adhesion molecule L1 and dipeptidyl peptidase 4.

Influence of skin ageing features on Chinese women's perception of facial age and attractiveness.

OBJECTIVES: Ageing leads to characteristic changes in the appearance of facial skin. Among these changes, we can distinguish the skin topographic cues (skin sagging and wrinkles), the dark spots and the dark circles around the eyes. Although skin changes are similar in Caucasian and Chinese faces, the age of occurrence and the severity of age-related features differ between the two populations. Little is known about how the ageing of skin influences the perception of female faces in Chinese women. The aim of this study is to evaluate the contribution of the different age-related skin features to the perception of age and attractiveness in Chinese women. METHODS: Facial images of Caucasian women and Chinese women in their 60s were manipulated separately to reduce the following skin features: (i) skin sagging and wrinkles, (ii) dark spots and (iii) dark circles. Finally, all signs were reduced simultaneously (iv). Female Chinese participants were asked to estimate the age difference between the modified and original images and evaluate the attractiveness of modified and original faces. RESULTS: Chinese women perceived the Chinese faces as younger after the manipulation of dark spots than after the reduction in wrinkles/sagging, whereas they perceived the Caucasian faces as the youngest after the manipulation of wrinkles/sagging. Interestingly, Chinese women evaluated faces with reduced dark spots as being the most attractive whatever the origin of the face. The manipulation of dark circles contributed to making Caucasian and Chinese faces being perceived younger and more attractive than the original faces, although the effect was less pronounced than for the two other types of manipulation. CONCLUSION: This is the first study to have examined the influence of various age-related skin features on the facial age and attractiveness perception of Chinese women. The results highlight different contributions of dark spots, sagging/wrinkles and dark circles to their perception of Chinese and Caucasian faces.

MC1R major variants are a risk factor of sleep lines in Caucasian women.

BACKGROUND: Sleep lines are caused by individual's sleeping positions and should be differentiated from expression wrinkles. OBJECTIVE: The aim of the study was to investigate possible risk factors for sleep lines on a sizeable sample of middle-aged Caucasian women. METHODS: This study involved a sample of 542 French middle-aged women (44 to 70 years old) from Paris area. Three standardized facial photographs (face and profiles) were examined independently by two dermatologists allowing the identification of sleep lines and the evaluation of the severity of several facial skin features. Possible impacts of MC1R gene polymorphisms were tested using logistic regression models. RESULTS: Sixty women (11%) had facial sleep lines and showed generally more than one sleep line. The sleep lines were often located on the forehead, along the nose, on the cheeks and under the eyes, and more rarely on the chin. As expected, the sleep lines were associated with age, and the women with sleep lines showed also more severe signs of skin ageing. After adjustment on possible confounders, the presence of two major diminished function variants of the MC1R gene was identified as a strong risk factor for sleep lines [adjusted odds ratios (AOR) (95% CI): 8.25 (2.62-25.97)]. DISCUSSION/CONCLUSION: The data in the literature are scarce and this study is the first to be conducted on a sizeable sample of women. Our results suggest that genetic variations of MC1R are important determinants of the development of sleep lines.

Histamine suppresses epidermal keratinocyte differentiation and impairs skin barrier function in a human skin model.

BACKGROUND: Defects in keratinocyte differentiation and skin barrier are important features of inflammatory skin diseases like atopic dermatitis. Mast cells and their main mediator histamine are abundant in inflamed skin and thus may contribute to disease pathogenesis. METHODS: Human primary keratinocytes were cultured under differentiation-promoting conditions in the presence and absence of histamine, histamine receptor agonists and antagonists. The expression of differentiation-associated genes and epidermal junction proteins was quantified by real-time PCR, Western blot, and immunofluorescence labeling. The barrier function of human skin models was tested by the application of biotin as tracer molecule. RESULTS: The addition of histamine to human keratinocyte cultures and organotypic skin models reduced the expression of the differentiation-associated proteins keratin 1/10, filaggrin, and loricrin by 80-95%. Moreover, the addition of histamine to skin models resulted in the loss of the granular layer and thinning of the epidermis and stratum corneum by 50%. The histamine receptor H1R agonist, 2-pyridylethylamine, suppressed keratinocyte differentiation to the same extent as did histamine. Correspondingly, cetirizine, an antagonist of H1R, virtually abrogated the effect of histamine. The expression of tight junction proteins zona occludens-1, occludin, claudin-1, and claudin-4, as well as that of desmosomal junction proteins corneodesmosin and desmoglein-1, was down-regulated by histamine. The tracer molecule biotin readily penetrated the tight junction barrier of skin cultures grown in the presence of histamine, while their diffusion was completely blocked in nontreated controls. CONCLUSIONS: Our findings suggest a new mechanism by which mast cell activation and histamine release contribute to skin barrier defects in inflammatory skin diseases.

