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.     

Assessment of gene expression levels of proopiomelanocortin (POMC) and melanocortin‐1 receptor (MC1R) in vitiligo

Proopiomelanocortin (POMC) and melanocortin 1 receptor (MC1R) are regulators of melanogenesis and pigmentation. Our objective was to estimate their levels, searching for a possible role of the melanocortin system in vitiligo. This study included 40 vitiligo patients and 40 controls. Skin biopsies were taken from lesional and non‐lesional skin of patients and from the non‐sun exposed skin of controls to detect the expression of POMC and MC1R using quantitative real‐time polymerase chain reaction. Both factors were significantly lower in lesional than non‐lesional skin and controls, while they were significantly higher in non‐lesional skin than in controls. There was a statistically significant positive correlation between lesional levels of POMC and MC1R, as well as between non‐lesional levels of POMC and MC1R in the patients. On the other hand, we found a statistically significant negative correlation between the lesional and non‐lesional levels of POMC, as well as between the lesional and non‐lesional levels of MC1R in the patients. As a conclusion, the melanocortin system could play a role in the pathogenesis of vitiligo or could be affected as the end result of the disease.     

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。     

Lymphangiogenesis and angiogenesis in non-phymatous rosacea

BACKGROUND: Our study evaluated the expression of vascular endothelial growth factor (VEGF), CD31 and D2-40 in involved and uninvolved skin of 18 patients with rosacea. METHODS: Immunostaining of facial skin specimens with VEGF, CD31 and D2-40 was compared between the lesional and the non-lesional skin of patients with erythemotelangiectatic and papulopustular rosacea. RESULTS: Significantly increased dermal expression of VEGF in lesional vs. non-lesional skin (88.9% and 55.6%) was observed. Dermal expression of CD31 and D2-40 was also increased in lesional vs. non-lesional skin. There was no statistically significant difference in cutaneous expression of VEGF, CD31 and D2-40 between patients with papulopustular and erythemotelangiectatic rosacea, and no correlation was found between disease duration and immunoreactivity of VEGF, CD31or D2-40. CONCLUSIONS: Our study showed marked immunostaining of lesional skin with VEGF, CD31 and D2-40 compared with non-lesional skin. Increased immunoreactivity of D2-40 in lesional skin is interesting, given that none of the patients had facial edema. There are no published data regarding the role of lymphangiogenesis in patients with non-phymatous rosacea; thus, our study represents a new understanding of its pathogenesis. Lack of correlation between D2-40 expression and disease duration suggests that lymphatics are involved early in the pathogenesis of rosacea and do not constitute a late event.     

采用生物信息学方法筛选和分析白癜风差异表达基因

目的:通过生物信息学方法探索与白癜风进展相关的信号通路和基因。方法:从GEO数据库中下载白癜风芯片检测数据集GSE75819,利用R语言软件中limma包的LMFit和eBayes函数筛选15例印度白癜风患者皮损与非皮损组织间的差异表达基因（DEG）。通过京都基因与基因组数据库（KEGG）、基因本体论（GO）分析和基因...     

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.     

银屑病患者角质形成细胞VEGF表达的研究

目的　研究银屑病发病与血管内皮生长因子 (VEGF)的关系 ,探讨银屑病可能的发病机制。方法　①用免疫组化法检测银屑病患者皮损和非皮损处皮肤、正常健康人皮肤及体外培养的银屑病患者和正常人角质形成细胞(KC)VEGF的表达 ;②用双抗体夹心ELISA法检测银屑病患者及正常人KC培养上清液中VEGF含量。结果　①银屑病皮损处VEGF表达明显高于非皮损处和正常人皮肤 (P均 <0 .0 0 1) ,非皮损处与正常人皮肤VEGF表达也有显著性差异 (P <0 .0 5 ) ;②体外培养的银屑病皮损处和非皮损处KCVEGF表达明显高于正常人 (P均 <0 .0 0 1) ;银屑病皮损处KC与非皮损处KC相比VEGF表达也有显著性差异 (P <0 .0 5 ) )。结论　VEGF可能参与银屑病的发病。     

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.     

