Role of In Vivo Reflectance Confocal Microscopy in Determining Stability in Vitiligo: A Preliminary Study

Background:

Vitiligo is an acquired pigmentary disorder. In vivo reflectance confocal microscopy (RCM) reproducible imaging technique has already been reported to be useful in the diagnosis of other skin diseases.

Objective:

To define RCM features of vitiligo on different clinical stages.

Materials and Methods:

A total of 125 patients with a clinical diagnosis of vitiligo were included in this study. After informed consent, lesional skins of those vitiligo patients were characterized by using RCM. Five patients with inflammatory cell infiltration observed at the edge of skin lesions and another 5 patients without inflammatory cell infiltration were selected. Biopsies were performed at same sites of the RCM examination areas for histological and immune-histological analysis.

Results:

In the active stage of vitiligo, the RCM examination revealed that the bright dermal papillary rings presented at the dermoepidermal junction level in normal skin lost their integrity or totally disappeared, border between vitiligo lesion and normal skin became unclear, and highly refractile cells that referred to infiltrated inflammatory cells could be seen within the papillary dermis at the edge of the lesions. In the stable stage of vitiligo, the RCM showed a complete loss of melanin in lesional skin and a clear border between lesional and normal skin.

Conclusion:

A simple clinical examination with RCM may reliably and efficiently allow evaluation of the stability status of vitiligo lesions.     

In vivo reflectance confocal microscopy imaging of vitiligo,nevus depigmentosus and nevus anemicus

Background/purpose: Hypopigmentary skin disorders such as vitiligo, nevus depigmentosus and nevus anemicus are common diseases in clinic. The lesions of these diseases could be similar to some extent, although each of them has its own characteristic clinical appearance and histological features. Clinically, the atypical lesions are often difficult to be differentiated. In vivo reflectance confocal microscopy (RCM) is a non‐invasive, repetitive imaging tool that provides real‐time images at a nearly cellular histological resolution. Our aim was to investigate the RCM features of vitiligo, nevus depigmentosus and nevus anemicus. Subjects and Methods: A total of 135 patients with a clinical diagnosis of the aforementioned diseases were included in this study. The RCM images from depigmented skin, border of the white macules, adjacent normal‐appearing skin and distant normal skin for all patients at the dermo‐epidermal junction (DEJ) level were investigated. Results: In the active phase of vitiligo (AVP), the RCM demonstrated a complete loss of melanin in lesional skin in eight (53; 15.1%) patients. In 45 patients (53; 84.9%) of the AVP, part of the bright dermal papillary rings normally seen at the DEJ level disappeared or part of the rings lost their integrity and the content of melanin decreased obviously. In 20 patients (53; 37.7%) of the AVP, highly refractile inflammatory cells could be seen within the papillary dermis in the lesional and adjacent normal‐appearing skin, which may indicate the lesion progresses. In addition, part of the dermal papillary rings showed lack of integrity or their brightness decreased in adjacent normal‐appearing skin in all the patients of the AVP. It is important to know that the RCM demonstrated an ill‐defined border. In the stable phase of vitiligo (SPV), the RCM demonstrates a complete loss of melanin in lesional skin and a clear border in 31 (41; 75.6%) patients; the content of melanin and dermal papillary rings in adjacent normal‐appearing skin show no changes. In 10 (41; 24.4%) patients, the dendritic and highly refractile melanocytes arose in the recovery phase of vitiligo, which may indicate the repigmentation of vitiligo. There are three kinds of repigmentation patterns under RCM: marginal, perifollicular and diffuse. Distant normal skin showed no difference from controls in both the active and the SPV. In all the patients with nevus depigmentosus, the content of melanin decreases obviously but the dermal papillary rings are intact. The dermal papillary rings show no differences between lesional skin and adjacent normal‐appearing skin of nevus anemicus. Conclusion: Considering our results, RCM may be useful to non‐invasively discriminate vitiligo, nevus depigmentosus and nevus anemicus in vivo.     