Freckles and solar lentigines have different risk factors in Caucasian women.

BACKGROUND: To date, few epidemiological data on the relationships between solar lentigines, freckles and behavioural and constitutional risk factors in Caucasian populations exist. OBJECTIVES: To investigate the potential impact of behavioural and phenotypic variables, as well as the MC1R genetic background, on the history of facial freckles and the severity of solar lentigines in Caucasian women. METHODS: The severity of solar lentigines was graded from facial digital images of 523 French middle-aged women by a dermatologist and summarized by a score afterwards. The history of facial freckles was assessed and the sun-exposure behaviour was characterized using a six-category typology. Risk factors including MC1R polymorphism were evaluated using logistic regression models. RESULTS: Two constitutive host factors were found to be independently associated with a history of facial freckles: frequent sunburns and the presence of diminished function variants of the MC1R gene. In addition to age, five factors were independently associated with solar lentigines: constitutive host factors (dark skin colour and tanning capacity), a history of freckles, sun-exposure behaviour and current intake of oral contraceptive or progestogen treatments. CONCLUSION: These results strengthen the hypothesis that solar lentigines are markers of photoaging, whereas freckles are mainly determined by genetic factors. The finding that hormonal treatment is associated with a higher risk for solar lentigines merits further investigations.

Impact of filaggrin mutations on Raman spectra and biophysical properties of the stratum corneum in mild to moderate atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is associated with null mutations in the filaggrin (FLG) gene. OBJECTIVE: To assess the impact of FLG null mutations on biophysical properties and the molecular composition of the stratum corneum (SC) in healthy individuals and AD patients. METHODS: A total of 196 French adults, including 97 with a history of mild to moderate AD, were genotyped for the three major European FLG mutations. Components of the natural moisturizing factor (NMF), lipids and water content in the SC were determined using Raman spectroscopy. In addition, trans-epidermal water loss, capacitance and pH of the SC were measured. RESULTS: Stratum corneum concentrations of total NMF, water, ornithine and urocanic acid (UCA) were significantly lower in AD patients than in healthy controls. Null mutations of FLG were detected in 4% of controls and 10% of AD patients. FLG mutations were associated with increased SC levels of lactate, reduced concentrations of most other NMF components and higher disease severity in AD patients. In AD patients without FLG mutations, the content of NMF constituents decreased with increasing disease severity. The concomittant presence of low concentrations of histidine, alanine and either glycine or pyrrolidone-5-carboxylic acid (PCA) in the SC was associated with FLG mutations with 92% specificity. CONCLUSIONS: Our findings suggest a low prevalence of FLG mutations in mild AD and support an important role for filaggrin in determining the physicochemical parameters of the SC. The combined measurement of several filaggrin breakdown products in the SC may be useful to specifically predict the presence of FLG mutations.

[MC1R polymorphisms and facial photoaging]. / Polymorphismes du MC1R et photovieillissement du visage.

BACKGROUND: The objective of this study was to assess the association between melanocortin-1 receptor (MC1R) variants and the severity of facial skin photoaging. METHODS: The study population comprised 530 French middle-aged women between 44 and 70 years. A trained dermatologist graded the severity of facial skin photoaging from photographs using Larnier's global scale. Logistic regressions were performed to assess the influence of MC1R polymorphism on severe photoaging (grades 1-3 vs. 4-6), with adjustment for possible confounders (demographic and phenotypic data, and sun exposure intensity). RESULTS: Overall, 35% of the women were wild-type homozygotes, 49% had one variant, 15% had two variants, and 1% had at least one rare variant. After adjustment for possible confounders, the presence of two major diminished function variants was found to be a risk factor for photoaging (adjusted odds ratio=5.61; 95% confidence interval [1.43-21.96]). DISCUSSION: Our results suggest that genetic variations of MC1R are important determinants for severe photoaging.