Over-expression of Mn-superoxide dismutase as a marker of oxidative stress in lesional skin of chronic idiopathic urticaria

We studied the involvement of oxidative stress in chronic idiopathic urticaria (CIU), assessing the activities of superoxide dismutase (SOD) and glutathione and the levels of malondialdeyde (MDA), a marker of lipid peroxidation, in samples taken from lesional skin (n = 16) and nonlesional skin (n = 11) of CIU patients. The activity of SOD and glutathione and the levels of MDA were markedly increased in lesional skin as compared with skin of healthy subjects, whereas no differences were detected between nonlesional skin of CIU patients and control samples. Immuno-dot blot assay revealed an up-regulation of Mn-SOD expression in lesional skin. These findings show that oxidative stress is crucially involved in CIU. The evidence of lipid peroxidation and compensatory increase of Mn-SOD and glutathione activities in lesional skin, in the absence of any alteration in uninvolved skin, suggests that oxidative stress is secondary to the development of inflammation.     

Activation/inactivation of the classical pathway of complement in non-lesional skin of patients with systemic lupus erythematosus

BACKGROUND: The link between the lupus band and pathogenesis remains controversial, because immunoglobulins and complement components, including the membrane attack complex, can be found in both lesional and non-lesional skin of patients with systemic lupus erythematosus (SLE). The expression of proteins that regulate complement has not been previously investigated in the skin of patients with SLE. AIM: The aim of this study is to compare the expression of protectin (CD59), which demonstrates the activation of the classical pathway of complement, in non-lesional skin obtained from patients with SLE with its expression in normal skin. This may help us explain the link between the lupus band and pathogenesis of cutaneous lupus erythematosus. METHODS: An indirect immunofluorescence technique was performed in order to provide unequivocal evidence for the activation of complement via the classical pathway and to compare the expression of CD59 in non-lesional skin from patients with SLE with normal skin samples obtained from healthy people. RESULTS: The activation of the classical pathway of complement was demonstrated in non-lesional skin in more than 90% of SLE patients investigated in this study. Staining intensity of the complement regulatory protein CD59 was markedly increased in the majority of non-lesional skin samples obtained from patients with SLE, compared to that from normals. Conclusions: CD59 is overexpressed in non-lesional skin in which complement activation has occurred. It seems likely that an increased and continuous CD59 expression may be important for maintaining the integrity of the skin BMZ during inflammatory responses involving complement activation in SLE skin. Alahlafi A, Wordsworth P, Wojnarowska F. Activation/inactivation of the classical pathway of complement in non-lesional skin of patients with systemic lupus erythematosus.     

Evaluation of skin expression profiles of patients with vitiligo treated with narrow-band UVB therapy by targeted RNA-seq

Background: Vitiligo is characterized by a lack of pigmentation in the skin. To date, there are no studies that analyze the changes in gene expression in the skin of vitiligo patients in response to narrow-band ultraviolet B (nb-UVB) phototherapy treatment.Objective: Explore the usefulness of new generation RNA sequencing in the identification of gene expression changes in the skin of vitiligo patients treated with nb-UVB phototherapy.Methods: Four skin biopsies (4mm in diameter) were collected from 45 Mexican vitiligo vulgaris patients, 2 specimens before and 2 after treatment with nb-UVB phototherapy, obtained from pigmented and non-pigmented tissue. RNA extracted from the biopsies was analyzed using the Illumina TruSeq Targeted RNA Expression protocol to study the expression of genes that participate in pathways of skin homeostasis. The 2 groups were compared using Student’s t-test and the Mann-Whitney U-test.Results: The expression analysis identified differences in 12 genes included in this study after comparing the samples obtained before and after treatment: 5 genes involved in skin pigmentation, 2 genes involved in apoptosis, 2 genes involved in cell survival, 2 genes involved in oxidative stress responses and 1 gene involved in signal transduction mechanisms (p<0.05).Study limitations: The small size of skin biopsies limits the amount of RNA obtained, the number of genes to be analyzed and the use of conventional techniques such as RT-qPCR.Conclusion: We demonstrated usefulness of new generation RNA sequencing in the identification of gene expression changes, in addition to identifying new targets in the study of vitiligo.     

