H2 O2 -induced oxidative stress disrupts mitochondrial functions and impairs migratory potential of human epidermal melanocytes.

Reactive oxygen species (ROS) have already been demonstrated to impede the migratory ability in non-melanocytic cell lines by depleting mitochondrial ATP production. Therefore, understanding the mitochondrial metabolic response to migration in the presence of ROS should be a key to understanding repigmentation in vitiligo. This study aimed to investigate the energy mechanism associated with the ROS-mediated attenuation of melanocyte migration. After melanocytes were pretreated with H2 O2 , their ATP production, migratory ability, ultrastructural changes and Mitochondrial Permeability Potential were analysed. The results showed that, in parallel with the decreased ATP production, the migratory ability of melanocytes was significantly inhibited by oxidative stress. Supplementation with exogenous ATP reversed the suppressed ATP-dependent migration of melanocytes. Melanocytes were then stressed with H2 O2 and Agilent Whole Human Genome microarray analysis identified 763 up-regulated mRNAs and 1117 down-regulated mRNAs. Among them, 11 of the encoded proteins were involved in mitochondrial ATP production and their expression levels were verified. The decreased expression of NADH dehydrogenase 2(ND2) , cytochrome c oxidase 1(COX1) and cytochrome c oxidase 3(COX3) was shown to be involved in the depletion of mitochondrial ATP production, which was coupled with the impaired migratory potential. These results indicate that the migration of melanocytes relies heavily on an inexhaustible supply of ATP from mitochondria.

Light-emitting diode 585nm photomodulation inhibiting melanin synthesis and inducing autophagy in human melanocytes.

BACKGROUND: Melasma is a common hyperpigmentation skin disease on face. Light-emitting diode (LED) photomodulation (585nm) is reported to be effective for the treatment of melasma. However, whether and how LED photomodulation would influence melanogenesis of human epidermal melanocytes (HEMs) is unknown. OBJECTIVE: To evaluate the effects of LED photomodulation (585nm) on melanogenesis in HEMs. METHODS: HEMs were irradiated with fluences of 0, 5, 10 and 20J/cm2 585nm LED light. After 5-day treatment, cell viability was analyzed by CCK-8 assay, and apoptosis was assessed by Annexin V APC assay. Melanin content and tyrosinase activity were measured by spectrophotometer. Melanosome stage and autophagosomes were determined under transmission electron microscope (TEM). The formation of autophagic punctate structures was observed under confocal microscope. RT-PCR and western blotting were used to assess the expression of relative mRNA and protein levels. RESULTS: Yellow light LED 585nm had no effects on HEMs cell viability and apoptosis. Treatment with LED 585nm from 5J/cm2 to 20J/cm2 inhibited melanosome maturation, decreased melanin content and tyrosinase activity. Inhibition was accompanied by the decreased expression of tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1) and microphthalmia-associated transcription factor (MITF) on both mRNA and protein levels. Autophagosomes were observed under TEM. Autophagic punctate structures of microtubule-associated protein light chain 3 (LC3) proteins were induced by LED 585nm light. The configuration change of LC3 from LC3-I to LC3-II, and the degradation of p62 protein were observed after LED 585nm. Furthermore, we also revealed that the anti-melanogenic effect of LED 585nm photomodulation was reversed by 3-Methyladenine (3-MA), which inhibits autophagy by blocking autophagosome formation via the inhibition of type III Phosphatidylinositol 3-kinases (PI-3K). CONCLUSIONS: Our finding demonstrated that LED photomodulation with 585nm wavelength suppressed melanin content in HEMs, and the effect was caused by its dose-dependent inhibition on melanogenesis and the induction of HEMs autophagy. This may provide new insights into the efficacy of LED photomodulation in the treatment of hyperpigmentation disorders.

Vitiligo, NB-UVB and tacrolimus: our experience in Naples.

