Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

Vitiligo is an acquired pigmentary disorder resulting from selective destruction of melanocytes. Emerging studies have suggested that T helper cell 17 (Th17) is potentially implicated in vitiligo development and progression. It was recently discovered that metabotropic glutamate receptor 4 (mGluR4) can modulate Th17-mediated adaptive immunity. However, the influence of mGluR4 on melanogenesis of melanocytes has yet to be elucidated. In the present study, we primarily cultured mouse bone marrow-derived dendritic cells (BMDC) and then knocked down and over-expressed mGluR4 using transfection. Transduced BMDC were co-cultured with CD4+ T cells and the expression of Th17-related cytokines were measured. The morphology and melanogenesis of B16 cells were observed after being treated with co-culture medium of CD4+ T cells and transduced BMDC. We found that mGluR4 knockdown did not affect the co-stimulatory CD80 and CD86 upregulation after lipopolysaccharide stimulation but did increase the expression of Th17-related cytokines, and further down-regulated the expression of microphthalmia-associated transcription factor (MITF) and the downstream genes, decreased melanin production, and destroyed the morphology of B16 cells. Conversely, over-expression of mGluR4 reduced the expression of CD80 and CD86, suppressed the production of Th17-related cytokines, increased the expression of MITF, and did not destroy the morphology of B16 cells. Our study confirmed that mGluR4 modulated the Th17 cell polarization and resulted in the alteration of melanogenesis and morphology of B16 cells. Collectively, these findings suggest mGluR4 might be a potent target involved in the immune pathogenesis of vitiligo.

Melanin bleaching with potassium permanganate for melanocytic lesions diagnosis in veterinary medicine / Despigmentação de melanócitos com permanganato de potássio para diagnóstico de lesões melanocíticas na medicina veterinária

The aim of this study was to microscopically re-evaluate the melanocytic lesions diagnoses established by the Animal Pathology Laboratory of the Federal University of Uberlândia, in the state of Minas Gerais, Brazil, over a period of eleven years; in addition, to perform a comparative analysis between the conventional histopathological (CH) method and the use of the melanin bleaching (MB) technique with potassium permanganate, sulfuric acid, and oxalic acid solutions. The results of the MB method presented a disagreement in 24.32% of the diagnosis previously by CH, with low agreement (61.0%) and low Kappa coefficient (0.2267). Melanoma was the most frequent lesion, more frequent in elderly and non-breed female dogs. The most frequent melanoma location was in the cutaneous tissue. The presence or absence of a pagetoid spread in cutaneous samples, distribution of melanin, pattern of cell layout, cell morphology, degree of cellular atypia, and the number of mitoses verified after MB were the most important criteria to confirm the diagnosis of malignancy or benignity of the lesions. Evaluating pathologists considered MB to be essential for the majority of diagnoses and an efficient complementary method for the diagnosis of melanocytic lesions, even in cases with a moderate degree of pigmentation.(AU)

Este estudo objetivou reavaliar microscopicamente os diagnósticos de lesões melanocíticas estabelecidos pelo setor de Patologia Animal da Universidade Federal de Uberlândia, em um período de 11 anos, e, com base nesse levantamento, realizar uma análise comparativa entre o método histopatológico convencional (HC) e o método de despigmentação de melanócitos (DM) com permanganato de potássio, ácido sulfúrico e ácido oxálico. A DM revelou discordância em 24,32% dos diagnósticos previamente estabelecidos por HC, apresentando baixa concordância (61,0%) e baixo valor de coeficiente Kappa (0,2267). A alteração mais frequente foi o melanoma, com maior ocorrência em cadelas idosas sem raça definida (SRD). A localização mais frequente dos melanomas foi cutânea. A presença ou ausência de disseminação pagetoide nos casos cutâneos, a forma de distribuição da melanina, o padrão de disposição das células, a morfologia celular, o grau de atipia celular e a quantidade de mitoses verificada após a despigmentação foram critérios de elevada importância para firmar o diagnóstico quanto à malignidade ou benignidade da lesão. A despigmentação foi considerada pelos patologistas avaliadores como essencial para o diagnóstico na maioria dos casos, o que leva a concluir que ela constitui um método complementar eficiente no diagnóstico das lesões melanocíticas, mesmo em casos com grau moderado de pigmentação.(AU)

Estudo de distribuição e morfologia dos melanócitos em pele com e sem exposição solar / Melanocytes distribution and morphology analysis in skin with and without sun exposure

