Epidermal changes in active vitiligo.

Light and electron-microscopic studies were performed on the vitiligo and adjacent, normal appearing skin from 97 patients with actively spreading vitiligo and 19 patients with stable vitiligo. The vitiliginous skin revealed complete loss of pigment and melanocytes. In addition to degenerative changes in melanocytes, vacuolar changes of basal cells, epidermal infiltration of lymphocytes, dermal infiltration of lymphocytes, and melanophages in the upper dermis were also seen in the normal appearing skin adjacent to vitiliginous skin. These epidermal and dermal changes are more prominent in the skin of actively spreading vitiligo than in stable vitiligo. These findings suggest that the adjacent, normal appearing skin of actively spreading vitiligo shows some characteristic histopathologic findings, especially in the epidermis, indicating that cellular immunity could be involved in the pathogenesis of vitiligo.     

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.     

白癜风患者皮损中CD4~+,CD8~+T淋巴细胞及朗格汉斯细胞的检测

目的探讨白癜风患者皮损中CD4+,CD8+T淋巴细胞、朗格汉斯细胞(LC)及黑素细胞(MC)在白癜风发病中的作用。方法采用Envision免疫组化染色法,对12例白癜风进展期患者和9例稳定期患者皮损处CD4+,CD8+T淋巴细胞、LC及MC进行检测,并与10例正常人皮肤进行对照。结果白癜风患者皮损中CD4+,CD8+T淋巴细胞、LC表达较对照组显著增多(P<0.01),而MC表达较对照组显著减少(P<0.01)。结论白癜风患者皮损中LC,CD4+,CD8+T淋巴细胞异常表达可能参与白癜风的发病,其作用模式可能是LC抗原递呈,CD4+,CD8+T淋巴细胞浸润破坏或攻击MC,从而引起白癜风患者表皮基底层的MC减少或消失,导致白癜风的发生。     

白癜风患者表皮内郎格罕细胞观察

用改良的Ｊｕｈｌｉｎ－ＳｈｅｌｌｅｙＡＴＰ酶细胞化学染色法检查了１３例白癜风（进展期）患者的白斑、白斑边缘区及对侧相应正常皮肤中郎格罕细胞情况，结果白斑部位表皮郎格罕细胞数目较对应正常皮肤表皮变化不大，而白斑边缘部皮肤郎格罕细胞数目较正常皮肤显著增多（Ｐ＜０．００５）。在白斑及白斑边缘区还可见到印格罕细胞的形态学变化：胞体变大、深染、胞突消失以及细胞积聚现象，在白斑边缘部尤其明显。本研究显示郎格罕细胞数目和形态学的变化与病情活动有关。提示郎格罕细胞在白癜风发病机制中起一定的作用。     

Role of hair follicles in the repigmentation of vitiligo.

Vitiligo is a common pigment disease that is difficult to treat. The mechanism of repigmentation is not known. We combined Dopa-Toluidine Blue complex stain, hair follicle split-Dopa stain, and hair follicle split-scanning electron microscope (SEM) to observe the changes of melanocytes in 23 normal, 24 vitiliginous, and 36 repigmented skin specimens. We found that only active (Dopa-positive) melanocytes existed in the epidermis of normal skin. There were some inactive (Dopa-negative) melanocytes in the outer root sheaths of normal hair follicles, which form the melanocyte reservoir in human skin. In the patients with vitiligo the active melanocytes in the epidermis were totally missing, whereas the inactive melanocytes in the outer root sheaths of hair follicles were not affected. Treatment stimulated the inactive melanocytes in the middle and/or lower parts of the outer root sheaths of hair follicles to divide, proliferate, and migrate upward along the surface of the outer root sheath to the nearby epidermis, where the melanocytes continued to migrate radially to form the pigmented island visible clinically in repigmented vitiligo lesions. During the migration to the epidermis, the melanocytes matured gradually from an inactive phase to an active condition. In conclusion, the existence of these inactive melanocytes provided the melanocyte sources for repigmentation of vitiligo.     