Aberrant expression of complement regulatory proteins, membrane cofactor protein and decay accelerating factor, in the involved epidermis of patients with vitiligo

BACKGROUND: Vitiligo is a pigmentary disorder of the skin characterized by the complete absence of melanocytes from the lesion. Complement-activating antimelanocyte antibodies have been implicated in vitiligo pathogenesis. As membrane regulators of complement activation, membrane cofactor protein, decay accelerating factor and CD59 protect cells from elimination by autologous complement, their absence or downregulation on melanocytes may be associated with autoantibody and complement-mediated melanocyte destruction in vitiligo. OBJECTIVES: We studied the expression of these regulatory proteins in non-lesional, perilesional and lesional vitiligo skin compared with those of control specimens. METHODS: We used immunohistochemistry to study the expression of the regulatory proteins, and flow cytometric analysis of cultured melanocytes to investigate possible constitutive changes in the expression levels of these molecules. We also investigated whether melanocytes can influence keratinocyte susceptibility to autologous complement by regulating keratinocytic decay accelerating factor and membrane cofactor protein expression levels. RESULTS: Immunohistochemical data showed that expression of membrane cofactor protein and decay accelerating factor in whole epidermis was lower in lesional and perilesional skin in comparison with non-lesional skin. The reduced in situ expression appeared to be specific to vitiligo. However, coculture experiments indicated that melanocytes do not influence keratinocyte susceptibility to autologous complement. Further, flow cytometric analysis of cultured melanocytes convincingly demonstrated that non-lesional vitiligo and control melanocytes have comparable decay accelerating factor, membrane cofactor protein and CD59 expression levels. CONCLUSIONS: It is therefore concluded that there is no constitutive melanocyte defect per se that could be related to the in vivo expression of these molecules in vitiligo. Nevertheless, the present data suggest that both keratinocytes and melanocytes in the involved vitiliginous whole epidermis express lower levels of decay accelerating factor and membrane cofactor protein compared with controls that could render them more vulnerable to autologous complement attack.     

Expression and modulation of apoptosis regulatory molecules in human melanocytes: significance in vitiligo

Although the aetiology of the hypopigmentary disorder vitiligo is ill understood, it is clear that pigment producing cells are absent from vitiliginous lesional skin. The present study was designed to investigate the possible role of melanocyte-expressed apoptosis regulatory molecules in melanocyte disappearance. Flow cytometric evaluation of p53, p21, Bcl-2 and Bax revealed no differences in in vitro expression levels between normal control and non-lesional melanocytes. Moreover, no in situ immunohistological differences were observed in melanocytes present in control, non-lesional and perilesional skin. However, an enhanced number of p53+ nuclei, in the absence of detectable p21 expression, was detected in involved areas. The observed p53 expression pattern did not involve melanocytes and could be the result of ultraviolet (UV) A irradiation. Further, we showed that UVB is capable of modulating melanocyte-expressed apoptosis regulatory molecules. Consequently, a lethal dose of UVB was given to two groups of cultured normal control and non-lesional melanocytes. No significant differences were found when comparing the percentages and kinetics of UVB-induced apoptosis in these groups. In conclusion, our results indicate that the relative apoptosis susceptibility of melanocytes in vitiligo is comparable with that of normal control cells. It is therefore unlikely that vitiligo is causally related to dysregulation of apoptosis regulatory molecules.     