INTRODUÇÃO E OBJETIVOS: Acredita-se que a exposição solar possa alterar número, distribuição e morfologia dos melanócitos na pele humana, muitas vezes dificultando a interpretação de biópsias de pele, principalmente para o diagnóstico de lesões melanocíticas iniciais e para a avaliação precisa de margens de ressecção. O objetivo deste trabalho foi avaliar os melanócitos da pele humana em área exposta e não exposta ao sol. MÉTODOS: Realizada análise histológica de 60 fragmentos de biópsias de pele obtidas do antebraço (área fotoexposta) e região glútea (área coberta) de cadáveres do Serviço de Verificação de Óbitos de Recife-PE. A estatística foi realizada com o SPSS Windows versão 12.0. RESULTADOS: Observou-se um número bastante variável de melanócitos nos fragmentos de pele, com maior concentração destes na região do antebraço (área de maior fotoexposição) (p < 0,001). Notou-se também uma distribuição irregular dos melanócitos ao longo da camada basal da epiderme, por vezes com células dispostas lado a lado. Essa confluência foi identificada com maior frequência na área fotoexposta (p = 0,035), não se observando mais que quatro melanócitos adjacentes. A atipia citológica foi encontrada em 40 por cento das amostras de pele da área fotoexposta, estando ausente na área fotoprotegida. Não se verificou em nenhuma das amostras a formação de ninhos ou a presença de disseminação pagetoide. CONCLUSÃO: Há grande variabilidade de densidade, morfologia e distribuição dos melanócitos na pele humana, principalmente em área fotoexposta. COMENTÁRIO: A presença de atipia citológica e confluência celular não devem ser usadas como critério isolado para a definição de uma lesão neoplásica.

INTRODUCTION AND OBJECTIVES: It is believed that sun exposure can change the number, distribution and morphology of melanocytes in human skin, which often hinders the interpretation of skin biopsies, mainly as to diagnosis of initial melanocytic lesions and accurate assessment of resection margins. Our objective was to evaluate melanocytes in sun-exposed and non-exposed skin. METHODS: It was conducted the histological analysis of 60 skin biopsy samples resected from cadaver forearm (sun-exposed skin) and cadaver buttock (non-exposed skin) from the Death Verification Service (Serviço de Verificação de Óbitos) of Recife, state of Pernambuco. The statistical analysis was performed with SPSS Windows version 12.0. RESULTS: There was considerable variability in melanocyte density, with a higher concentration of these cells in sun-exposed areas (p < 0.001). There was also an irregular distribution of melanocytes along the epidermal basal layer, occasionally with cells arranged side by side. This confluence was identified with a higher frequency in sun-exposed areas (p = 0.035) and did not exceed more than 4 adjacent melanocytes. Cytological atypia was found in 40 percent of sun-exposed skin samples and was absent in non-exposed areas. Neither nests of melanocytes nor pagetoid spread was observed. CONCLUSION: There is a great variability in the density, distribution and morphology of melanocytes in human skin, mostly in sun-exposed areas. COMMENT: The presence of cytological atypia and cell confluence should not be used as isolated criteria for the definition of a neoplastic lesion.

Bee venom stimulates human melanocyte proliferation, melanogenesis, dendricity and migration

Pigmentation may result from melanocyte proliferation, melanogenesis, migration or increases in dendricity. Recently, it has been reported that secreted phospholipase A2(sPLA2) known as a component of bee venom (BV), stimulates melanocyte dendricity and pigmentation. BV has been used clinically to control rheumatoid arthritis and to ameliorate pain via its anti-inflammatory and antinociceptive properties. Moreover, after treatment with BV, pigmentation around the injection sites was occasionally observed and the pigmentation lasted a few months. However, no study has been done about the effect of BV on melanocytes. Thus, in the present study, we examined the effect of BV on the proliferation, melanogenesis, dendricity and migration in normal human melanocytes and its signal transduction. BV increased the number of melanocytes dose and time dependently through PKA, ERK, and PI3K/Akt activation. The level of cAMP was also increased by BV treatment. Moreover, BV induced melanogenesis through increased tyrosinase expression. Furthermore, BV induced melanocyte dendricity and migration through PLA2activation. Overall, in this study, we demonstrated that BV may have an effect on the melanocyte proliferation, melanogenesis, dendricity and migration through complex signaling pathways in vitro, responsible for the pigmentation. Thus, our study suggests a possibility that BV may be developed as a therapeutic drug for inducing repigmentation in vitiligo skin.