Quantitative analysis of tryptase- and chymase-containing mast cells in atopic dermatitis and nummular eczema

The distribution of mast cells (MCs) containing tryptase (T) and chymase (C) was studied in the non-lesional and lesional skin of 26 patients with atopic dertnatitis (AD) and 23 patients with non-atopic nummular eczema (NE). and in the skin of eight healthy controls. T and C activities were demonstrated enzymehistochemically using 2-Gly-Pro-Arg-MNA and Suc-Val-Pro-Phe-MNA as substrates, respectively. The T- and C-containing MCs were counted separately in the epidermis, in contact with the basement membrane. In the papillary dermis and in different dermal levels (0·2 mm each). Also, the C protein was determined immunohistochemically. T-positive MCs were similarly distributed in non-lesional and lesional skin of both AD and NE. The MC number was relatively high in the upper dermis (papillary dermis and levels I and I!) of non-lesional and lesional skin of AD. In the upper dermis of non-lesional AD and NE skin and in normal skin, about 50% of T-positive MCs displayed C activity, whereas the percentage in lesional AD and NE skin was only about 30%. hi this respect, the non-lesional and lesional samples differed significantly froLu each other in both dennatoses (in AD p = 0%005, in NEP = 0·002. Students' t-test). In all samples the MC number decreased in the deeper dermal levels, although numerous T-containing MCs were still counted in the deeper dermis (dermal levels IV-VII) of lesional AD and NE skiti. differing significantly from the MC number in normal skin (In ADp = 0·005. in NE p=0·041). In the deeper dermis. the percentage of MCs containing active C was about 70% in non-lesional and lesional AD and NE. and about 90% in normal healthy skin. However, in the upper dermis of non-lesional and lesional skin of both AD and NE. about H()% of all MCs contained the C protein, which differed significantly from the value of 100% in normal skin (p<0·5). In conclusion, the increased number of T-positive MCs in the upper dermis of non-lesional and lesional AD contributes to promoting inflammation. C apparently loses its activity in the upper dermis of lesional AD and especially in NE. Thus. Ihe enzyme partially lacks its capability to suppress inflammation, such as degradation of neuropeptides and proteins. The dysregulation of these proteinases exists already in non-lesional skin of AD and NE.     

Gene expression study of IL10 family genes in vitiligo skin biopsies, peripheral blood mononuclear cells and sera

Background Vitiligo is a pigmentation disorder, the cause of which is complex and not yet fully understood. There is a significant change of epidermal cytokines in involved skin of patients with vitiligo compared with uninvolved skin and skin of healthy controls, thus suggesting a possible involvement of cytokines in the pathogenesis of vitiligo. Objectives To evaluate potential roles of IL10 family cytokines (IL10, IL19, IL20, IL22 and IL24) in vitiligo. Along with the selected cytokines, we investigated subunits of the receptors (IL10RA, IL10RB, IL20RA and IL22RA1) which are involved in the signalling pathway of the cytokines. Methods Quantitative real‐time polymerase chain reaction was used to detect mRNA expression levels in samples extracted from skin biopsies and peripheral blood mononuclear cells and an enzyme‐linked immunosorbent assay was used to measure protein concentrations in serum from patients with vitiligo and healthy controls. Results IL22 is significantly associated with vitiligo, especially with the active stage of vitiligo, as shown by results of mRNA expression and supported by results of protein level in sera. IL22 may provoke inflammation which leads to destruction of melanocytes. Conclusions The actual role of IL22 during pathogenesis of vitiligo remains to be better characterized. Signal transductions of other investigated cytokines seem to be regulated on the expression level of their receptor complex subunits.     