AIM: The aim of the present study was to evaluate the efficacy and safety of narrowband UVB (NB-UVB) compared with tacrolimus ointment 0.1% in patients with bilateral vitiligo. METHODS: In this comparative study, four groups of patients were randomized. Each group was composed by 12 patients with bilateral vitiligo; in each group, every patient was irradiated with NB-UVB (length: 311 nm) twice a week for 9 months and applied tacrolimus ointment 0.1% twice a day on the other area in the same period. Before starting therapy and after 3, 6 and 9 months of therapy, a clinical and photographic evaluation of percentage of repigmentation was performed and Dermatology Life Quality Index Questionnaire was fulfilled. RESULTS: A repigmentation at least partial occurred in 71% of patients after 36 weeks of treatment with tacrolimus ointment 0.1%; in the whole sample, 14 patients (29%) showed no repigmentation at all, with 2 of them discontinuing the therapy because of side effects (erythema and folliculitis-like manifestations). A homogeneous repigmentation at least partial occurred in 69% of patients after 36 weeks of treatment with NB-UVB; in the whole sample 15 patients (31%) showed no repigmentation at all, with 1 of them discontinuing the therapy because of side effects. CONCLUSION: The present study confirmed that the efficacy of NB-UVB phototherapy in vitiligo is comparable to tacrolimus ointment 0.1% therapy. On the basis of our study, we may suggest tacrolimus ointment 0.1% as an alternative to NB-UVB therapy for treating vitiligo.

Bimodal effect of retinoic acid on melanocyte differentiation identified by time-dependent analysis.

Retinoic acid (RA) is considered to control melanocytes; however, its precise mechanism remains unclear because of a bimodal effect, which promotes or inhibits melanin synthesis depending on the cell type, culture condition of melanocytes and skin conditions. In this study, we examined the effects of RA throughout each stage of differentiation of melanocytes using a mouse embryonic stem cell culture system to induce melanocytes. The results showed that RA has significantly different effects depending on the stage of differentiation of melanocytes. More specifically, RA promoted differentiation in earlier stages, wherein embryonic stem cells became melanoblasts via neural crest cells, and inhibited differentiation in later stages, wherein melanoblasts became melanocytes. It was revealed for the first time that melanocytes show markedly different reactions to RA depending on the stage of differentiation.

Effect of PUVA therapy on melanocytes and keratinocytes in non-segmental vitiligo: histopathological, immuno-histochemical and ultrastructural study.

BACKGROUND AND AIMS: Psoralen ultraviolet A (PUVA) is an important modality in treating vitiligo. Its effect on melanocytes and keratinocytes is not sufficiently studied. In this work, we investigated 30 cases of non-segmental vitiligo regarding the changes of melanocytes and keratinocytes in both vitiliginous and nearby areas before and after PUVA therapy. METHODS: Three skin biopsies were obtained from each patient from the vitiliginous, marginal and perilesional areas before and after 12 months of PUVA. Biopsies were examined histologically using haematoxylin and eosin, Masson-Fontana stains and 3,4-dihydroxyphenylalanine (DOPA) reaction and histochemically using human melanoma black-45 (HMB-45) antibody while ultrastructural examination was performed on six patients. Control biopsies were taken from five healthy volunteers. RESULTS: In 10% of pretreated biopsies from the centre of vitiligo lesions, scanty melanocytes were detected histologically and ultrastructurally, while they did not stain with DOPA or HMB-45 antibody suggesting that these melanocytes were inactive. Moreover, degenerative changes were detected by electron microscopy in both melanocytes and keratinocytes in all areas. After PUVA therapy, obvious improvement of the histopathological changes occurred with significant increase in active melanocytes. The degeneration of melanocytes and keratinocytes was also reduced at the ultrastructural level. CONCLUSION: Vitiligo affects both melanocytes and keratinocytes causing degenerative changes. These changes were present in both the leucodermic and the apparently normal perilesional skin. PUVA increases the number of active epidermal melanocytes in the three tested areas and recovers the melanocyte and keratinocyte degeneration.

In vivo microscopic approaches for facial melanocytic lesions after quality-switched ruby laser therapy: time-sequential imaging of melanin and melanocytes of solar lentigo in Asian skin.