Model for human skin reconstructed in vitro composed of associated dermis and epidermis

CONTEXTO E OBJETIVO: A técnica para obtenção de pele humana que apresente derme e epiderme, reconstruída a partir de células isoladas de pacientes, pode possibilitar a realização de enxertos autólogos de pele reconstruída em laboratório em pacientes com áreas doadoras escassas, além de permitir ensaios com substâncias químicas e drogas in vitro e não mais in vivo. O objetivo do trabalho é demonstrar um método de obtenção de pele humana reconstruída in vitro composta de derme e epiderme associadas. TIPO DE ESTUDO E LOCAL: Estudo experimental laboratorial realizado no Laboratório de Cultura de Células da Pele da Faculdade de Ciências Médicas da Universidade Estadual de Campinas, Campinas, São Paulo, Brasil. MÉTODOS: A partir da cultura de fibroblastos humanos, é possível obter um número suficiente de células que podem ser injetadas em uma matriz de colágeno bovino tipo I que, mantida imersa em meio de cultura específico para fibroblastos, permite a formação de uma derme humana reconstruída in vitro. Sobre essa derme, por meio de cultura de queratinócitos e melanócitos humanos, forma-se epiderme diferenciada, levando à formação de pele humana reconstruída in vitro, composta de derme e epiderme associadas. RESULTADOS: Demonstramos que é possível reproduzir pele humana reconstruída in vitro, composta de derme e epiderme associadas. Essa pele humana formada é, histologicamente, semelhante à pele humana in vivo. Na derme, identifica-se o tecido colágeno, com suas células, e a matriz extracelular organizados paralelamente à epiderme. Esta se desenvolve em várias camadas. CONCLUSÃO: É possível obter pele humana reconstruída in vitro, completamente diferenciada, composta de derme e epiderme, associadas, a partir da injeção de fibroblastos humanos em uma matriz de colágeno bovino tipo I e da cultura seqüencial de queratinócitos e melanócitose humanos sobre essa matriz contendo fibroblastos em seu interior.

Melanin containing cells of the uterine cervix and a possible histogenesis--a case report.

A 30 year old nulligravidfemale attended gynaecological OPD for investigation of primary infertility. Local examination revealed presence of a dark pigmented area in the posterior lip of the cervix. The biopsy from cervix showed, squamous metaplasia of the lining epithelium with presence of granules of melanin pigment in the basal layer. Schmorl's stain for melanin and immunohistochemical staining for S-100 and HMB-45 showed strong positivity in these cells. Melanosis of the uterine cervix is usually an incidental finding in females with uterine prolapse in their fifth and sixth decade. The origin of melanin containing cells in the uterine cervix is debatable till date. Amongst the various possibilities for the origin of these cells in the uterine cervix, neural origin is probably more acceptable than epithelial cell origin. The combined expression of melanocytic and Schwanian markers in the index case, suggest a biphasic differentiation of melanin containing cells in the uterine cervix. Although the exact histogenesis and clinical significance of these are still unknown, a long term follow-up is needed to study the nature of these lesions to look for any precursor lesion for development of malignant melanoma.

Acquired, Bilateral Nevus of Ota-like Macules (ABNOM) Associated with Ota's Nevus: Case Report

Ota's nevus is mongolian spot-like macular blue-black or gray-brown patchy pigmentation that most commonly ocurrs in areas innervated by the first and second division of the trigeminal nerve. Acquired, bilateral nevus of Ota-like macules (ABNOM) is located bilaterally on the face, appears later in life, is blue-brown or slate-gray in color. It is not accompanied by macules on the ocular and mucosal membranes. There is also debate as to whether ABNOM is part of the Ota's nevus spectrum. We report an interesting case of ABNOM associated with Ota's nevus. A 36-yr-old Korean women visited our clinic with dark bluish patch on the right cheek and right conjunctiva since birth. She also had mottled brownish macules on both forehead and both lower eyelids that have developed 3 yr ago. Skin biopsy specimens taken from the right cheek and left forehead all showed scattered, bipolar or irregular melanocytes in the dermis. We diagnosed lesion on the right cheek area as Ota's nevus and those on both forehead and both lower eyelids as ABNOM by clinical and histologic findings. This case may support the view that ABNOM is a separate entity from bilateral Ota's nevus.

Donor Specific Response of Estrogen and Progesterone on Cultured Human Melanocytes

The mechanisms of estrogen and progesterone in human cutaneous pigmentation are largely unknown. The molecular identification of estrogen receptor (ER) and progesterone receptor (PR) in the human melanocytes is of great importance to understand the mechanisms. We performed immunocytochemistry analysis and demonstrated that ER and PR were expressed in the cytoplasms and nuclei of human melanocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis confirmed the expression of ER and PR at the transcriptional level. Despite of the presence of ER and PR, the physiological and pregnant levels of estrogen and progesterone showed inconsistent effects on the proliferation and tyrosinase activity of cultured human melanocytes. These results suggest that human melanocytes express ER and PR, which have a donor-specific action in human pigmentation. Further studies are needed to elucidate the induction mechanism and functions of these receptors, and the role of estrogen and progesterone in melanocytes.

UV-dependent melanocyte plasticity--the structure-function relationship.