Psoriasin (S100A7) is a major Escherichia coli-cidal factor of the female genital tract.

The female urogenital tract requires an efficient defense against bacteria, potentially derived from the adjacent intestinal tract. We have thus sought to identify the factors that protect against Escherichia coli (E. coli) in the female genital tract. Vaginal fluid from healthy human donors consistently killed E. coli in vitro and vaginal epithelium strongly expressed and secreted psoriasin. Psoriasin was constitutively produced in an organotypic vaginal epithelium model, and exposure of these cells to supernatants of E. coli cultures led to an enhanced psoriasin expression. Secreted psoriasin in vaginal fluids accounted for approximately 2.5-3% of total protein. Fractionation of vaginal fluids by high performance liquid chromatography (HPLC) showed that psoriasin co-eluted with a peak of E. coli killing activity. Our data show that normal vaginal fluid contains a powerful intrinsic antimicrobial defense against E. coli and that psoriasin contributes to the innate immune response of the female genital tract.

Immunosuppression and atypical infections in CML patients treated with dasatinib at 140 mg daily.

BACKGROUND: The multikinase inhibitor dasatinib exerts growth-inhibitory effects in patients with imatinib-resistant chronic myeloid leukaemia (CML). In first clinical trials, side effects of dasatinib, 140 mg daily, were reported to be mild and tolerable. PATIENTS AND METHODS: We examined the side effect profile in 16 patients with imatinib-resistant CML who received 140 mg dasatinib daily in our center. RESULTS: Dasatinib produced substantial and sometimes severe or even life-threatening side effects with > or = 10% body weight loss (6/16 patients), pleural effusions grade II or higher (12/16) and infectious complications (12/16), including atypical infections not seen in imatinib-treated patients. One patient developed Epstein-Barr-Virus-positive mucosal leucoplakia, one died from pneumonia caused by pneumocystis carinii and three patients developed a skin-cancer. Most events were recorded within the first 2 years of therapy, only skin tumours developed after the second year. In ex vivo experiments performed in dasatinib-treated patients, transient suppression of IgE-dependent activation of blood basophils and TcR-dependent activation of T-lymphocytes was found. Moreover, in drug-binding studies, dasatinib was found to bind to several key kinase-targets of the immune system including Lyn and Btk, in mast cell, basophil, B-cell and T-cell lines. CONCLUSION: Dasatinib acts not only anti-neoplastic in CML but may also act as an immunosuppressive agent when applied at 140 mg daily, and produces frequent pleural effusions and weight loss in advanced CML.

DNase1L2 suppresses biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus.

BACKGROUND: The formation of biofilms, which is an important step in bacterial colonization, can be inhibited by deoxyribonuclease (DNase)-mediated breakdown of extracellular DNA. We have recently demonstrated that epidermal keratinocytes strongly express DNase1-like 2 (DNase1L2) in a differentiation-associated manner. OBJECTIVES: To determine whether enzymatically active DNase1L2 is present in human stratum corneum and whether it is able to suppress bacterial biofilm formation. METHODS: DNase1L2 was extracted from normal human stratum corneum, immunocaptured and incubated with plasmid DNA. DNA hydrolysis was monitored by gel electrophoresis and ethidium bromide staining. The effect of DNase1L2 on biofilm formation was assayed by cultivation of Pseudomonas aeruginosa and Staphylococcus aureus in the presence or absence of purified recombinant DNase1L2 in microtitre plates and subsequent quantification of biofilm-forming bacteria by crystal violet staining. RESULTS: DNase1L2 was found to be present in an enzymatically active form in the stratum corneum of human skin. In an in vitro assay, purified recombinant DNase1L2 efficiently suppressed the formation of biofilms by P. aeruginosa and S. aureus. CONCLUSIONS: Our data suggest that DNase1L2 is a novel component of the innate antimicrobial defence of the epidermis.

Reference ranges of skin micro-relief according to age in French Caucasian and Japanese women.

BACKGROUND/PURPOSE: The variation of skin surface morphological indicators according to age has not been frequently studied. The aim of this work was to establish French Caucasian and Japanese reference ranges of these indicators according to age. METHODS: Two studies were performed simultaneously in Paris and Sendai on 356 Caucasian and 120 Japanese healthy women aged from 20 to 80 years. Skin replicas were obtained from the volar forearm and analysed by interferometry. This analysis yielded 16 morphological indicators. Reference ranges according to age were established using the statistical methodology defined by Royston. RESULTS/DISCUSSION: Reference ranges were found for 15 out of the 16 parameters for the French women as well as for the Japanese women. The models' truthfulness will have to be confirmed using new samples, larger if possible. Moreover, non-parametric methods will be used in order to compare the results provided by these approaches.