Decreased lysyl oxidase-like 2 expression in mid-dermal elastolysis

Mid-dermal elastolysis (MDE) is a rare disorder that is histopathologically characterized by selective loss of elastic fibres in the mid-dermis. Aetiopathogenesis of MDE is still obscure. We report for the first time on the expression of lysyl oxidase-like (LOXL) proteins in lesional and non-lesional skin of a patient with reticular variant of MDE. Real-time RT-PCR and immunohistochemistry were performed for matrix metalloproteinase-2 (MMP2), MMP7, MMP9, LOXL1, LOXL2, and LOXL3. LOXL1 and LOXL3 mRNA levels in lesional skin did not substantially differ from mRNA levels measured in healthy skin. For LOXL2, however, we found decreased mRNA expression in lesional skin as compared to healthy skin. mRNA expression of MMP2 and MMP7 of lesional skin did not substantially differ from healthy skin. However, MMP9 mRNA expression was massively increased in lesional skin when compared to healthy skin. Immunohistochemistry confirmed the altered expression of LOXL2 and MMP9 in lesional skin. In conclusion, our preliminary data suggest that not only increased elastolytic activity (e.g. MMP9 up-regulation) but also affected elastin renewal due to reduced LOXL expression may contribute to the pathogenesis of MDE. More research is warranted in MDE especially with regard to the LOX family, fibulins, fibrillins, and desmosines.     

Interleukin-6 expression in the skin of patients with lupus erythematosus

Abstract It has been proposed that interleukin-6 may play a role in the pathogenesis of autoimmune diseases like lupus erythematosus. We have therefore investigated the immunoreactivity of IL-6 in 32 skin biopsies of 23 patients suffering from chronic discoid lupus erythematosus (n=16), subacute cutaneous lupus erythematosus (n=5) and systemic lupus erythematosus (n = 5) as well as in uninvolved skin (n = 6) and in normal skin from healthy volunteers (n = 3). Increased immunohistochemical staining was detectable in 14 of 26 biopsies from lesional skin. The remaining biopsies from lesional, non-lesional and normal skin displayed only minimal or no reactivity, but 8 out of 12 lupus erythematosus patients had been pretreated with local or systemic antiinflammatory drugs. Irrespective of the LE subtype, immunolabelling was generally most intense in the basal layer of the epidermis, with additional intense suprabasal staining in sections from 2 of 5 SLE patients. Preferential production of IL-6 in the lower parts of the epidermis was confirmed by RNA in situ hybridization. No correlation was found between the deposition of immuno-globulins and complement at the dermo-epidermal junction and IL-6 expression in keratinocytcs. These data suggest that IL-6 may be involved in LE although its exact role in the pathogenesis of the disease needs to be further elucidated.     

Increased expression of adhesion receptors in both lesional and non-lesional psoriatic skin

Adhesion receptors and their ligands play a vital role in the immune system. We studied the expression of different adhesion receptors, using single- and double-staining immunohistochemical techniques, in both lesional and non-lesional skin specimens from seven psoriasis patients and in skin biopsy specimens from eight normal healthy controls. Our results showed an overall increased expression of several adhesion receptors in both lesional and non-lesional psoriatic skin. We consistently found an increased expression in particular of ICAM-1 and E-selectin on endothelial cells, and ICAM-1 on T cells and Langerhans cells. In contrast, a weak expression of VCAM-1 was found on endothelial cells and mononuclear cells in lesional psoriatic skin specimens alone. Interestingly, LFA-1 was also expressed on Langerhans cells, with a greater frequency in skin from lesional than from non-lesional sites, but was never expressed in skin from normal healthy individuals. Furthermore, significantly increased numbers of Langerhans cells and T cells with a positive reactivity for MAb HECA-452 were found in both lesional and non-lesional psoriatic skin. We hypothesize that the enhanced expression of adhesion receptors on migrating immunocompetent cells and endothelial cells of psoriatic skin in general facilitates the increased influx of activated T lymphocytes and other immunocomponent cells into the skin, and thus underscores the generalized character of the disease.     

