Excellent color-matched repigmentation of human vitiligo can be obtained by mini-punch grafting using a machine in combination with ultraviolet therapy

A new method is reported here for treatment of vitiligo by using mini-punch grafting using a machine (mMG) in combination with ultraviolet light B (UVB) therapy. After UVB had been repeatedly irradiated to the lesional facial skin of a patient for 1 month, many mini-punch grafts were removed from the lesional skin and the same numbers of punch biopsies taken from normal scalp skin were placed into the holes of the recipient skin. Repigmentation started soon, and excellent color-matched repigmentation was observed after 12 months. Our easy and speedy mMG seems to be useful for treatment of vitiligo.     

白癜风表皮黑素细胞超微结构及小眼畸形相关转录因子转录调控研究

目的 研究白癜风黑素细胞超微结构和小眼畸形相关转录因子 （MITF）及其转录调控的酪氨酸酶相关蛋白（TRP）与白癜风临床类型与病程的相关性。方法 选择不同病程的寻常型白癜风（VV）12例和节段型白癜风（SV）8例,分别取白斑区、白斑边缘正常肤色区和远离白斑正常肤色区的表皮片,经组织学确定其表皮的完整性。透射电镜观察10例患者（VV 6例,SV 4例）不同区表皮黑素细胞的超微结构特点。对所有20例远离白斑正常肤色区的表皮片黑素细胞进行培养,应用免疫印迹方法检测 MITF及其转录调控的酪氨酸酶（TYR）、酪氨酸酶相关蛋白1（TYRP1）和酪氨酸酶相关蛋白2（TYRP2）的表达水平。结果 白癜风表皮黑素细胞超微结构病理改变：10例中7例白斑区表皮内未见黑素细胞,1例短病程和2例长病程VV分别可见少量黑素体显著减少或缺失的黑素细胞;白斑边缘正常肤色区,6例VV中,3例病程小于15个月者可见黑素细胞超微结构异常,而4例SV中仅1例异常;远离白斑正常肤色区,10例黑素细胞超微结构均正常。白癜风表皮黑素细胞MITF及其转录调控TRP的表达：VV的MITF表达下调与TYR、TYRP1、TYRP2的表达下调一致;SV存在MITF显著表达下调,而TYR、TYRP1、TYRP2几均正常表达。结论 VV和SV可能存在不同的表皮黑素细胞超微结构病理改变和MITF转录调控机制。     

体外培养节段型白癜风样无色素痣皮损黑素细胞及其临床意义

【摘要】 目的 评价负压吸疱表皮黑素细胞培养对节段型白癜风样无色素痣的辅助诊断价值。方法 收集2019年6月至2020年3月于杭州市第三人民医院皮肤科依据Coupe标准临床诊断的8例节段型白癜风样无色素痣患者,进行伍德灯、反射式共聚焦显微镜（RCM）检查,308 nm准分子激光试验,负压吸疱获取表皮黑素细胞并培养,记录结果。结果 8例患者中,6例皮损伍德灯下可见荧光,4例RCM下可见色素环完整性缺失,5例经308 nm准分子激光照射试验后未见复色反应。8例患者体外培养的皮损黑素细胞硫酸亚铁染色均阳性,经消化离心后沉淀呈黄白色,电镜下黑素细胞胞质内可见Ⅰ~ Ⅲ期黑素小体;皮损对侧同一解剖部位正常皮肤黑素细胞沉淀则呈黑色,电镜下黑素细胞胞质内可见Ⅰ~ Ⅳ期黑素小体。结论 负压吸疱表皮黑素细胞培养有助于诊断节段型白癜风样无色素痣。     

Tumour necrosis factors and several interleukins inhibit the growth and modulate the antigen expression of normal human melanocytes in vitro

In the present study the action of various cytokines as regulators of human melanocyte growth and differentiation was examined in vitro. Primary melanocyte cultures were obtained in complete medium free of 12-O-tetradecanoylphorbol-13-acetate or serum. First passage melanocytes were treated with various concentrations of recombinant tumour necrosis factor alpha and beta (rTNF-alpha, rTNF-beta), as well as with various recombinant interleukins (rIL-1a, rIL-1b, rIL-2, rIL-3, rIL-4 and rIL-6) for 6 days in complete medium and for 6 and 12 days in a mitogen-reduced medium variant. The 4-methylumbelliferyl heptanoate fluorometric microassay and Ki-67 staining were used for assessing cell proliferation, and the immunophenotype was evaluated using various monoclonal antibodies. Melanocyte proliferation in complete medium was inhibited by rTNF-alpha (–24%), rTNF-beta (–17%), rIL-1a (–21%), rIL-1b (–18%) and rIL-6 (–29%); in contrast, rIL-2, rIL-3 and rIL-4 had no antiproliferative effect. Measurements of Ki-67-positive nuclei confirmed these results. In the reduced medium variant, none of the above cytokines inhibited melanocyte proliferation. Recombinant TNF-alpha and rTNF-beta markedly reduced the expression of the pigment cell-associated antigens HMB-45 and K.1.2, and they enhanced the expression of VLA-2, ICAM-1 and HLA class I antigens and strongly induced HLA-DR. Similar changes were induced by rIL-1a, rIL-b and rIL-6, and rIL-2 decreased the expression of HLA class I antigens and of ICAM-1. In conclusion, several cytokines inhibited the growth and modulated the phenotype of melanocytes in vitro. Since these cytokines are major mediators of inflammatory processes, they may cause the pigmentary alterations seen after cutaneous inflammatory processes.Presented in part at the 23rd meeting of the European Society for Dermatological Research (ESDR). Amsterdam, 3–6 April 1992     

