Efficacy of quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro

Please cite this paper as: Efficacy quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro. Experimental Dermatology 2010; 19 : e282–e285. Abstract: In this study, we describe a simple, specific, reproducible and quantitative assay system to assess melanosome transfer. We first established a co‐culture model of normal human epidermal melanocytes and HaCaT keratinocytes. The cells were co‐cultured for 72 h in a serum‐free keratinocyte growth media and double labelled with Fluorescein isothiocyanate (FITC)‐conjugated antibody against the melanosome‐specific protein gp100, and with Phycoerythrin (PE)‐conjugated antibody against the keratinocyte‐specific marker cytokeratin. Then, the cells were examined using co‐focal microscope and flow cytometry. The increased melanosome transfer from melanocytes to HaCaT keratinocytes was observed in a time‐dependent manner. To verify the accessibility of this method, two known melanosome transfer inhibitors and two known melanosome transfer stimulators were applied. Consistent with previous investigation, soybean trypsin inhibitor (STI), niacinamide inhibited melanosome transfer, α‐melanocyte stimulating hormone (α‐MSH) and keratinocyte growth factor (KGF) increased melanosome transfer, respectively, in a dose‐dependent manner. The model used in this study could thus represent a rapid and reliable tool to identify modulators of human melanosome transfer.     

CLINICAL OBSERVATIONS ON A CASE OF IMMUNODEFICIENCY AND THYMOMA (GOOD'S SYNDROME) ASSOCIATED WITH CHRONIC MUCOCUTANEOUS CANDIDIASIS

A 50 year-old woman with immunodeficiency and thymoma (Good's syndrome) associated with chronic muco-cutaneous candidiasis (CMCC) was observed. Immunological treatment with transfer factor (TF), supplements of γ-globulin and anti-candidal agents was maintained for six years. Because of the association of pure red cell aplasia in the clinical course, systemic steroid therapy was added to the treatment. Although the patient gradually improved, further immunological therapy was considered to be necessary for the tongue lesions due to candidiasis and the agammaglobulinemia.     

Provoking factors,including chemicals,in Dutch patients with vitiligo

Background In vitiligo, many provoking factors have been described, but epidemiological data, especially on the role of contact with chemicals, are scarce. Objective To obtain an insight into the patient‐reported factors provoking vitiligo, including contact with chemicals. Methods A retrospective cohort study was conducted on all 1264 patients with vitiligo who visited the Netherlands Institute for Pigment disorders from January 2003 to December 2007. Patients for whom an exogenous provoking factor was recorded were sent a questionnaire. Subsequently, patients who mentioned a chemical provoking factor were contacted to elucidate the alleged causal relationship between exposure to the chemical and the onset of vitiligo. Results A total of 300 out of the 1264 patients indicated that provoking factors had played a role in their disease. Two hundred and forty‐six patients were sent a questionnaire, which was returned by 177 (response rate of 72%). Emotional stress was indicated as a provoking factor in 98 patients (55·4%), 51 patients (28·8%) recorded sunburn, 34 patients (19·2%) recorded mechanical factors and 20 patients (11·3%) other factors. Of 29 patients (16·4%) who indicated a chemical factor, a presumed causal relationship could be corroborated in four. The chemicals involved were para‐tertiary butylphenol (n = 2), captan (n = 1) and diphencyprone (n = 1). Conclusion The majority of the patients with vitiligo from this study did not mention provoking factors, but the ones who did point to emotional stress in more than half of the cases. Of the 29 patients who assigned chemical provoking factors, solvents were mainly indicated. However, a presumed relationship with the chemical could be corroborated in only four patients.     

