Autoregulation of endothelin-1 secretion by cultured human keratinocytes via the endothelin B receptor.

We investigated endothelin-1 (ET-1) receptor expression on normal human keratinocytes (HK). We show that HK express the ETB receptor isoform and respond to ET-1 with a 2.7-fold increase in intracellular free calcium. HK did not respond to ET-1 with increased proliferation; however, 30 nM ET-1 caused a 51.7% decrease in ET-1 accumulation in HK-conditioned medium. We propose that HK ET-1 receptors function in autocrine regulation of ET-1 secretion.

Melanocyte movement in vitro: role of matrix proteins and integrin receptors.

During the repigmentation of vitiliginous skin, melanocytes migrate from the outer root sheath of the hair follicle into the depigmented skin. We hypothesize that this requires changes in the local microenvironment that are conductive to melanocyte migration. One important change in the microenvironment could be the localized production of matrix proteins. We have previously employed time-lapse photography to evaluate the effect of inflammatory mediators and cytokines on melanocyte movement. We have adapted this system to study the effect of matrix proteins on melanocyte movement in vitro. Type IV collagen significantly increases melanocyte migration, whereas laminin and fibronectin have no effect. Cell/matrix interactions are in part controlled by cell-surface integrins. Integrins have been demonstrated to be important in controlling the migration of many cell types. We demonstrate that melanocytes express cell-membrane alpha 2, alpha 3, and alpha 5 integrins and that the enhanced melanocyte migration on type IV collagen is inhibited by specific function-blocking antibodies to integrins alpha 2 and alpha 3, but not to alpha 5 integrins.

Cultured human melanocytes from black and white donors have different sunlight and ultraviolet A radiation sensitivities.

Short-term and long-term survival of cultured neonatal foreskin melanocytes from black and white individuals were assessed following a single exposure to simulated sunlight or ultraviolet A (UVA) radiation. Melanocytes from black individuals contained significantly more melanin than melanocytes from white individuals (p less than 0.05). Black and white melanocytes had similar survival profiles following simulated sunlight exposure, whereas black melanocytes were significantly more resistant to UVA cytotoxicity than melanocytes from white subjects (p less than 0.05) at UVA doses above 15 J/cm2. There was no difference in unscheduled DNA synthesis in the black or white melanocytes following simulated sunlight exposure and no unscheduled DNA synthesis was measurable following melanocyte exposure to UVA radiation. Low-dose UVA (1 or 5 J/cm2) was mitogenic to both black and white melanocytes. By analysis of co-variance, the melanin content of melanocytes of black and white subjects was significantly (p less than 0.05) associated with susceptibility to UVA killing; melanocytes with high melanin content had high resistance to UVA cytotoxicity and those with low melanin content had low resistance to UVA cytotoxicity. From these data we suggest that the higher melanin content of melanocytes of black subjects confers increased resistance to UVA damage. This is likely to be of importance in epidermal photodamage.

Modulation of melanocyte intercellular adhesion molecule-1 by immune cytokines.

Human melanocyte expression of intercellular adhesion molecule-1 (ICAM-1) with or without stimulation by interferon gamma (IFN-G), tumor necrosis factor alpha (TNF-alpha), or interleukin-1-alpha (IL-1 alpha), was measured utilizing direct immunofluorescence and fluorescence-activated cell sorting (FACS). Melanocytes grown in vitro expressed low levels of ICAM-1, which could be increased by exposing the cells to IFN-G, TNF-alpha, or IL-1 alpha. Each cytokine caused an enhancement of melanocyte ICAM-1 expression in a dose-dependent fashion. The lowest dose necessary to cause a significant increase in melanocyte ICAM-1 expression was 1 U/ml IFN-G, 0.3 ng/ml TNF-alpha, or 3 U/ml IL-1 alpha. Melanocytes were most sensitive to TNF-alpha stimulation, with the greatest levels of ICAM-1 expression following 30 ng/ml or more TNF-alpha. When IFN-G was added to melanocyte cultures in combination with TNF-alpha or IL-1 alpha, there was an additive increase in ICAM-1 expression but no synergy was noted with the combined cytokines. To our knowledge, this is the first report of melanocyte ICAM-1 induction by TNF-alpha and IL-1 alpha and by physiologically relevant doses of IFN-G. Because of the importance of ICAM-1 in the regulation of immune cell-target interactions, the study of ICAM-1 expression by melanocytes may help us to better understand immune mechanisms of melanocyte injury.