Investigation by in vivo reflectance confocal microscopy: melanocytes at the edges of solar lentigines

In vivo reflectance confocal microscopy (RCM) provides high-resolution, real-time optical sections of the skin in a non-invasive manner, allowing visualization of the skin in its native state. Highly reflective skin components including melanin, collagen and keratin appear bright (white) in RCM images. RCM examination of solar lentigines is known to show features that correlate well with histologic findings such as supranuclear melanin caps, but there are a limited number of reports on melanocyte dendrites. In this study, we utilized RCM to investigate the melanocyte dendricity and distribution within solar lentigines. Seventeen healthy Japanese females who had fairly large solar lentigines on their faces were recruited to join our clinical study, and we examined them by using RCM on their non-lesional areas, and the inside and the outer rim of the lesional areas. As a result, we discovered that dendritic melanocytes were rarely seen in the center of a solar lentigo (SL), but were seen at a very high frequency in the outer rim of a SL. The results suggest that the melanocytes are more active at the edge of a SL, produce more melanin, and often spread their dendrites widely in a horizontal direction. The findings in this report might shed light on the dynamic pathomechanisms of solar lentigines in vivo.     

Expressional changes in the intracellular melanogenesis pathways and their possible role in the pathogenesis of vitiligo

BACKGROUND: Main pathway in human melanocytes through which signal from the melanocortin system reaches the melanogenesis enzymes is cAMP/PKA pathway and it is modulated by Wnt and MAPK pathways. In our previous study we established significant increase of melanocortin receptor expression in unaffected skin of vitiligo patients compared to healthy subjects. OBJECTIVE: The aim of this study was to assess the gene expression profile of the intracellular signalling pathways linking melanocortin system with enzymes involved in melanogenesis. METHODS: Using QRT-PCR method, mRNA expression levels of eight genes related to signal transduction from the melanocortin system to melanogenesis enzymes was measured in lesional and non-lesional skin of vitiligo patients and in the skin of healthy control subjects. Following genes were analyzed in the study: MITF, CREB1, p38, USF1, PIK3CB (PI3K), RPS6KB1, LEF1 and BCL2. RESULTS: The mRNA levels of MITF, LEF1, p38, PIK3CB and RPS6KB1 were decreased in lesional skin of vitiligo patients compared to skin of healthy control subjects. We also found increased expression of USF1 and BCL2 in non-lesional skin of vitiligo patients compared to skin of healthy control subjects. mRNA levels of MITF and BCL2 were decreased in lesional skin of vitiligo patients compared to non-lesional skin of vitiligo patients. CONCLUSIONS: Present study indicates increased expression of the genes of the intracellular melanogenesis pathway in the non-lesional skin of vitiligo patients. This finding suggests activation of melanogenesis pathway in the non-lesional skin of vitiligo.     

BIOLOGIC CHARACTERISTICS OF CULTURED HUMAN VITILIGO MELANOCYTES

Background. Vitiligo is a pigmentary disorder of unknown cause characterized by depigmented patches due to destruction of melanocytes. Recently, the inherent cellular defect theory has been discussed. To investigate the biologic characteristics of cultured melanocytes from normal and vitiligo subjects, this study had the purpose to examine the functional and ultrastructural characteristics of these melanocytes and to observe the morphologic and functional changes of melanocytes in response to ultraviolet B irradiation. Methods. Melanocytes were isolated and cultured from foreskin and arm skin of normal and vitiligo subjects. The DNA synthesis, tyrosinase activity assay, transmission and scanning electron microscopic examination, and the effects of ultraviolet B(uvB)-irradiation on cultured melanocytes were studied. Results. Vitiligo melanocytes showed no significant differences in DNA synthesis and tyrosinase activity compared with normal melanocytes, but the vitiligo melanocytes contained dilated and/or circular rough endoplasmic reticulum (RER) on transmission electron microscopic examination. Exposure of the cultured melanocytes to UVB resulted in increased protein synthesis and tyrosinase activity. Morphologic alterations and changes in DNA synthesis were also noted. Compared with normal melanocytes, the responses of vitiligo melanocyte to UVB showed no significant difference. Conclusions. Normal and vitiligo melanocytes showed similar biologic characteristics except in the changes of RERS in the vitiligo melanocytes. The ultrastructural aberrations in vitiligo subjects do not seem to be directly related to the biologic characteristics and the responses to UVB irradiation in vitiligo melanocytes.     