Clinical, power Doppler sonography and histological assessment of the psoriatic plaque: short-term monitoring in patients treated with etanercept

Background To date, the diagnosis of psoriasis is based on both clinical history and physical examination, and its severity is assessed by the Psoriasis Area and Severity Index (PASI). Continuous technological advances in the field of sonography have led to the development of equipment with high power Doppler frequency, which allows for very detailed morphological information regarding the dermal blood flow. Objectives To compare power Doppler sonography (PDS) with clinical and histological findings before and after etanercept treatment in patients with psoriasis. Methods Twelve patients with a clinical diagnosis of psoriasis were enrolled in this study. The PASI, PDS and histological examinations were assessed in all patients on the same day at baseline, and after 12 weeks of biological treatment. PDS examination was performed by an experienced sonographer, using a sonographic system equipped with transducer ranging from 6 to 18 MHz and Doppler frequency ranging from 7 to 14 MHz. Results At follow up there was a significant decrease in PASI. A significant change was also detected for the PDS findings (P = 0·005). At baseline the median value for factor VIII staining was 1·5, and the median value for vascular endothelial growth factor (VEGF) staining was also 1·5. At follow up there was a significant decrease in both factor VIII and VEGF staining scores. Moreover, a positive correlation between reduction in PDS score and improvement in clinical and histological scores was found: Spearman’s ρ = 0·639, P = 0·0022; Spearman’s ρ = 0·619, P = 0·0013; Spearman’s ρ = 0·765, P = 0·0002, respectively. Conclusions Our results show a significant correlation between PDS findings and both PASI and histological degree of vascularization before and after etanercept treatment. These data provide evidence in favour of the validity of PDS in the assessment of dermal perfusional changes in patients with psoriatic plaques.     

Propionibacterium acnes (P. acnes) resistance and antibiotic use in patients attending Australian general practice

Background/Objectives: Antibiotic resistance in the community, including transfer between bacteria, is a growing concern for clinicians. Acne is commonly treated in general practice, sometimes with antibiotics. The aim of this study is to measure the rate of carriage of antibiotic resistant Propionibacterium acnes 10 years apart in general practice and the relationship of resistance to type of treatment, as well potential effects on other flora. Methods: Patients (N = 215) with acne presenting to Australian Capital Territory and south‐eastern New South Wales general practices were swabbed for P. acnes in 1997–1998 and 2007. Clinical details were collected with questionnaires. In 2007 swabs were also taken for Staphylococcus aureus and Streptococcus pneumoniae. GP's diagnostic classification of acne was tested using a set of standard photographs. Results: Resistant P. acnes was isolated from 20 patients (9%) and the proportion that was resistant was the same in 1997–1998 and in 2007. Antibiotic use, particularly topical, was associated with P. acnes resistance. Resistance rates declined with the time elapsed since ceasing antibiotics. Use of retinoids was associated with a decreased chance of growing P. acnes (P = 0.008) but not with decreased resistance. Simultaneous resistance with S. aureus was not detected, but only in 30 patients was S. aureus isolated. Conclusions: P. acnes resistance was similar in 1997–1998 and in 2007.The chance of a patient carrying a resistant strain of P. acnes is significantly greater with recent exposure to antibiotics, and clinicians should limit prescribing where possible. Resistance disappears rapidly after ceasing antibiotics. Cross resistance with other organisms was not detected in this study.     

Z‐ligustilide ameliorated ultraviolet B‐induced oxidative stress and inflammatory cytokine production in human keratinocytes through upregulation of Nrf2/HO‐1 and suppression of NF‐κB pathway

Ultraviolet B (UVB), a harmful environmental factor, is responsible for a variety of skin disorders including skin inflammation through reactive oxygen species (ROS) and inflammatory mediator production. Here, we investigated the effect of Z‐ligustilide (Z‐lig), an active ingredient isolated from the medicinal plants Cnidium officinale and Angelica acutiloba, on UVB‐induced ROS generation and inflammatory mediator production in normal human epidermal keratinocytes (NHEKs) as well as its underlying mechanisms. Z‐lig significantly rescued UVB‐induced NHEKs damage in a dosage‐dependent manner. Pretreatment of NHEKs with Z‐lig inhibited UVB‐induced ROS production in NHEKs. Both silencing the nuclear factor E2‐related factor 2 (Nrf2) and the supplement of tin protoporphyrin IX (SnPP), a haeme oxygenase‐1 (HO‐1) inhibitor, cancelled the inhibitory effect of Z‐lig on UVB‐induced ROS upregulation in NHEKs. Moreover, pretreatment of NHEKs with Z‐lig reduced UVB‐induced nuclear factor kappa B (NF‐κB)‐dependent inflammatory mediators (IL‐6, IL‐8 and MCP‐1) production at both mRNA and protein level. In the presence of Z‐lig, UVB‐induced NF‐κB subunit p65 nuclear translocation was abolished, and the IκBα degradation was suppressed. Taken together, these findings suggest that Z‐lig can suppress UVB‐induced ROS generation through Nrf2/HO‐1 upregulation and inflammation by suppressing the NF‐κB pathway, suggesting that Z‐lig may be beneficial in protecting skin from UVB exposure.     