Leukotriene C4 and TGF-alpha are stimulators of human melanocyte migration in vitro.

Human vitiligo is a disease of melanocyte destruction that leads to areas of depigmentation in the skin. The major form of treatment for vitiligo is photochemotherapy using psoralens and UVA radiation (PUVA), which induces the slow migration of pigment cells from hair follicles and normal skin into the depigmented areas. Our hypothesis is that immune cytokines and inflammatory mediators released as a result of the photochemotherapy are signals for melanocyte migration. We have developed an in vitro assay that quantitates the movement of individual cultured melanocytes over a 72-h period using time-lapse photography. Using this assay we found that both LTC4 and TGF-alpha were stimulators of melanocyte migration in vitro. The LTC4 effect was greater and lasted for the entire 72-h experimental period, whereas the TGF-alpha effect was significant only during the first 24 h of the experiment.

Leukotrienes C4 and D4 as potent mitogens for cultured human neonatal melanocytes.

Arachidonic acid and its metabolites (eicosanoids) are membrane-derived inflammatory mediators with a diverse set of biologic properties affecting numerous cells and organ systems, including the skin. They have been implicated in the pathogenesis of inflammatory skin disease and post-inflammatory hyperpigmentation. We have studied the ability of arachidonic acid, prostaglandin D2, prostaglandin E2, leukotriene B4, leukotriene C4, leukotriene D4, and leukotriene E4 to enhance the growth of cultured human melanocytes. Of these compounds, only leukotriene C4 and leukotriene D4 were capable of stimulating melanocyte proliferation. In addition, cultured melanocytes metabolized leukotriene C4 to leukotriene E4 with greater than 60% conversion in less than three hours. Melanocytes grown on suboptimal media (doubling time 12-20 days) respond in a dose-dependent fashion to leukotriene C4, with a significant difference from control noted at 28 days with a concentration of LTC4 of 30 nM and a doubling time of 5-8 days. We feel that leukotriene C4 and D4 could play an important role in post-inflammatory melanocyte hyperplasia.

Melanocyte mitogens induce both melanocyte chemokinesis and chemotaxis.

It is believed that during repigmentation of vitiligo, inactive melanocytes in the outer root sheath of the hair follicle become activated, proliferate, and migrate into the depigmented skin. However, the mechanisms controlling melanocyte migration remain to be elucidated. In this study, we investigated the effects of well-described melanocyte growth factors on melanocyte migration. Using time-lapse photography, we demonstrated that melanocyte chemokinetic movement was induced by basic fibroblast growth factor, stem cell factor, and endothelin-1, with the greatest effect noted using 100 nM endothelin-1. Similar results were reported previously with leukotriene C4. When surrounded by these stimuli, melanocytes moved in a random, nonlinear fashion and showed no desensitization at the concentrations studied. In Boyden chamber checkerboard analysis, basic fibroblast growth factor, leukotriene C4 and endothelin-1 were chemotactic. They produced directional migration and showed desensitization at higher concentrations. The greatest effect again was seen with 100 nM endothelin-1. Stem cell factor showed no effect in this assay system at the concentrations tested. The four melanocyte mitogens--leukotriene C4, endothelin-1, basic fibroblast growth factor, and stem cell factor--stimulate melanocyte migration, and this migration may be either chemokinetic (activated random movement) or chemotactic (requiring a gradient, directional, and showing desensitization), depending on the conditions used. We believe that these factors may be effective in stimulating vitiligo repigmentation by inducing proliferation and migration of hair-follicle outer-root-sheath melanocytes into the depigmented epidermis.

Ultraviolet radiation can either suppress or induce expression of intercellular adhesion molecule 1 (ICAM-1) on the surface of cultured human keratinocytes.