BACKGROUND: The quality-switched ruby laser (QSRL) has been widely used for the treatment of pigmented lesions, but clinical evaluations in most studies have been conducted on macroscopic skin color observation comparing the laser-treated skin with its nontreated surrounding area. A few investigations examined skin changes after laser therapy at a cellular level, but almost none did so noninvasively. OBJECTIVE: To elucidate the dynamic changes after QSRL irradiation of facial solar lentigo using noninvasive optical techniques. MATERIALS AND METHODS: Time-sequential imaging of Japanese female patients with a clinical diagnosis of solar lentigo was performed using ultraviolet photography, high-magnification videomicroscopy, and reflectance-mode confocal microscopy to examine pigmentary change after QSRL irradiation. RESULTS: The present study showed that remaining melanocytes were visible in the solar lentigo of all subjects when crusts peeled off, despite hardly observable skin pigmentation to the naked eye. Moreover, noninvasive confocal imaging revealed that pigmented melanocytes varied in each solar lentigo after QSRL treatment, as indicated by melanin reflection level. CONCLUSIONS: Optical techniques facilitate the evaluation of the in vivo dynamics of epidermal-melanocytic changes in solar lentigo after QSRL therapy and may be useful for monitoring outcomes after laser irradiation.

Stimulation of the proliferation and differentiation of mouse pink-eyed dilution epidermal melanocytes by excess tyrosine in serum-free primary culture.

The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mouse at the pink-eyed dilution (p) locus (black, C57BL/10JHir-P/P) and their congenic mutant mouse (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with a serum-free melanocyte growth medium supplemented with additional L-tyrosine (Tyr) from initiation of the primary culture. L-Tyr inhibited the proliferation of P/Pmelanocytes in a dose-dependent manner, whereas L-Tyr stimulated the proliferation of p/p melanoblasts and melanocytes regardless of dose. On the other hand, L-Tyr stimulated (P/P) or induced (p/p) the differentiation of epidermal melanocytes in a dose-dependent manner. In both P/P and p/p melanoblasts and melanocytes cultured with 2.0 mM L-Tyr for 14 days, slight increases in contents of eumelanin marker, pyrrole-2,3,5-tricarboxylic acid (PTCA) and pheomelanin marker, aminohydroxyphenylalanine (AHP) were observed. The average number of total melanosomes (stages I, II, III, and IV) per P/P melanocyte was not changed by L-Tyr treatment, but the proportion of stage IV melanosomes in the total melanosomes was increased. On the contrary, in p/p melanoblasts and melanocytes L-Tyr increased dramatically the number of stage II, III, and IV melanosomes as well as the proportion of stage III melanosomes. Contents of PTCA and eumelanin precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) of cultured media in p/p melanocytes were much more greatly increased than in P/P melanocytes. However, contents of AHP and pheomelanin precursor, 5-S-cysteinyldopa (5-S-CD) of cultured media in p/p melanocytes were increased in a similar tendency to P/Pmelanocytes. These results suggest that p/p melanocytes in the primary culture are induced to synthesize eumelanin by excess L-Tyr, but difficult to accumulate them in melanosomes.

Estudio clínicopatológico de nevus melanocíticos en distintos grupos de edad / Clinical-pathological study of melanocytic nevi in diferent age groups