几种色素减退性皮肤病的共聚焦激光扫描显微镜图像特点

目的 观察白癜风、无色素痣、进行性斑状色素减少症、贫血痣的活体共聚焦激光扫描显微镜（CLSM）特征。方法 用CLSM观察同一层面（基底层真表皮交界处）皮损处、交界处及白斑周边正常皮肤的镜下特征。结果 进展期白癜风白斑区部分区域色素完全缺失,部分区域可见残存色素环,残存之色素环结构欠完整且色素含量降低;交界处界限模糊;白斑周边正常皮肤可见部分色素环失去完整性。稳定期白癜风白斑处色素完全消失;交界处界限清晰;白斑周边正常皮肤色素环完整,折光明亮;恢复期可见到树突状、折光明亮的黑素细胞。无色素痣和进行性斑状色素减少症的CLSM表现相似：白斑处色素环结构完整,色素含量降低,折光减弱。贫血痣白斑处色素环结构和色素含量与周边正常皮肤无明显差异。结论 结合临床表现,CLSM可以作为鉴别诊断白癜风、无色素痣、进行性斑状色素减少症、贫血痣的一种辅助方法。     

Gene expression analysis of melanocortin system in vitiligo

BACKGROUND: The melanocortin system in the skin coordinates pigmentation and immune response and could be implicated in the pathogenesis of vitiligo. OBJECTIVES: We aimed to analyze changes in expression of genes involved in skin pigmentation (melanocortin system and enzymes involved in melanin synthesis). METHODS: With quantitative RT-PCR we measured the mRNA expression levels of eight genes from the melanocortin system and two enzymes involved in melanogenesis. RNA was extracted from both lesional and non-lesional skin of vitiligo patients and in non-sun-exposed skin of healthy subjects. RESULTS: POMC (proopiomelanocortin) expression was lower in lesional skin compared to non-lesional skin. Expression of melanocortin receptors was increased in unaffected skin of vitiligo patients compared to healthy subjects and decreased in lesional skin compared to uninvolved skin of vitiligo patients, the differences were statistically significant in the cases of MC1R (melanocortin receptor 1) and MC4R (melanocortin receptor 4). TRP1 and DCT genes were down-regulated in lesional skin compared to non-lesional vitiligo skin or skin of healthy controls and up-regulated in uninvolved vitiligo skin compared to healthy control samples. In non-lesional skin, POMC expression was not elevated, possibly indicating that systemic influences are involved in up-regulation of MC receptor genes. Decreased expression of the analyzed genes in the lesional skin is not surprising, but statistically significant increased expression of studied genes in non-lesional skin from vitiligo patients is not described previously. CONCLUSION: In our mind, up-regulation of melanocortin system in non-lesional skin could be systemic compensation to restore normal pigmentation in lesions.     

UVB 311 nm tolerance of vitiligo skin increases with skin photo type

It is assumed that skin is protected against sunburn by melanin. In patients with vitiligo, there are white patches in the normal pigmented skin. We noticed that there is a difference in burning capacity of these white patches between people with different skin types. With UVB 311 nm lamps, we irradiated both lesional and non-lesional skin with increasing doses in 33 patients with vitiligo, divided into 5 groups according to skin type (II-VI). Twenty-four hours later we assessed the minimal erythema dose and found a correlation between skin type and UV sensitivity in both lesional skin and normal skin. We suggest that there must be a protection mechanism, other than that offered by melanin pigmentation. The antioxidant status may play a role in this phenomenon.     