Interactions of the ligand/receptor pair LFA-1(CD11a/CD18) and ICAM-1(CD54) initiate and control the cell-cell interactions of leukocytes and interactions of leukocytes with parenchymal cells in all phases of the immune response. Induction of the intercellular adhesion molecule 1 (ICAM-1) on the surface of epidermal keratinocytes has been proposed as an important regulator of contact-dependent aspects of cutaneous inflammation. Ultraviolet radiation (UVR) also modifies cutaneous inflammation, producing both up- and down-regulation of contact hypersensitivity. We have found that UVR has a biphasic effect on the induction of keratinocyte CD54. Using immunofluorescence and FACS techniques to quantitate cell-surface CD54 staining, we have shown that UVR (100 mJ/cm2 of UVB) significantly (p less than 0.01) inhibits keratinocyte CD54 induction by gamma interferon 24 h after irradiation. However, at 48, 72, and 96 h after UVR (10 to 100 mJ/cm2), CD54 expression is significantly induced (p less than 0.01 to p less than 0.001) to levels even greater than are induced by gamma interferon (20 U/ml). In addition, at 48, 72, or 96 h following UVR (30-100 mJ/cm2), the gamma-interferon-induced CD54 expression on human keratinocytes is also strongly (p less than 0.05 to p less than 0.001) enhanced. In this cell-culture system, gamma interferon and TNF-alpha are both strong CD54 inducers and are synergistic, but GM-CSF, TFG-beta, and IL-1 have no direct CD54-inducing effects. Thus the effects of UVR on CD54 induction are biphasic, producing inhibition at 24 h and induction at 48, 72, and 96 h. This effect on CD54 may contribute to the biphasic effects of UVR on delayed hypersensitivity in vivo. The early inhibition of ICAM-1 by UVR may also contribute to the therapeutic effects of UVR. We also speculate that the late induction of ICAM-1 by UVR might be an important step in the induction of photosensitive diseases such as lupus erythematosus.

Ras mutations in human melanoma: a marker of malignant progression.

In this study we address whether there is an association between ras mutations and disease progression in malignant melanoma. DNA was extracted from 100 paraffin-embedded melanomas and sequences around the 12th, 13th and 61st codons of N-, H-, and K-ras were amplified using the polymerase chain reaction and probed for single base pair mutations using synthetic oligonucleotide probes. Thirty-six melanomas contained mutations, which in 25 cases (69%) occurred at the 61st codon of N-ras. The results from dot blot hybridizations were confirmed by subcloning and sequencing the polymerase chain reaction products from two tumors. No ras mutations were found in Clark's level I melanomas, whereas 19% of level II and 45% of the more advanced primary tumors contained ras mutations (Chi squared test: p < 0.05). The median Breslow thickness of primary melanomas with ras mutations was 0.72 mm, significantly thicker than the 0.42 mm of melanomas without mutations (Mann-Whitney U test, p = 0.042). Ras mutations were found more frequently in primary tumors from continuously exposed skin (56%) than tumors from intermittently or non-sun exposed sites (21%). Fifty percent of locally recurrent and 47% of metastatic melanomas had ras mutations. We conclude that ras mutations occur in a subset of melanomas from sun-exposed skin as a feature of tumor progression.

Disparate antioxidant enzyme activities in cultured human cutaneous fibroblasts, keratinocytes, and melanocytes.

Antioxidant enzyme activities of cultured human foreskin fibroblasts, keratinocytes, and melanocytes from healthy black and Caucasian donors were measured and compared. Fibroblasts had more (p less than 0.05) peroxidase, catalase, glutathione peroxidase, and superoxide dismutase activity than keratinocytes. Keratinocytes had more (p less than 0.05) peroxidase, catalase, glutathione peroxidase, and superoxide dismutase activity than melanocytes. No differences in antioxidant enzyme activities were observed between the cells of any type taken from black or Caucasian people. Antioxidant enzyme activities may affect resistance to damage by oxidants induced by ultraviolet radiation and inflammation.

Cultured human keratinocytes synthesize and secrete endothelin-1.

The human epidermal-melanin unit exists as a complex interplay of cell-cell interactions. Melanocytes synthesize melanin and transfer it to the surrounding keratinocytes, which, in turn, produce factors that affect melanocyte homeostasis, growth, and melanization. Endothelin-1 (ET-1), a vasoconstrictor peptide produced by endothelial cells, has recently been shown to stimulate human melanocyte proliferation and tyrosinase activity. To investigate the possibility that keratinocytes synthesize and secrete ET-1, we grew human keratinocytes in a defined serum-free medium and measured ET-1 levels in the keratinocytes and the keratinocyte-conditioned medium. Northern analysis of keratinocyte total RNA also was performed. We found that human keratinocytes express preproET-1 mRNA and translate the message to ET-1 protein, which is secreted into the keratinocyte medium. Human keratinocytes produced ET-1 in a time-dependent manner with total production of 20.1 +/- 1.1 pg ET-1/10(6) cells at 24 h (n = 7). Although total ET-1 production (secreted plus cell-associated ET-1) was similar, the proportion of secreted versus cell-associated ET-1 varied widely among the different donors. We have found that human keratinocytes synthesize and secrete ET-1 in vitro. From these data we believe that the keratinocyte could be an in vivo epidermal source of this melanocyte growth and pigmentation factor.