Improvement in skin wrinkles using a preparation containing human growth factors and hyaluronic acid serum

Background: Skin aging is accompanied by wrinkle formation. At some sites, such as the periorbital skin, this is a relatively early phenomenon. Objective: We evaluated the anti-wrinkle effect of a preparation containing human growth factor and hyaluronic acid serum on periorbital wrinkles (crow's feet). Materials and methods: In total, 23 Korean women (age range: 39–59 years), who were not pregnant, nursing, or undergoing any concurrent therapy, were enrolled in this study. All the patients completed an 8-week trial of twice-daily application of human growth factor and hyaluronic acid serum on the entire face. Efficacy was based on a global photodamage score, photographs, and image analysis using replicas and visiometer analysis every 4 weeks. The standard wrinkle and roughness parameters used in assessing skin by visiometer were calculated and statistically analyzed. Results: Periorbital wrinkles were significantly improved after treatment, with improvements noted both by physician's assessment and visiometer analysis. Conclusion: Topical application of human growth factor and hyaluronic acid was beneficial in reducing periorbital wrinkles.     

Epidermal growth factor receptor gene numerical aberrations are frequent events in actinic keratoses and invasive cutaneous squamous cell carcinomas

Please cite this paper as: Epidermal growth factor receptor gene numerical aberrations are frequent events in actinic keratoses and invasive cutaneous squamous cell carcinomas. Experimental Dermatology 2010; 19: 151–153. Abstract: Epidermal growth factor receptor (EGFR) gene amplification and protein overexpression are common in several cancers. EGFR status has seldom been studied in cutaneous squamous carcinomas (SCCs), or their precursors, actinic keratoses (AKs). We evaluated the presence of EGFR genomic aberrations and EGFR protein overexpression in 25 AKs and 35 invasive SCCs by means of fluorescence in situ hybridization (FISH) and immunohistochemistry. EGFR numerical aberrations were detected in 52% of AKs and 77.1% of SCCs (P = 0.042). EGFR amplification was identified in 12% of AKs and 20% of SCCs. No differences regarding EGFR numerical aberrations were observed when AKs with high‐grade dysplasia were compared with SCCs. A good correlation was observed between EGFR numerical aberrations and EGFR overexpression. Our results suggest that EGFR numerical aberrations occur in the early stages of epithelial carcinogenesis in skin, not playing a role in the progression from low‐grade SCCs into more aggressive phenotypes.     

A rare heterozygous TRAF6 variant is associated with hypohidrotic ectodermal dysplasia

Summary Background Mutations in the genes encoding components of the tumour necrosis factor (TNF)‐α‐like pathway cause hypohidrotic ectodermal dysplasia (HED). It has been postulated that the TNF receptor‐associated factor 6 (TRAF6) is also involved in this pathway. Objectives To investigate mutations in the TRAF6 gene in an individual with HED. Methods Genetic analysis was performed on TRAF6 in a patient with HED, her parents, her sister and 150 ethnically matched, healthy individuals. Results In the patient, sequencing analysis of one DNA strand revealed a deletion of eight nucleotides (c.1074–1081delCAATTTG) in the 5′ fragment of the last exon of TRAF6, while no deletion was detected in the other DNA strand indicating a heterozygous mutation. No such sequence abnormality was detected in the patient’s parents and her sister. Conclusion This is the first report of a heterozygous TRAF6 sequence variant associated with symptoms typical of HED.     