The expression of the c-kit receptor by epidermal melanocytes may be reduced in vitiligo

Summary The proto-oncogene c-kit encodes the transmembrane tyrosine kinase receptor that has a role in the growth regulation of various cell types including melanocytes. In the present study we have examined the expression of the c-kit protein in the skin of seven patients with vitiligo. Melanocytes positive for c-kit protein were observed in the basal layer in non-lesional skin and the mean number of 25.8 ± 5·2 (per 200 basai ceils) compared with that of 21·8 ± 3·5 from six control subjects. In perilesional skin there was a reduction in the numbers of c-kit positive melanocytes (6·7±2·6) and this was especially noticeable in six of the seven patients. Such a reduction was less obvious following staining with MEL-5 and in only two subjects were the numbers of melanocytes below the normal range. This suggests that the reduction in c-kit staining was the result of decreased expression of the protein rather than a loss of melanocytes. No melanocytes. positive for c-kit protein, or after staining with MEL-5. were identified in lesional skin although isolated tyrosinase-positive melanocytes were seen in one subject. There was no apparent change in the numbers of mast ceils expressing c-kit protein and the intensity of staining in the dermis even in lesional skin was similar to that in the controls. These results demonstrate that c-kit protein is present on melanocytes in adult human skin and that in perilesional skin of some vitiligo patients there is a reduction in the numbers of melanocytes expressing this receptor. Whether this may contribute to the defective melanocyte growth and/or survival that occurs in vitiligo or whether it is a consequence of melanocyte damage remains to be seen.     

Probable Mechanisms of Loss of Merkel Cells in Completely Depigmented Skin of Stable Vitiligo

Vitiligo is frequently associated with segmental involvement. It spares paralyzed limbs in transverse myelitis. There is spontaneous repigmentation of vitiligo lesional skin (VL) in diabetic neuropathy. Increased neuropeptide in VL and adjacent normal skin of vitiligo (ANS) along with thickened nerve fibers showing ultrastructural abnormalities all indicate a neural pathogenesis (1). Loss of Merkel cells has been observed in early vitiligo lesional skin (2). Recently, catecholamines have been found to play a major role in initiating the cascade of events leading to loss of melanocytes (1, 3, 4). To investigate the presence of Merkel cells in the completely depigmented skin of stable vitiligo (SV), a study was undertaken using TROMA 1, a monoclonal antibody specific for adult Merkel cells. No TROMA 1-positive cells were observed in SV, whereas normal numbers of these cells were seen in ANS. This new finding further supports the hypothesis of neural involvement in vitiligo.     

难治部位白癜风的治疗进展

白癜风是一种与黑素细胞缺失有关的色素脱失性皮肤病,易诊难治,尤其对难治部位,诸如嘴唇、生殖器、头皮、眼睑、肘膝部及四肢、掌跖足底等疗效更差.就近年来对难治部位的有效疗法,如药物、外科移植术、光疗及联合疗法等进行概述.根据难治部位的临床特点,选用合理的治疗方案,以提高难治部位的疗效,指导临床治疗.     

难治部位白癜风的治疗进展

白癜风是一种与黑素细胞缺失有关的色素脱失性皮肤病,易诊难治,尤其对难治部位,诸如嘴唇、生殖器、头皮、眼睑、肘膝部及四肢、掌跖足底等疗效更差.就近年来对难治部位的有效疗法,如药物、外科移植术、光疗及联合疗法等进行概述.根据难治部位的临床特点,选用合理的治疗方案,以提高难治部位的疗效,指导临床治疗.     

难治部位白癜风的治疗进展

白癜风是一种与黑素细胞缺失有关的色素脱失性皮肤病,易诊难治,尤其对难治部位,诸如嘴唇、生殖器、头皮、眼睑、肘膝部及四肢、掌跖足底等疗效更差.就近年来对难治部位的有效疗法,如药物、外科移植术、光疗及联合疗法等进行概述.根据难治部位的临床特点,选用合理的治疗方案,以提高难治部位的疗效,指导临床治疗.     