Melasma: histopathological characteristics in 56 Korean patients

BACKGROUND: Melasma is a common acquired symmetrical hypermelanosis characterized by irregular light to dark brown macules and patches on sun-exposed areas of the skin. Its histopathological characteristics are not fully understood. OBJECTIVES: To characterize the histopathological features of facial melasma skin in comparison with adjacent normal skin. METHODS: Biopsies were taken from both melasma lesional skin and adjacent perilesional normal skin in 56 Korean women with melasma. The sections were stained using haematoxylin and eosin, Fontana-Masson, diastase-resistant periodic acid-Schiff, Masson trichrome and Verhoeff-van Gieson stains, and immunostaining for melanocytes. Data on the changes in number of melanocytes and melanin contents of the epidermis were analysed by a computer-assisted image analysis program. The ultrastructure of the skin was also examined. RESULTS: The amount of melanin was significantly increased in all epidermal layers in melasma skin. The staining intensity and number of epidermal melanocytes increased in melasma lesions. Lesional skin showed more prominent solar elastosis compared with normal skin. Melanosomes increased in number and were more widely dispersed in the keratinocytes of the lesional skin. Lesional melanocytes had many more mitochondria, Golgi apparatus, rough endoplasmic reticulum and ribosomes in their cytoplasm. A dihydroxyphenylalanine reaction was apparent in the cisternae and vesicles of the trans-Golgi network in melanocytes from lesional skin. CONCLUSIONS: Melasma is characterized by epidermal hyperpigmentation, possibly caused both by an increased number of melanocytes and by an increased activity of melanogenic enzymes overlying dermal changes caused by solar radiation.     

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。     

Study of CCN3 (NOV) and DDR1 in normal melanocytes and vitiligo skin

We have hypothesised that melanocytes disappear in vitiligo because they are weakly attached to the epidermal basal membrane (melanocytorrhagy). In the epidermis, attachment of melanocytes to collagen IV is mediated through DDR1, which is under the control of CCN3. DDR1 genetic variants have been associated with vitiligo in patients of different ethnic origin. In vitro studies have shown that inhibition of CCN3 induces the detachment of melanocytes. We have studied in parallel the expression of CCN3 and DDR1 in lesional and perilesional skin of patients with vitiligo and the impact of the silencing of CCN3 and DDR1 in normal human melanocytes on their behaviour in epidermal reconstructs. Our in vivo study provides evidence of a dysregulation of the DDR1-CCN3 interaction in vitiligo skin as melanocytes remaining in perilesional skin did not express CCN3. Expression of DDR1 was decreased in lesional versus perilesional vitiligo skin in the majority of patients, and the expression of collagen IV was found decreased in all patients. Silencing of CCN3 in melanocytes induced a significant inhibition of cell adhesion to collagen IV whereas melanocytes transduced with shDDR1 still adhered well on collagen IV and did not increase melanocyte loss in epidermal reconstructs as compared with normal melanocytes. Melanocyte detachment was observed but not in all reconstructs using CCN3 silenced melanocytes. Overall, our study confirms that a downregulation of CCN3 is implicated in melanocyte adhesion in part through DDR1. In vitiligo skin, the interaction of CCN3 with other molecules, such as TGFβ and CCN2, needs to be addressed.     

Epidermal reconstructs in vitiligo: an extrinsic factor is needed to trigger the disease

The primary cellular or molecular targets accounting for melanocyte loss in vitiligo are not clearly identified. To study a putative latent epidermal defect in the epidermis of vitiligo patients, we performed in vitro studies using cultured vitiligo melanocytes and keratinocytes transplanted on to a dead de-epidermized dermis according to a variant of Pruniéras technique. Control autologous constructs were made with keratinocytes and melanocytes of normal adult epidermis and vitiligo epidermis from perilesional skin. For heterologous reconstructs we combined vitiligo-derived melanocytes or keratinocytes with their normal phototype-matched counterpart. After 15 days of culture at the air-liquid interface, epidermal reconstructs were studied macroscopically and microscopically. Immunohistochemistry was performed using antibodies to TRP-1 and NKI-beteb. All heterologous and autologous reconstruets made with melanocytes and keratinocytes from vitiligo patients had a normal histology and ultrastructure. For vitiligo melanocytes or normal melanocytes submitted to the influence of vitiligo keratinocytes, immunophenotype and function (pigment production and transfer) were similar to normal controls. So, without additional noxious stimuli, we could not discriminate between melanocytes and keratinocytes as inducers of the disease.Our data suggest that the basic abnormality in vitiligo vulgaris needs extrinsic factors to be macroscopically revealed or requires a longer period of culture to develop. Our model will allow analysis of the various pathophysiological mechanisms of vitiligo. e.g. autoantibodies or oxidative stress, at the cellular, biochemical or molecular level.     