Correlation among metallothionein expression,intratumoural macrophage infiltration and the risk of metastasis in human cutaneous malignant melanoma

Background The formation of metastases and the efficacy of systemic therapies in cutaneous malignant melanoma (CMM) depend on the characteristics of the tumour cells and the host immune response. Aberrant expression of metallothionein (MT) has been observed in several types of cancers with poor prognoses. Objective To perform an immunohistochemical study on primary CMM comparing the MT expression of tumours without metastases (n = 23) to that of samples with haematogenous metastases (n = 23) and to examine the correlation between MT staining and immunological markers relevant in CMM progression. Methods The immunohistochemical labelling of different tumour sections was analysed using tissue microarrays for the evaluation of the suitability of this method in future studies. Results Our results suggest that MT overexpression is significantly more frequent in primary CMM with haematogenous metastases (P = 0.018) and that the overexpression is independent of the Breslow tumour thickness (R = 0.102, P = 0.501). Interestingly, MT overexpression of the tumour cells was correlated with the presence of tumour‐infiltrating CD68⁺ macrophages (P = 0.003), a known predictive factor for melanoma progression, thereby suggesting a role for MT in the development of a defective host immune response. Furthermore, the presence of CD163⁺ macrophages infiltrating the tumours correlated with metastasis formation (P < 0.001), whereas the presence CD1a⁺ dendritic cells surrounding the tumours was associated with a lower risk of haematogenous spread (P = 0.003). Conclusion Our results demonstrate that MT may represent a suitable prognostic factor that can characterize the metastasising ability of CMM and the tumour‐promoting host immune response.     

Elevation of serum epidermal growth factor and interleukin 1 receptor antagonist in active psoriasis vulgaris

Background Psoriatic plaques present a complex expression profile, including high levels of cytokines, chemokines and growth factors. Circulating cytokines have been suggested to reflect the activation status of the inflammatory process. Objectives To analyse 20 cytokines, chemokines and growth factors in 14 patients with psoriasis vulgaris at the start and during the course of ultraviolet B treatment. Methods A multiplex cytokine assay was used. Results We identified increased serum levels of epidermal growth factor (EGF) (mean 323 vs. 36·6 pg mL^?1, P =0·0001), interleukin (IL)‐1 receptor antagonist (mean 39·1 vs. 14·6 pg mL^?1, P =0·02) and tumour necrosis factor‐α (mean 7·5 vs. 4·5 pg mL^?1, P =0·04) at baseline in patients with psoriasis compared with matched controls. None of these cytokines was correlated to the severity of the disease (Psoriasis Area and Severity Index) or decreased with phototherapy, suggesting that sources other than lesional skin contribute to the production of these cytokines. Using cluster analysis, we observed coordinate upregulation of EGF, IL‐6, macrophage inflammatory protein‐1β and vascular endothelial growth factor. Conclusions The sustained high expression of inflammatory circulating cytokines is a potential mechanism linking psoriasis with its extracutaneous comorbidities.     

Melanoma‐derived IL‐1 converts vascular endothelium to a proinflammatory and procoagulatory phenotype via NFκB activation

Spreading of melanoma is associated with efficient extravasation of circulating tumor cells from the vascular system into distant target organs. This process is accompanied and supported by proinflammatory and procoagulatory conditions. In this study, we analysed the ability of human melanoma cell lines to activate endothelial cells (ECs) in vitro. Some melanoma cells, that is, MV3, were shown to trigger an prompt calcium‐flux‐dependent, procoagulatory endothelial response that was accompanied by luminal release of ultra‐large von Willebrand factor (ULVWF) fibres that were immobilized to the endothelial surface layer. In contrast to MV3‐derived supernatant, prolonged treatment of ECs with WM9‐derived supernatant mediated a pronounced activation of nuclear factor kappa B (NFκB). NFκB activation in ECs was dependent on both IL‐1α and IL‐1β secreted from melanoma cells. Melanoma‐derived IL‐1 mediated an upregulation of proinflammatory cytokines IL‐6 and IL‐8, the intercellular adhesion molecule‐1 (ICAM‐1), the vascular cell adhesion molecule‐1 (VCAM‐1) and the procoagulatory tissue factor (TF) in ECs. Our data show that melanoma cells activate ECs either directly and within seconds or by an IL‐1‐mediated NFκB activation. Both pathways of EC activation convert the regular repressive function of ECs on inflammation and coagulation to a proinflammatory and procoagulatory surface that supports tumor progression.     