Self-reported skin sensitivity in a general adult population in France: data of the SU.VI.MAX cohort.

OBJECTIVE: This study aimed to examine the frequency of self-assessed facial skin sensitivity and its different patterns, and the relationship with gender and sun sensitivity in a general adult population. METHODS: A standardized 11-item questionnaire investigating reactions experienced during the past year was developed. The questions explored different patterns of skin sensitivity: pattern I (blushing related to vascular reactivity), pattern II (skin reactions to certain environmental conditions), pattern III (skin reactions after substance contact), and for women pattern IV ('breakout of spots' related to menstrual cycle). Additional items were addressed for women and men, including sun sensitivity. The questionnaire was administered to a large middle-aged population involved in the 'Supplément en Vitamines et Minéraux Antioxydants' (SU.VI.MAX) cohort. RESULTS: Sensitive facial skin was reported by 61% of the women (n = 5074) and 32% of the men (n = 3448), and the frequency decreased with age. The frequency of patterns I, II and III was greater for women (78, 72 and 58%, respectively) than for men (56, 48 and 28%) of comparable classes of age. The frequency of pattern IV was reported by 49% of premenopausal women, and skin reactions after shaving by 41% of the men. Sun sensitivity was found to be a major component of skin sensitivity. Factor analysis showed that individuals with fair phototype frequently evoked reactions associated with pattern I, and skin redness and burning sensations were related to certain environmental conditions (pattern II). CONCLUSION: Skin sensitivity is a common concern that declines with age and is relevant for men as well as for women.

[Phototype, vitamin D status and bone mineral density among women at risk of osteoporosis]. / Phototype, statut en vitamine D et densité minérale osseuse chez des femmes à risque d'ostéoporose.

PURPOSE: The aim of this study was to test the influence of phototype and vitamin D status feature on the bone mineral density (BMD) of the femoral neck in a group of middle-aged women considered at risk of osteoporosis (low levels of vitamin D [25(OH)D3<78 nmol/L] and hyperparathyroidism [parathormone level>36 pg/mL]). METHODS: This two-step study was conducted on 122 French women enrolled in the SUVIMAX (supplémentation en vitamines et minéraux antioxydants: antioxidant vitamin and mineral supplementation) cohort. The impact of various variables on BMD, including age, body mass index (BMI), vitamin D status, alcohol intake, sun exposure intensity and phototype was investigated using regression models. RESULTS: No statistical link was found between BMD and the variables documenting vitamin D status and parathormone levels, nor phototype. Nevertheless, fair phototypes tended to be associated with lower BMD values. However, BMD decreased with age and increased with BMI and physical activity level. CONCLUSIONS: Whatever their phototype, adult women concerned about precarious vitamin D status should undergo a vitamin D supplementation in combination with an adequate calcium intake all year long and a proper sun protection. Moreover, a physical activity maintenance should provide an additional benefit for prevention of osteoporosis.

Inhibitors of differentiation/DNA binding proteins Id1 and Id3 are regulated by statins in endothelial cells.

Id proteins (inhibitors of differentiation), which are involved in the control of cell cycle progression, can delay cellular differentiation and senescence and have been implicated in angiogenesis. The regulation of Id proteins in endothelial cells (ECs) by proangiogenic statins has not been investigated yet and remains unresolved. In this study, human dermal microvascular ECs (HDMECs) were stimulated with fluvastatin, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and serum in vitro. The regulation of Id1, Id3, p21, p27, and p53 and the phosphorylation of AKT was investigated by Western blotting. Id1 was up-regulated by fluvastatin and serum, but not by VEGF and HGF. Fluvastatin did not regulate p21 and p27, but down-regulated Id3 and p53 slightly. In contrast to VEGF and HGF, fluvastatin did not result in AKT phosphorylation, indicating that this pathway is not involved in the control of endothelial Id1 expression. These experiments demonstrate for the first time that Id1 can be up-regulated and p53 down-regulated by a statin in HDMECs. Regulation of these proteins in ECs may account for the proangiogenic effect of statins.