窄谱中波紫外线围白斑照射治疗难治性白癜风的临床疗效观察

【摘要】 目的 探讨窄谱中波紫外线围白斑照射治疗难治性白癜风的临床疗效。方法 回顾2019年6月至2020年11月南京医科大学第一附属医院皮肤科治疗的126例难治性白癜风,分别采用遮盖白斑、窄谱中波紫外线照射围白斑区域皮肤和常规照射白斑治疗,每周2次,持续3个月。治疗结束后评估2组的疗效。运用倾向性评分匹配分析,按1∶1匹配。采用单因素和多因素Logistic回归分析、分层分析围白斑照射法对于难治性白癜风的临床疗效。结果 采用围白斑照射组皮损420处,常规照射组257处,倾向性评分匹配后每组各190处,匹配前后,围白斑照射组有效率（71.9%、67.9%）均高于常规照射组（31.9%、30.0%,均P < 0.05）。倾向性评分匹配后单因素Logistic回归分析显示,围白斑照射与常规照射对疗效的影响差异有统计学意义（OR = 4.9,95% CI：3.2,7.6,P < 0.001）;多因素分析显示,围白斑照射与常规照射对疗效的影响差异亦有统计学意义（OR = 12.0,95% CI：6.5,22.3,P < 0.001）。对不同毛发类型与照射方法对白斑疗效的影响进行分层分析,匹配前,毛白白斑采用常规照射187处,围白斑照射246处,毛黑白斑采用常规照射70处,围白斑照射174处;匹配后,毛白白斑两照射组各140处,毛黑白斑各50处。对于毛白白斑,匹配前后围白斑照射组的有效率（77.6%、72.8%）均好于常规照射组（19.3%、20.7%,P < 0.01）。对于毛黑白斑,匹配后两组疗效差异无统计学意义（P = 0.908）。结论 围白斑窄谱中波紫外线对于治疗难治性白癜风尤其是白斑处毛发变白的皮损疗效优于一般照射方法。     

白癜风皮损边缘黑素细胞线粒体超微结构的电镜观察

【摘要】 目的 探讨白癜风患者皮损边缘黑素细胞线粒体结构的变化。方法 在透射电镜下观察健康对照、进展期白癜风及稳定期白癜风患者皮损边缘黑素细胞形态,体视学方法测量线粒体体密度（Vv）、表面积密度（Sv）、数密度（Nv）等参数。结果 健康对照组黑素细胞可见大量黑素小体（28.57 ± 3.21）,以Ⅲ、Ⅳ期为主,线粒体规则分布在细胞内,结构正常、嵴密集,部分细胞胞质内可见自噬小体。进展期和稳定期白癜风黑素细胞内黑素小体数量减少,单位细胞内黑素小体数量分别为22 ± 6.16和17.43 ± 6.24,其中,Ⅲ期黑素小体显著减少,线粒体大小不一、形态多样,大部分线粒体明显肿胀,嵴模糊、排列紊乱甚至断裂,呈空泡状改变,未见线粒体自噬现象。线粒体形态结构定量研究显示,健康对照组Nv、Vv、Sv分别为（7.194 ± 1.434） μm－3、（4.8 ± 1.2）%、（2.42 ± 0.86） μm－1;进展期白癜风组Nv、Vv、Sv分别为（4.055 ± 0.906） μm－3、（7.4 ± 2.1）%、（3.58 ± 1.15） μm－1;稳定期白癜风组Nv、Vv、Sv分别为（5.311 ± 0.873） μm－3、（6.5 ± 1.4）%和（2.82 ± 0.94） μm－1,组间差异有统计学意义（P ＜ 0.05）。结论 白癜风皮损边缘黑素细胞线粒体受损,且进展期损伤程度大于稳定期。 【关键词】 白癜风; 黑素细胞; 线粒体; 显微镜检查,电子,透射     

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.     

Trichrome vitiligo in segmental type

Trichrome vitiligo consists of an intermediate zone of hypopigmentation located between the depigmentation center and the normal unaffected skin. Previously, trichrome vitiligo was described in non-segmental vitiligo. Here, we report two cases of trichrome vitiligo that showed a poor response to phototherapy or systemic steroid. These findings suggest that trichrome vitiligo in segmental type seems to be an active lesion resistant to medical treatment.     

308nm准分子激光对寻常型稳定期白癜风患者皮肤组织液TNF-α、bFGF水平的影响

目的:评价308 nm准分子激光对寻常型稳定期白癜风患者皮肤组织液肿瘤坏死因子(TNF-α)、碱性成纤维细胞生长因子(bFGF)水平的影响。方法:对28例寻常型稳定期白癜风患者进行负压吸疱表皮移植,先将白斑、围白斑、正常皮肤分为非激光区和激光区。在移植前,激光区接受308 nm准分子激光治疗10次;非激光区不作处理。吸疱移植时收集各区疱液,采用酶联免疫吸附法(ELISA)测定皮肤组织液中TNF-α和bFGF水平。结果:白斑、围白斑、正常皮肤的激光区分别与各自的非激光区比较,皮肤组织液的TNF-α均有不同程度的降低,而bFGF升高,差异均有统计学意义(P值均<0.05)。白斑、围白斑的非激光区和激光区分别与正常皮肤的非激光区和激光区比较,TNF-α较高,bFGF较低,差异均有统计学意义(P值均<0.05);而白斑非激光区、激光区的TNF-α、bFGF水平分别与围白斑非激光区、激光区比较,差异均无统计学意义(P值均>0.05)。结论:308 nm准分子激光能下调皮肤组织液内的TNF-α水平,提高bFGF的水平,可能是其治疗白癜风的机制之一。     