Cultivation of keratinocytes derived from epidermal explants of sheep skin and the roles of growth factors in the regulation of proliferation

Summary Procedures to promote the growth of primary cultures of keratinocytes derived from sheep epidermis through several passages are described. Rapid epithelial outgrowth was obtained from explants of epidermis isolated from trypsinised inguinal skin biopsy specimens. Following initiation and attachment, cells displayed the polygonal morphology typical of keratinocytes in culture and survived a number of passages before terminally differentiating and sloughing from the surface of the culture vessel. Proliferation occurred in the absence of a feeder layer and was attained in a medium supplemented with foetal bovine serum and hydrocortisone or cholera toxin. Growth was stimulated by the addition of epidermal growth factor or fibroblast growth factor (FGF) to the culture medium. The detection of basic-FGF immunoreactivity in Western immunoblats of extracts of fresh tissues suggests a role for this factor in autocrine or paracrine growth regulation of skin cell populations in vivo.     

白癜风自体表皮移植处内皮素与干细胞因子的检测

目的 探讨白癜风自体表皮移植的疗效与局部细胞因子变化的相关性。方法 对稳定期白癜风患者进行负压吸疱移植治疗,部分患者移植前白斑曾进行窄谱中波紫外线照射,共57例患者成功收集到白斑及非白斑区疱液,并完成3个月随访,判断其疗效,其中照光者17例。用ELISA方法测定皮肤组织液中内皮素-1、干细胞因子的水平。结果 白斑区与非白斑区组织液自身对照比较,移植成功患者45例白斑区皮肤组织液内皮素-1、干细胞因子浓度分别为（728.97 ± 286.12） ng/L、（329.97±114.13） ng/L,非白斑区分别为（503.16 ± 251.44） ng/L、（224.73 ± 107.91） ng/L,白斑区与非白斑区比较,t值分别为5.44、5.90,P < 0.05。12例移植失败患者白斑区干细胞因子为（309.00 ± 163.89） ng/L,非白斑区为（204.22 ± 83.25） ng/L,两组比较,t ＝ 3.03,P < 0.05;而内皮素-1两组差异无统计学意义。移植成功患者在白斑区与非白斑区内皮素-1浓度明显高于移植失败患者,分别为（507.52 ± 283.31） ng/L和（344.91 ± 156.18） ng/L,t值分别为2.39,2.70,P < 0.05,干细胞因子浓度差异则无统计学意义。紫外线照射患者白斑区内皮素-1浓度与未照光患者比较,t ＝ 1.44,P > 0.05。在移植成功者中,行紫外线照射的15例内皮素-1为（548.48 ± 230.22） ng/L,未照光组为（794.60 ± 278.72） ng/L（P < 0.05）;干细胞因子浓度差异则无统计学意义。结论 内皮素-1、干细胞因子在白癜风的色素恢复中起作用,其中内皮素-1的作用可能更为重要。     

Cytokines and growth factors influence hair growth in vitro. Possible implications for the pathogenesis and treatment of alopecia areata