Objetivo: Conocer la frecuencia de presentación, las características clínicas y la correlación clinicopatológica de los nevus melanocíticos en distintos grupos de edad. Material y Métodos: Realizamos el estudio en 2247 lesiones melanocíticas correspondientes a 461 pacientes. En 90 individuos pudimos realizar un protocolo clínico en el que se recogían los datos sobre tipo de piel, color de ojos y pelo, hábitos de exposición solar, localización, número y características de los nevus melanocíticos y antecedentes familiares. De las 2247 lesiones estudiadas clínicamente, 593 fueron extirpadas y estudiadas histopatológicamnete. Resultados: Estudiamos 461 pacientes (68,1 por ciento mujeres y 31,9 por ciento hombres), de edades comprendidas entre 4 y 97 años (Media= 33,02 años). El tipo medio de piel fue el III (48,1 por ciento). Respecto a los hábitos de exposición solar, el 62,4 por ciento había sufrido alguna vez quemaduras solares, y un 20 por ciento se quemaba a menudo. La localización de los nevus más frecuente fue el tronco (34,5 por ciento de los nevus), la cabeza y el cuello (16,3 por ciento). El número total de nevus fue de 2247 (media=21.1 nevus y SD=20,3 nevus). Encontramos una asociación significativa del número de nevus con el sexo masculino (p<0.003), (media=24,2 nevus) y entre el tipo de piel (fototipo claro) y el número de nevus melanocíticos (p<0,05). Según la clasificación ABCD para los nevus atípicos, el 23,8 por ciento de los individuos estudiados presentaban un nevus con alguna de estas características. El estudio anatomopatológico se realizó sobre 593 nevus: nevus intradérmicos (30,5 por ciento), compuestos (26,8 por ciento) y congénitos, tanto intradérmicos como compuestos (22,6 por ciento). La correlación con la edad fue: nevus de unión en edades inferiores a 15 años, nevus compuestos entre 15 y 24 años, nevus intradérmicos a partir de los 45 (p<0.001), los nevus congénitos se diagnosticaban preferentemente en hombres menores de 24 años. Desde el punto de vista microscópico, el 11 por ciento de las lesiones eran nevus displásicos, asociándose significativamente a hombres entre 15 y 24 años (p<0,001). La correlación clinicopatológica fue del 100 por ciento en aquellos nevus que reunían todas las características clínicas atípicas (ABCD) e histopatológicas del displásico. Conclusión: El fototipo cutáneo representa la sensibilidad a la radiación solar: los tipos I y II presentan mayor número de nevus melanocítico y y riesgo de padecer melanoma, lo cual debe condicionar el comportamiento frente a la exposición solar desde la infancia. Además, los programas de prevención deberían combinar una alta sensibilidad, para extirpar los melanomas en un estadio inicial y una especificidad elevada, para extirpar el menor número de nevus comunes (AU)

A role of SH-compounds in maturation of melanosomes in cells cultured from nevus Ota.

A role of SH-compounds such as cysteine and glutathione in melanogenesis in dermal melanocytes cultured from Ota's nevus tissue was demonstrated in relation to another substrate, dihydroxyphenylalanine (DOPA). Chemical analysis of eumelanin and pheomelanin was performed in addition to the conventional electron microscopic observation. Supplements of the culture medium with each of these compounds separately for two weeks gave rise to the formation of pre-pheomelanosomes and secondary lysosomes or myelinosome-like inclusions. When DOPA and glutathione were added to the medium together, the maturation of melanosomes was promoted. This was proven by the increase in electron-density of pre-melanosomes observed as well as by the content of pheomelanin and eumelanin. However, mature melanosomes were not formed when each of these chemicals was added to the medium individually for the same periods. The melanosome maturation seemed to occur via a process involving secondary lysosomes or myelinosomes, in which more electron-dense particles accumulated in the presence of both reagents. The pheomelanosomal process was also observed, but typical eumelanosome-related processes were not observed in this culture system.

Melanosomal defects in melanocytes from mice lacking expression of the pink-eyed dilution gene: correction by culture in the presence of excess tyrosine.

Mutations in the murine pink-eyed dilution (p) gene, or its human homologue P, result in oculocutaneous albinism. Melanocytes cultured from mice lacking p gene expression exhibit defective melanogenesis, but following culture in the presence of high concentrations of L-tyrosine, increased melanin deposition is observed. Electron microscopy and image analysis demonstrated that untreated p mutant melanocytes exhibited small melanosomes, largely of stages I-II. Following tyrosine treatment, increased proportions of stage III-IV melanosomes, almost normal in size, were observed. Levels of tyrosinase protein and to a lesser extent of tyrosinase-related protein-1 (TRP-1) were subnormal but rose dramatically following stimulation by tyrosine. Levels of TRP-2 and Pmel17/silver gene product were not altered, nor were the levels of mRNA for tyrosinase, TRP-1, TRP-2, or the Pmel17/silver gene product. As expected, the 110-kDa product of the p gene was absent from both stimulated and unstimulated p mutant cells. In a melanoblast line derived from the same mice, excess tyrosine failed to stimulate visible melanogenesis or increase the low levels of tyrosinase. The melanosomes in these cells were smaller still than those in the mutant melanocytes even when cultured in the presence of excess tyrosine. Thus, absence of the p gene product affects melanosomal structure and protein composition at the posttranscriptional level. These defects are correctable at least in part by supplementation with L-tyrosine.