Vitiligo as a systemic disease

Vitiligo is an acquired depigmentary skin disorder of unknown etiology. Vitiligo is not only a disease of melanocytes of the skin. Human melanocytes are derived from the neural crest and are located on various parts of the body. The involvement of skin melanocytes is the most visible one, but a systemic involvement of melanocytes can be observed. Some types of vitiligo (nonsegmental vitiligo) may also be associated with various diseases, mainly with autoimmune pathogenesis. Vitiligo represents a spectrum of many different disorders with different etiologies and pathogeneses, causing a common phenotype: the loss of melanocytes and/or their products. This phenotype is always consistent with a systemic involvement.     

Molecular and cellular basis of depigmentation in vitiligo patients

Vitiligo is a chronic skin disease characterized by the appearance of zones of depigmentation. It is mostly described as an autoimmune disease in which the immune system destroys the melanocytes. Consistent with this origin, genetic studies have implicated genes encoding proteins mediating the immune response targeting melanocytes in the aetiology of this disease, together with proteins specific to these cells. However, the destruction of melanocytes by the immune system is neither global nor complete, because the patients do not display total depigmentation. The etiopathology of vitiligo is clearly complex and cannot be simply reduced to an autoimmune reaction directed against pigmented cells. Intrinsic changes have been observed in the melanocytes, keratinocytes and dermal cells of vitiligo patients. Identification of the molecular and cellular changes occurring in normally pigmented skin in vitiligo patients, and an understanding of these changes, is essential to improve the definition of trigger events for this disease, with a view to developing treatments with long‐term efficacy. This review focuses on the early events identified to date in the non‐lesional regions of the skin in vitiligo patients and discusses the process of repigmentation from melanocyte stem cells.     

干细胞因子及其受体在寻常型白癜风表皮中的表达

目的 探讨SCF/c-kit信号通路在白癜风发病中的作用。方法 采用免疫组化和RT-PCR法检测17例寻常型稳定期白癜风患者和10例正常对照标本中表皮角质形成细胞的干细胞因子表达及基底层黑素细胞c-kit的表达情况。结果 白癜风非皮损区干细胞因子、c-kit蛋白表达与正常对照无明显差异(P>0.05),皮损区干细胞因子表达显著高于正常对照皮肤(P<0.05),而c-kit表达显著低于正常对照皮肤(P<0.05)。白癜风非皮损区表皮干细胞因子、c-kit mRNA表达平均水平与正常对照近似(P>0.05);皮损区干细胞因子mRNA表达水平高于非皮损区及正常对照组差异有统计学意义(P<0.05);皮损区c-kit mRNA表达水平显著低于非皮损区及正常对照组(P<0.05)。结论 SCF/c-kit的异常表达可能与白癜风的发病有关。     

特殊部位白癜风的治疗进展

白癜风的治疗近年有了长足的进展,医生可以依据中国《白癜风治疗共识2009版》,根据患者的病期、型别、皮损面积、病程等诸多因素制定安全有效、合理的方案。但是白癜风的病因及发病机制目前仍未完全明确,在白癜风治疗中依然存在很多难题,如黏膜部位、肢端部位、累积毛发部位的白癜风仍然很难治疗。本文针对以上难题进行了国内外文献综述,旨在为临床医生治疗难治部位白癜风提供更多的治疗对策。     

Vitiligo management update

Vitiligo is an acquired skin disorder caused by the disappearance of pigment cells from the epidermis, and results in well defined white patches that are often symmetrically distributed. The lack of melanin pigment makes the lesional skin more sensitive to sunburn. Vitiligo can be cosmetically disfiguring and is a stigmatizing condition, leading to serious psychological problems in daily life. It occurs worldwide in about 1% of the population, mostly between the ages of 10-30 years, and as often in males as in females. The cause is unknown, but might involve genetic factors, autoimmunity, toxic metabolites, and/or a higher vulnerability of melanocytes. Some new treatments for this condition include corticosteroid + UVA treatment, UVB narrow wave band (311 nm) irradiation, and transplantation of autologous pigment cells. In widespread vitiligo, residual pigment can be removed by depigmentation agents. Sunscreens, camouflage products and good guidance may help the patient to better cope with this disease.