Repigmentation in vitiligo universalis: role of melanocyte density, disease duration, and melanocytic reservoir

Recent reports suggest that vitiligo lesions are not totally devoid of melanocytes. We report an interesting observation made in the vitiliginous skin of an Indian patient being treated for pemphigus vulgaris with dexamethasone cyclophosphamide pulse therapy. Our observations indicate that melanocytes are never completely absent in the depigmented epidermis and that these melanocytes can recover their functionality in vivo and in vitro under an appropriate stimulus.     

Increased monoamine oxidase A activity in the epidermis of patients with vitiligo

Human keratinocytes under in vitro conditions synthesize norepinephrine and epinephrine, whereas melanocytes lack this capacity. Keratinocytes established from lesional and nonlesional skin of patients with vitiligo synthesized four and two times more norepinephrine, respectively, than controls. Epinephrine synthesis was similar in keratinocytes from uninvolved epidermis and controls, but cells from involved skin had 6.5-fold less epinephrine than controls, indicative of low phenylehtanolamine-N-methyl transferase (PNMT) activity. Similar results were obtained in five patients with vitiligo who showed low epinephrine levels in involved epidermis. Both human keratinocytes and melanocytes expressed significant levels of monoamine oxidase A (MAO-A) activities as shown using¹⁴C-labelled 5-hydroxytryptamine as substrate and immunohistochemical staining with mouse monoclonal antibody. MAO-A activities in the total epidermis of patients with vitiligo were increased five- to ten-fold compared with skin of type-matched controls. Similar increases in MAO-A activities were also found in both keratinocytes and melanocytes established in vitro from vitiliginous epidermis. Based on these results, it can be concluded that defective catecholamine synthesis in the epidermis of patients with vitiligo leads to increased levels of norepinephrine with a concomitant increase in MAO-A activity.     

Increased monoamine oxidase A activity in the epidermis of patients with vitiligo

Human keratinocytes under in vitro conditions synthesize norepinephrine and epinephrine, whereas melanocytes lack this capacity. Keratinocytes established from lesional and nonlesional skin of patients with vitiligo synthesized four and two times more norepinephrine, respectively, than controls. Epinephrine synthesis was similar in keratinocytes from uninvolved epidermis and controls, but cells from involved skin had 6.5-fold less epinephrine than controls, indicative of low phenylehtanolamine-N-methyl transferase (PNMT) activity. Similar results were obtained in five patients with vitiligo who showed low epinephrine levels in involved epidermis. Both human keratinocytes and melanocytes expressed significant levels of monoamine oxidase A (MAO-A) activities as shown using¹⁴C-labelled 5-hydroxytryptamine as substrate and immunohistochemical staining with mouse monoclonal antibody. MAO-A activities in the total epidermis of patients with vitiligo were increased five- to ten-fold compared with skin of type-matched controls. Similar increases in MAO-A activities were also found in both keratinocytes and melanocytes established in vitro from vitiliginous epidermis. Based on these results, it can be concluded that defective catecholamine synthesis in the epidermis of patients with vitiligo leads to increased levels of norepinephrine with a concomitant increase in MAO-A activity.     

Behavior of pigment cells on lesions of the pigmented venus with vitiligo.

When vitiligo occurred on lesions of the pigmented nevus, the behavior of pigment cells in this nevus was investigated. Three cases of giant hairy nevi, seven cases of moles, three cases of Mongolian spots and eleven specimens in nine cases of halo nevi were used. Giant hairy nevi combining with vitiligo showed intensive decreases in nevus cells, particularly superficial A and B-type nevus cells. The epidermal dopa-positive melanocytes and melanin granules in the epidermis decreased, but still remained. On the other hand, moles in vitiligo showed an almost complete disappearance of epidermal dopa-positive melanocytes and melanin granules in the epidermis; nevus cells in the dermis decreased only slightly. Mongolian spots with vitiligo showed an epidermis similar to vitiligo, but the dermal melanocytes were hardly changed. Halo nevi exhibited an intensive decrease and degeneration of nevus cells and marked lymphocytic infiltration. Some of them showed disappearance of epidermal dopa-positive melanocytes and melanin granules in the epidermis. The characteristic findings of vitiliginous skin are mostly restricted to epidermis. In contrast, however, it is interesting to note that, on the lesions of nevocellular nevi with vitiligo, the dermis also exhibited some decrease and degeneration of nevus cells and lymphocytic infiltration.