Initial lesion size as a predictive factor in determining the response of port-wine stains in children treated with the pulsed dye laser.

OBJECTIVE: To evaluate the response of facial port-wine stains in children to pulsed (450 microseconds) dye (577- or 585-nm) laser treatment based on the age of the patient and the size of the port-wine stain at the initiation of treatments. DESIGN: Case series. SETTING: Outpatient dermatology clinic at the University of Colorado School of Medicine, Denver. PATIENTS: A consecutive sample of 83 children who had facial port-wine stains that were treated in their entirety at each visit. INTERVENTION: Patients were treated with a pulsed (450 microseconds) dye (577- and 585-nm) laser. MAIN OUTCOME MEASURE: Decrease in size of the port-wine stain. RESULTS: Overall 18 (22%) of the 85 patients responded with complete clearing of their port-wine stains. The port-wine stains in 32% (7/22) of the patients who began treatment before 1 year of age responded with complete clearing of their port-wine stains compared with 18% (11/61) in older patients (P < .01). Fifteen (32%) of 47 patients with port-wine stains smaller than 20 cm2 at initial evaluation were totally cleared vs three (8%) of the 36 patients with port-wine stains larger than 20 cm2 (P < .05). This size effect is apparent at all ages. CONCLUSION: When considering treatment for facial port-wine stains with pulsed dye lasers, it is important to consider not only the age of the child at the beginning of treatments, but also the initial size of the lesion.

"Block the sun, not the fun": evaluation of a skin cancer prevention program for child care centers.

INTRODUCTION: This paper describes the evaluation of a skin cancer prevention program for preschools and daycare centers. The intervention was targeted primarily at staff of child care centers, with the aim of increasing use of sun protection practices for young children while attending these centers. Secondary target groups included parents and the children themselves. The intervention, which adopted the slogan, "Block the Sun, Not the Fun," included workshops for child care center staff, and information/activity packets for parents. METHODS: Twenty-seven preschools and daycare centers were randomly assigned to an intervention or wait-list control group. The intervention group received the intervention during the spring of 1994; the wait-list control group received the intervention during the spring of 1995. Evaluation consisted of interviews with center directors, observations of practices, and review of written policies before the intervention (in summer, 1993) and after the intervention (in summer, 1994). A survey of 201 parents was conducted during late summer 1994. RESULTS: While the intervention did not appear to change the sun protection attitudes or practices of parents, or use of clothing and shade at child care centers, results suggested significant changes in the sun protection knowledge/attitudes of center directors and the use of sunscreen at child care centers. Additionally, parents with children attending centers in the intervention group were more likely to be satisfied with sun protection practices at their centers. CONCLUSION: This low-intensity intervention appears to be effective at changing sun protection attitudes and sunscreen use at child care centers, and can be easily replicated. However, high staff turnover at child care centers would suggest that "boosters" will be necessary to sustain the impact. More intensive efforts directed at social norms are likely to be necessary to change clothing and outdoor play practices.

Serum melatonin levels in melanoma patients after repeated oral administration.

The goal of this study was to determine the effect of oral melatonin in divided doses on plasma melatonin levels in patients with metastatic melanoma. Hourly blood samples were obtained from five patients for 24 h prior to melatonin administration and for 24 h during oral administration of melatonin, 50 mg every 4 h. In two of the five patients, the expected nocturnal plasma melatonin peak was observed. Oral melatonin was well absorbed. Plasma melatonin levels exhibited six peaks and troughs, were two to four-fold higher during peaks than troughs, and remained more than 25 times higher than peak pretreatment melatonin levels, even during troughs. Divided oral doses of melatonin were well tolerated and maintained plasma melatonin levels 25-80 times higher than endogenous peak values.

Overexpression of mutant ras in human melanoma increases invasiveness, proliferation and anchorage-independent growth in vitro and induces tumour formation and cachexia in vivo.