Melanosome formation in cultured amelanotic melanophores of Rana brevipoda by a frog tyrosinase cDNA Transfection.

A cDNA encoding tyrosinase of Rana nigromaculata was introduced into cultured, tyrosinase-negative amelanotic melanophores of R. brevipoda by a calcium phosphate precipitation method. Within a few days following transfection, dark pigmentation became visible in a small number of cells. Light microscopic observation revealed that the morphology of these transformed cells was comparable to that of normal melanophores in culture, and their proliferative activity was lower than that of amelanotic cells. Ultrastructural examination verified that amelanotic melanophores possessed a relatively small number of premelanosomes while the transformants contained numerous melanosomes at various stages of pigment deposition. The result indicated that tyrosinase cDNA of R. nigromaculata was expressed in amelanotic melanophores of R. brevipoda including the maturation of premelanosomes. It was also suggested that the expression of transfected tyrosinase cDNA had promoted differentiation of the amelanotic cells into fully developed melanophores.

Effect of melanocyte stimulating hormone on human cultured choroidal melanocytes, uveal melanoma cells, and retinal epithelial cells.

PURPOSE: To establish methodology for the culture of human choroidal melanocytes to compare their responsiveness to melanocyte stimulating hormone (MSH) with that of their transformed melanoma counterparts and with that of the retinal epithelial cell. METHODS: Choroidal melanocytes from the choroid of eyes enucleated for the presence of malignant melanoma were cultured in MCDB 153 medium supplemented with insulin, transferrin, hydrocortisone, glutamine, nystatin, vitamin E, phorbol myristate acetate, bovine hypothalamic extract, cholera toxin, and chelexed fetal calf serum. RESULTS: High yields of pure spindle-shaped bipolar melanocytes were obtained with a doubling time of 3 to 4 days in nine consecutive eyes. Cells continued to divide after 4 months in culture. In contrast, uveal malignant melanoma cells grew rapidly in a relatively simple medium of Ham's F12:DMEM (1:1) supplemented with fetal calf serum, insulin, transferrin and glutamine. This medium was unable to support choroidal melanocytes. Choroidal melanocytes were DOPA-positive with appreciable tyrosinase activity that significantly increased with treatment with MSH. MSH also significantly altered the size, local density, and distribution of primary and mature melanosomes of ocular melanocytes. In contrast, uveal melanoma cells had a low level of tyrosinase activity and failed to respond to MSH with either an increase in enzyme activity or melanosome size. Retinal epithelial cells failed to show significant tyrosinase activity under the conditions studied or any increase in melanosome size in response to MSH. CONCLUSION: Ocular melanocytes show evidence of regulation by MSH and a range of mitogenic stimuli unlike the transformed melanoma cells, implying a loss of regulatory control in the latter.

Relación entre desplazamiento de melanosomas y cambios de color en lagartijas machos: liolaemus tenuis tenuis: un estudio ultraestructural / Relationship between melanosomes displacement and color change in male lizards: liolaemus tenuis tenuis: an ultrastructural study

La mayor parte de los organismos multicelulares se caracterizan por un patrón de color distintivo y, a menudo, particular. Uno de los efectores relacionados con el proceso corresponde a células especiales, los cromatóforos. En este trabajo se demuestra mediante técnicas de microscopía electrónica, que en el dermis de reptiles sometidos a 35§C por 12 min, se produce una fuerte concentración de melanosomas en torno al núcleo de los melanóforos. Por el contrario, a 0§ los melanóforos presentan expansiones citoplasmáticas que contienen numerosos melanosomas. Asociados a los melanosomas en dispersión, se observan microtúbulos. Esta característica permite plantear la posible existencia de algún tipo de relación entre microtúbulos y movimiento de melanosomas en este reptil, cuando es sometido a bajas temperaturas, momento en que se oscurecen. Se destacan también, gruesos manojos tridimensionales de fibras colágenas asociadas a fibroblastos, estructuras, que para algunos autores, representarían un primordio de esclerificación dérmica, fenómeno que predecería a la fase fibrilar de la osificación dérmica o directa. Otro tipo de célula, los iridóforos, contienen placas reflectantes que reflejan la luz en direcciones determinadas

Cutaneous pigmented melanocytic tumor in a mouse treated with psoralen plus ultraviolet A radiation.