Epidemiological patterns of perniosis,and its association with systemic disorder

Background. There are few studies exploring the epidemiological properties of perniosis (chilblains) and its association with systemic disorders. Aim. To collect epidemiological data for perniosis, to investigate any association with systemic disorders, and to identify markers for the differential diagnosis of idiopathic and secondary perniosis. Methods. This was a prospective study of 51 patients with perniosis [female : male ratio 2.64 : 1, mean ± SD age 24.6 ± 14.7 years, with 25 patients (49%) aged 0–18 years]. Each patient was interviewed, and signs suggestive of connective‐tissue disorders were recorded. Detailed laboratory investigations including autoimmune parameters were performed. Results. Significant proportions of the patients had both chronic and idiopathic perniosis (P < 0.001 for both). The mean age of the group with secondary perniosis was significantly higher than that of the idiopathic group (P < 0.01). There was no significant gender difference in the secondary perniosis group (P = 0.71). Clerking work was the most common occupation (37%, P = 0.01). Persistence beyond the cold seasons, and presence of photosensitivity, hypergammaglobulinaemia and rheumatoid factor were significant findings in the secondary group (P = 0.02, P = 0.02, P < 0.01, P < 0.05, respectively). Conclusions. Perniosis is not rare in children, but patients with secondary perniosis are more likely to be older. In terms of underlying systemic disorder, advanced age and male gender may be important demographic features. Measurement of cryoglobulin levels in the initial laboratory investigations of patients with perniosis is not necessary. Persistence beyond the cold seasons, and presence of photosensitivity, hypergammaglobulinaemia and rheumatoid factor may be useful in differentiating between idiopathic and secondary perniosis.     

Colorimetric measurements of iris colour and their significance in East Asian patients with skin cancer

Background. A light‐coloured iris is considered a risk factor for skin cancer in general. However, iris colour cannot be considered a plausible risk factor for skin cancer in East Asian populations because of the relative homogeneity of iris colours. Furthermore, subjective classifications of iris colour cannot distinguish between different East Asian individuals as to their likelihood of developing cancer. Aim. To measure human iris colours quantitatively and to assess the significances of iris colours with respect to skin cancer in Korean patients. Methods. Reference Commission Internationale d’Eclairage (CIE) L*a*b* coordinates on a ColorCheck chart were recorded using a reflectance spectrophotometer and compared with computed CIE L*a*b* coordinates from digital images to determine equations to calibrate CIE L*a*b* values. We then took iris images and measured iris colours and the colours of sun‐exposed and sun‐protected skin in 42 Korean patients with various cutaneous malignancies and nonmalignant dermatological diseases. Results were statistically analysed with regard to iris and skin colours in CIE L*a*b* coordinates. Results. Patients with skin cancer had significantly lighter irises or higher L* values than dermatological patients without a malignancy (P = 0.02). Colour differences (ΔE*ab) between sun‐exposed skin and sun‐protected skin were greater in men (P < 0.01) and in patients with skin cancer (P < 0.01), and the lightness (L*) values of sun‐exposed skins decreased with age (r = ?0.32, P < 0.05). Conclusions. Iris colour appears to be a possible skin cancer risk factor in East Asian populations. The larger colour differences seen between sun‐protected and sun‐exposed skin in men and in patients with skin cancer may have been due to chronic or excessive sun exposure.     

Evaluation of carotenoids and reactive oxygen species in human skin after UV irradiation: a critical comparison between in vivo and ex vivo investigations

UV irradiation is one of the most harmful exogenous factors for the human skin. In addition to the development of erythema, free radicals, that is reactive oxygen species (ROS), are induced under its influence and promote the development of oxidative stress in the skin. Several techniques are available for determining the effect of UV irradiation. Resonance Raman spectroscopy (RRS) measures the reduction of the carotenoid concentration, while electron paramagnetic resonance (EPR) spectroscopy enables the analysis of the production of free radicals. Depending on the method, the skin parameters are analysed in vivo or ex vivo. This study provides a critical comparison between in vivo and ex vivo investigations on the ROS formation and carotenoid depletion caused by UV irradiation in human skin. The oxygen content of tissue was also determined. It was shown that the antioxidant status measured in the skin samples in vivo and ex vivo was different. The depletion in the carotenoid concentration in vivo exceeded the value determined ex vivo by a factor of about 1.5, and the radical formation after UV irradiation was significantly greater in vivo by a factor of 3.5 than that measured in excised human skin, which can be explained by the lack of oxygen ex vivo.