Factors that influence the growth of the anagen hair follicle or initiate the switch to a catagen growth pattern have so far not been definitely determined, but there is increasing evidence that cytokines and growth factors play an important role during these processes. Recently we detected an aberrant in situ expression pattern of cytokines of the Th1 type (IFNγ, IL-2) plus IL-1β expression in untreated alopecia areata (AA), and a switch to high levels of IL-10 TGF-β1 expression after successful treatment with the contact allergen diphenylcyclopropenone (DCP). Hence the question arose as to whether cytokines are able to arrest hair growth and whether IL-10 or TGFβ1 have the capacity to antagonize this process. Using whole-organ cultures of microdissected human hair follicles we studied the effect of a panel of cytokines and growth factors on hair growth and on the gross morphology of the hair follicles in vitro. IL-2, IL-10 and IFN-γ had no effect in this regard, whereas TGFβ1 partially inhibited hair growth and EGF, TNFα and IL-1β completely abrogated it. EGF and TNFα induced the formation of a club-like hair follicle, similar to catagen morphology of the hair bulb, whereas hair follicles grown in the presence of IL-1β or TGFβ1 showed no particular morphological changes. We conclude that cytokines and growth factors are pivotal regulators of hair growth at least in vitro. IL-1 is suggested as playing an important role during the pathogenesis of AA. Possible mediators of therapeutic contact dermatitis (IL-10, TGFβ1, TNFα, PGE₂) are, at least in vitro, not able to antagonize the IL-1β-triggered hair growth inhibition. Therefore, we infer that these mediators rather ‘modulate’ the immune response in AA.     

Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin by Eisinger and Marko

Melanocytes are pigment producing cells that arise from the neural crest and migrate to the skin early in fetal development. The pigment that melanocytes synthesize, melanin, plays a critical role in protecting the skin from mutagenic ultraviolet irradiation. Melanocytes are also precursor cells for melanoma, a deadly form of skin cancer. Because melanocytes make up a minority population of cells in the epidermis they have been difficult to propagate in culture. The landmark paper by Eisinger and Marko, described below, was the first successful report of large scale propagation of pure cultures of melanocytes. This paper set the stage for an explosive growth in knowledge in the biology of human melanocytes and allowed scientists to begin dissecting the different oncogenic events involved in the transition of melanocytes to melanoma.     

Solar-simulating irradiation of the skin of human subjects in vivo produces Langerhans cell responses distinct from irradiation ex vivo and in vitro

It has been postulated that Langerhans cells (LC) provide tolerogenic signals in the local impairment of cutaneous immune functions and antigen-specific tolerance induced by UV radiation. Studies in vitro and ex vivo have indicated that UV radiation may down-regulate the expression of costimulatory molecules on LC, leading to reduced antigen-presenting function. In contrast, we recently observed an up-regulatory stage in the number of human epidermal LC with induced expression of B7 costimulatory molecules 12-24 h after solar-simulating UV radiation (SSR) in vivo. To examine the apparent discrepancy between the observed human LC responses in vitro, ex vivo and in vivo, we compared the three protocols in a parallel fashion. The intact skin as well as skin explants and epidermal cell suspensions from the same individuals were irradiated with a single erythematogenic dose of SSR. The expression of cell surface markers in the epidermal cells was analysed with flow cytometry 24 h later. The number of CD1a+/HLA-DR+ LC increased post-SSR in vivo by a factor of 2.8+/-0.4, whereas in irradiated skin explants ex vivo or in cell suspensions in vitro, reduced numbers were seen. HLA-DR expression intensities were found to have increased on DR+ and CD1a+/DR+ cells in vivo. Similarly, SSR induced B7-2 (CD86) expression in CD1a+ cells significantly in vivo (P=0.031) but reduced the expression ex vivo or in vitro. We conclude that the early up-regulatory stage of human LC number and membrane markers, recorded at 24 h after a single exposure to SSR, is exclusively an in vivo phenomenon.     

Cultivation of mesenchymal cells derived from the skin and hair follicles of the sheep: The involvement of peptide factors in growth regulation

Summary Mesenchymal components of skin and vibrissa follicles of the sheep have been introduced into culture. Outgrowths of cells were obtained from expiants of the dermal papilla, follicular capsule, dermal sheath and the reticular region of the dermis. Following trypsinization, the cells were successfully propagated as monolayers through several passages. As numbers increased, both the papilla and sheath cells displayed aggregative behaviour. Capsular and dermal fibroblasts did not aggregate but became aligned into polarized arrays, the cells appearing to exert tractional forces on each other and the surface of the culture dish. In general, cell proliferation was promoted by fetal bovine serum (FBS), epidermal growth factor (EGF) and fibroblast growth factor (FGF), although the extents of the responses varied amongst the different types. Dermal fibroblasts underwent the greatest increase in numbers in the presence of FBS. The sheath and papilla cells, by contrast, were more responsive to EGF than dermal fibroblasts, with capsular fibroblasts displaying an intermediate response. Intense EGF immunoreactivity was detected in Western immunoblots of freshly isolated capsular tissue. The presence of EGF-like activity in capsular extracts was confirmed by radioreceptor assay, suggesting a specific autocrine or paracrine function for the growth factor in the local follicular environment. Mitogenic responses to FGF were approximately equivalent in all cell types when compared with controls. The similarities in aggregative behaviour and proliferative responses displayed by the dermal sheath and papilla cells suggest that they may be members of a lineage which diverged from that giving rise to the other mesenchymal derivatives during early follicle development.