Malignant melanoma is the deadliest form of skin cancer. Previous studies have shown that the incidence of ras mutation increases with progression of melanoma, but that such mutations may not be present in the earliest radial growth phase melanomas. Recently it has been proposed that introduction of ras mutations into cells deficient in tumour suppressor genes such as p16 (INK4a) is sufficient to induce characteristics of cellular transformation such as anchorage-independent growth and tumour formation in vivo. To test this hypothesis in human melanoma, mutant N-ras, mutant H-ras or wild-type H-ras genes were transfected by electroporation into WM35 cells, a p16-deficient human melanoma cell line of low invasive potential. Increased expression of mutant ras p21 enhanced anchorage-dependent cell growth on tissue culture plastic. In addition, overexpression of mutant N-ras and H-ras, but not of wild-type H-ras, increased the experimental invasive potential, inducing anchorage-independent growth in soft agar, increasing cell motility measured by time-lapse video microscopy, and increasing invasiveness through reconstituted basement membranes. Finally, overexpression of mutant H-ras in melanoma cells was shown to increase tumorigenicity and to induce cachexia when H-ras transfected cell lines were injected subcutaneously in severe combined immunodeficiency (SCID) mice. Thus the addition of activating ras mutations to a melanoma cell line already deficient in p16 leads to enhanced proliferation, survival and migration in vitro and to enhanced subcutaneous tumour formation in vivo. This phenotype is typical of the behaviour of vertical growth phase (VGP) melanoma, and we propose that activation of the ras signalling pathway in the presence of deletions in p16 or related tumour suppressors can induce the VGP melanoma phenotype.

Malassezia folliculitis in immunocompromised patients.

Four cases of Malassezia folliculitis in immuno-compromised patients with leukemia, papillary adenocarcinoma of the lung, and chronic renal failure are reported. This condition manifests with multiple bland asymptomatic follicular papules of the trunk and arms. Biopsy specimens show dilated follicles containing unipolar budding yeast forms. Malassezia is a common infection that must be differentiated from the cutaneous manifestations of systemic candidiasis.

Lesion size is a factor for determining the rate of port-wine stain clearing following pulsed dye laser treatment in adults.

Seventy-four adult patients with facial, truncal, and extremity port-wine stains (PWS) were treated with the flashlamp-pumped pulsed dye laser (PDL) with laser output ranging from 6.0 to 7.5 J/cm2. Response to treatment was analyzed by comparing the area of involvement following each treatment with the area of involvement measured in the first treatment session. All the PWS responded with 25 to 90 percent lightening, and 85.1 percent of patients achieved 25 percent clearing. However, only 36.5 percent achieved 50 percent clearing, and none of the patients achieved 100 percent clearing. None of four patients with PWS greater than 100 cm2 achieved 50 percent clearing following a mean of 17.2 +/- 5.7 treatments. These data emphasize the importance of objectively documenting clinical response to PDL treatment of PWS. Adult patients need to be made aware that complete clearing may not be obtainable by PDL treatment alone. This is especially important for adult patients with PWS larger than 100 cm2.

Facial port wine stains in childhood: prediction of the rate of improvement as a function of the age of the patient, size and location of the port wine stain and the number of treatments with the pulsed dye (585 nm) laser.

In this study we examined the rate of decrease in size of facial port wine stains (PWS) as a function of number of treatments, lesion size, lesion location and patients' age. This study was performed at the University of Colorado Hospital Outpatient Dermatology Center, Denver, U.S.A. A consecutive sample of 91 patients 18 years of age or younger with facial PWS in which the entire lesion was treated at each visit were studied. Included were all patients who had a minimum of five treatments or complete clearance of their lesion in fewer than five treatments. Patients were evaluated following one, five and 10 treatments with the pulsed (450 s) dye (585 nm) laser. Improvement was defined as the percentage decrease in the size of the PWS. For all patients, the first five treatments resulted in a mean decrease in size of 55% while the second five treatments (38 patients) only improved the mean decrease in size by 18%. Grouped by location, the mean decreases in size from the first five and the second five treatments were as follows: central forehead = 100%, 0%; peripheral face = 58%, 28%; central face = 48%, 14%; and mixed (combination of peripheral and central face) = 21%, 9%. All central forehead PWS completely cleared within five treatments while none of the mixed PWS did so even with an average of 14 treatments. Grouped by size, mean decrease in size was highest for small lesions; < 20 cm2 = 67%, 21%; 20 to < 40 cm2 = 45%, 8%; and > 40 cm2 = 23%, 29%. Grouped by age, mean decrease in size was highest for young children: < 1-year-old = 63%, 33%; 1 to < 6 years = 48%, 15%; and older than 6 years = 54%, 10%. For all patients studied, maximal improvement was obtained in the first five treatments. Major determinants of treatment response in order of decreasing importance are PWS location, size and patients' age. The most successful responses are seen in young patients (less than 1 year old) with small PWS (under 20 cm2) that are located over bony areas of the face such as the central forehead. These three determinants may be useful tools to guide patient expectations and to predict the rate of improvement of PWS to pulsed dye laser treatment.