The Increased Expression of Matrix Metalloproteinase-9 Messenger RNA in the Non-lesional Skin of Patients with Large Plaque Psoriasis Vulgaris

Background

A difference of the interleukin-18 (IL-18) mRNA expression among several proinflammatory genes was previously observed between large plaque (LP) psoriasis patients (more than 5 cm lesions are typical) and small plaque (SP) psoriasis patients (1~2 cm lesions are typical). Therefore, it is necessary to test whether there is any difference in the expression of the genes that activate IL-18 or the expression of genes that are induced by IL-18.

Objective

To test the differential mRNA expressions of caspase-1, STAT-6, MMP-1, MMP-2, MMP-9 and TIMP-1 according to the clinical types of psoriasis vulgaris lesions in Korean patients, we have analyzed the skin samples of psoriasis vulgaris patients.

Methods

The total cellular RNA of skin samples from groups of patient with LP and SP psoriasis was analyzed by performing real-time PCR (the Taqman method) to compare the differences in the mRNA expressions.

Results

The caspase-1 and STAT-6 mRNA expression levels from the SP lesional skin of the patients were increased compared with the caspase-1 and STAT-6 mRNA expression levels from SP non-lesional skin or normal skin, but these expression levels from the SP non-lesional skin were not significantly different from those of the LP non-lesional skin. Among MMP-1, MMP-2, MMP-9 and TIMP-1, the expressions of MMP-1, MMP-2 and MMP-9 mRNA were increased in the SP lesional skin compared with those of the SP non-lesional skin. The MMP-1 mRNA expressions in both the LP and SP lesional skin were increased compared with those in the normal skin (p=0.028 and p=0.007 respectively). The MMP-9 mRNA expression in the LP non-lesional skin was elevated compared with the MMP-9 mRNA expression in the SP non-lesional skin (p=0.047). The TIMP-1 mRNA expression levels from the non-lesional skin and the lesional skin of the psoriasis patients and the normal skin samples were not significantly different.

Conclusion

The increased expression of MMP-9 mRNA in the LP non-lesional skin compared to that of the SP non-lesional skin in the psoriatic skin suggests that the increased MMP-9 mRNA expression is related to the large size type of lesion.     

Alterations of glucosylceramide-beta-glucosidase levels in the skin of patients with psoriasis vulgaris

Hydrolysis of glucosylceramides by the enzyme glucosylceramide-beta-glucosidase (GlcCer'ase) results in ceramide, a critical component of the intercellular lamellae that mediates the epidermal permeability barrier. A disturbance of ceramide formation is supposed to influence the transepidermal water loss in common skin diseases like atopic eczema or psoriasis. The aim of this study was to investigate whether GlcCer'ase levels were altered in the skin of subjects with psoriasis vulgaris. Skin punch biopsies were taken from lesional and non-lesional psoriatic skin and GlcCer'ase was evaluated both at the RNA and at the protein level. Normal skin from surgical patients provided the baseline GlcCer'ase expression in healthy subjects. Our results show that GlcCer'ase mRNA expression was decreased in psoriatic non-lesional skin compared to normal controls in all cases. Interestingly, in lesional psoriatic skin the level of GlcCer'ase was increased compared to non-lesional skin in all cases. For the immunohistochemical analysis, we used a newly synthesized monoclonal antibody anti-human GBC (GlcCer'ase-GST fusion protein). The results confirmed that GlcCer'ase, mainly present in the upper epidermis, was decreased in psoriatic skin compared to normal control and was increased in lesional compared to non-lesional psoriatic skin. Our findings support the concept that alteration in water permeability barrier in lesional psoriatic skin can serve as a trigger for the upregulation of the expression of enzymes like GlcCer'ase with consequent stimulation of ceramide generation.