The UV response of marginal melanocytes in vitiliginous skin was studied using a whole skin organ culture technique. This method assesses the plasticity of melanocytes in response to UV. It is observed that there is a sequential increase in catecholoxidase production and in the volume and dendricity of the melanocytes on exposure to a single pulse of UV, reaching a peak at 3 1/2 h. From this study it is evident that the melanocyte shows a prominent structural and functional plasticity in response to UV. Implicit is the utilisation and transduction of UV energy by the melanocyte, for transcriptional and translational activity, enhancing catecholoxidase and cell structural protein production.

Control of melanosome movements in isolated skin melanophores of a catfish Clarias batrachus (Linn.).

Adrenergic and cholinergic receptors have been studied in isolated skin melanophores of a catfish Clarias batrachus. Catecholamines induced a strong aggregatory effect on the melanophores. Melanosome aggregation induced by adrenaline and noradrenaline was partially blocked by alpha adrenergic receptor blockers and a beta receptor blocker. Cholinomimetic drugs aroused a significant dispersion of melanophroes. Atropine effectively blocked the dispersal, responses of melanophores to acetylcholine and carbachol, while, hexamethonium blocked the nocotine induced dispersal responses of the melanophores.

Melanomas multiples primitivos sincronicos / Synchronous primitive multiple melanomas

Comunicamos un caso en un paciente de 46 años del sexo masculino de tres melanomas multiples primitivos cutaneos sicronicos localizados todos en el dorso.Los niveles y espesores fueron:lV y 2,77 mm,"in siru" y lll Y 0,73 mm respectivamente.No presentaba nevos displasicos ni otros precursores y/o marcadores. Resaltamos la necesidad de comunicar estos casos excepcionales para adquirir experiencia en su comportamiento biologico que algunos autores consideran diferente y estable en su incidencia a pesar del aumento global en el numero de melanomas en todo el mundo.

Tratamiento de la melanosis del tejido gingival por la técnica de mucoabrasión / Treatment of melanosis of the gingival tissue by the muco-abrassion technique

Se realizaron 54 tratamientos de eliminación melánica fisiológica gingival; 10 pacientes mostraron repigmentación clínica leve al término de un año del tratamiento. Ocho de estos individuos resultaron ser fumadores crónicos. El método mucoabrasivo es una técnica quirúrgica sencilla sin grandes riesgos; se trató la región anterosuperior e inferior debido a que es el área de la sonrisa y afecta estética y psicológicamente.

Culture of melanocytes obtained from normal and vitiligo subjects

The development of human melanocyte culture in vitro from normal adult skin and uninvolved skin of vitiligo patients is essential to investigate the mechanism of depigmentation in vitiligo and other pigmentary dermatoses. By using selective growth and long-term maintenance conditions, we selectively cultured melanocytes derived from normal foreskins and arm skins, and uninvolved foreskins and arm skins of vitiligo patients. The melanocytes of the arm skins were successfully cultured from the roofs of suction blisters. Melanocyte Growth Media (MGM) consisting of MCDB-153 formulation with basic fibroblast growth factor (bFGF), bovine pituitary extract (BPE), insulin, hydrocortisone, phorbol 12-myristate 13-acetate (PMA) and 10% human AB serum was sufficient to grow the melanocytes from normal and vitiligo donors. Melanocytes from uninvolved skin of vitiligo donors showed no different morphologic features, initial seeding capacity and population doubling time compared with those from normal skin. Melanocytes from both cell types grew without any lag period for more than 6 months (6-11 passages). Melanocytes obtained from foreskins had higher initial seeding capacity and shorter population doubling time than those obtained from arm skins using suction-blistered roofs. Our results suggest that the culture method using suction blisters may be a simple and easy way to obtain melanocytes. In addition, vitiligo melanocytes can be successfully cultured with appropriate growth conditions and may show no defective growth patterns. This culture system will be applied to investigate the basic pathophysiology of vitiligo and other various pigmentary dermatoses.

Observations on iris melanocytes implanted in the cornea

The pattern and morphology of cellular infiltration of iris melanocytes implanted into the corneal stroma were studied with a rabbit corneal model. Iris melanocytes are transformed into fibroblast-like cells with a loss of pigment granules, which may reflect the in vivo characteristics of iris melanocytes under pathologic conditions. The metaplastic chararter of iris melanocytes appears to be related to the formation of retrocorneal pigmentation and fibrous membrane.

Mancha mongólica y otras melanocitosis dérmicas / Mongolic stain and others dermal melanocytosis

Los autores comunican un caso de mancha mongólica en una niña de dos años de edad. La paciente presentó manchas azules en la espalda y en región lumbar. El examen clínico no reveló alteraciones y el diagnóstico histológico reafirmó la impresión clínica. Se efectúa una revisión de la literatura