A pigmented melanocytic tumor was induced on the treated site of a C3H/HeN female mouse given repeated topical applications of 8-methoxy-psoralen and subsequent exposures to ultraviolet A radiation (PUVA) over a 7-month period. The tumor invaded the subcutaneous tissue and muscle but produced no distant metastases. To our knowledge, this is the first report suggesting a direct relationship between PUVA treatment and the induction of cutaneous melanocytic tumor in mice.

Acquired pigmented macules in human piebald lesions. Ultrastructure of melanocytes in hypomelanotic skin.

Two cases of piebaldism are reported. The first patient was a 9-month-old girl with inborn hypopigmented areas on the frontal region of the scalp and both knees. There were no melanocytes in the lesions. In the second case, we observed the patient from 2 months of age for a period of 9 years. Many hyperpigmented spots appeared on the hypomelanotic areas on the frontal region of the scalp, abdomen and both knees. Electron-microscopic examinations of the hypomelanotic skin disclosed an area with regularly distributed melanocytes as well as an area with no melanocytes. Most of the melanosomes were ellipsoidal and lamellar. They were in stage II to III, which signified delayed pigmentation. Hyperpigmented spots were slightly enlarged following PUVA treatment.

Photochemotherapy-induced Lentigines on a Vitiliginous Patch Electron Microscopic Observations

Patients with vitiligo seem be less prone to the development of lentigines as a side effect of long-term photochemotherapy than do psoriatics. An 8-year-old boy who had a vitiliginous patch on his left thigh, had been receiving photochemotherapy since he was 2 years old. At the age of 3, multiple star-shaped brownish macules developed at the site of treatment. Photochemotherapy was continued until the patient was 6 year old, at which time no improvement in the vitiligo was seen, so photochemotherapy was discontinued. Now 2 years after treatment the lentigines still persist. On electron microscopic examination, the melanocytes showed two patterns of cell death: coagulative necrosis and apotosis together with atypical cytoplasmic and melanosomal alterations.

Establishment of proliferative, pure cultures of pigmented chicken melanocytes from neural tubes.

In order to obtain pure cultures of chicken melanocytes, neural tubes were excised from 22-somite stage embryos and placed in culture dishes to allow melanoblasts to migrate out and proliferate. The growth of contaminating cells was inhibited by maintaining the primary cultures in low-calcium and low-magnesium medium supplemented with 32 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). Subsequently the pure cultures of melanocytes were maintained in Ham's F-10 medium supplemented with TPA. The population doubling time was approximately 12 h. The cell density at confluency in medium containing 32 nM TPA, 80 nM TPA, or 32 nM TPA plus 1 nM cholera toxin was 3.4, 5.6, or 8.3 X 10(4) cells/cm2, respectively. The melanocytes were highly pigmented and had tyrosinase activities ranging from 0.7-5.0 mU/mg protein.

Morphologic alterations of epidermal melanocytes and melanosomes in PUVA lentigines: a comparative ultrastructural investigation of lentigines induced by PUVA and sunlight.

Ultrastructural studies were conducted in order to determine morphologic and functional differences in melanocytes and melanosomes in PUVA lentigines and solar lentigines, and light-protected buttock skin. Compared to melanocytes in solar lentigines from 7 subjects and light-protected buttock skin from 5 subjects (none of these subjects had received UV radiation therapy), melanocytes in PUVA lentigines from 6 subjects generally had longer and more numerous dendrites, and showed more active melanogenesis. Basal keratinocytes in PUVA lentigines had a significantly increased frequency of large, single melanosomes, and revealed significantly larger individual melanosomes within compound melanosomes. Other findings in some PUVA lentigines included the close apposition of Langerhans cells to melanocytes, and atypical nuclear, cytoplasmic and melanosomal alterations, including melanosomal pleomorphism and melanin macroglobules. The presence of relatively large and predominantly single melanosomes in basal keratinocytes of PUVA lentigines suggests more active melanogenesis and/or an irreversible somatic alteration. It will be important to determine the clinical course and ultrastructural findings of PUVA lentigines that persist long after PUVA is discontinued.