Quantification of mitochondrial DNA damage and copy number in circulating blood of patients with systemic sclerosis by a qPCR-based assay

BackgroundAlthough not fully understood, oxidative stress has been implicated in the pathogenesis of different autoimmune diseases such as systemic sclerosis. Accumulating evidence indicates that oxidative stress can induce mitochondrial DNA (mtDNA) damage and variations in mtDNA copy number (mtDNAcn).ObjectiveThe aim of this study was to explore mtDNAcn and oxidative DNA damage byproducts in peripheral blood of patients with systemic sclerosis and healthy controls.MethodsForty six patients with systemic sclerosis and forty nine healthy subjects were studied. Quantitative real-time PCR used to measure the relative mtDNAcn and the oxidative damage (oxidized purines) of each sample.ResultsThe mean mtDNAcn was lower in patients with systemic sclerosis than in healthy controls whereas the degree of mtDNA damage was significantly higher in cases as compared to controls. Moreover, there was a negative correlation between mtDNAcn and oxidative DNA damage.Study limitationsThe lack of simultaneous analysis and quantification of DNA oxidative damage markers in serum or urine of patients with systemic sclerosis and healthy controls.ConclusionThese data suggest that alteration in mtDNAcn and increased oxidative DNA damage may be involved in the pathogenesis of systemic sclerosis.     

Increased basic fibroblast growth factor levels in serum and blister fluid from patients with vitiligo

Basic fibroblast growth factor (bFGF) is a pleiotropic growth factor which has a high capacity for stimulating normal melanocyte proliferation and suppressing melanogenesis. The close and complicated relationship between bFGF, melanocyte proliferation and melanogenesis raises the theoretical possibility that bFGF may also be involved in the pathomechanism leading to vitiligo. The aim of this study was to compare the serum and suction blister fluid bFGF levels of vitiligo patients (9 females, 11 males) with those of healthy controls (3 females, 8 males). Vitiliginous skin-blister fluid bFGF levels and serum levels were significantly higher in vitiligo patients compared with healthy normal controls. Our data indicate that bFGF might be involved in the pathogenetic chain of events leading to vitiligo. Further studies are needed to define the exact role of bFGF and various other melanocytic mitogens in this disease.     

Increased interleukin-6 and granulocyte-macrophage colony stimulating factor levels in the sera of patients with non-segmental vitiligo

BACKGROUND: Although the cause of vitiligo is unknown, an autoimmune theory has been proposed, and there is now convincing evidence that cytokines have an important role in pathogenesis of autoimmunity. OBJECTIVE: To study the possible role of interleukin-1, beta (IL-1 beta), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony stimulating factor (GM-CSF) in the pathogenesis of vitiligo. METHODS: The authors measured the serum levels of the above-mentioned cytokines from 50 patients with the vitiligo compared with 20 healthy volunteers, employing the method of radioimmunoassay. RESULTS: The results showed that the serum levels of both IL-6 and GM-CSF of the patients with both focal type and generalized type of vitiligo, and the serum level of IL-1 beta of the generalized type,were significantly, higher than those of normal controls in the patients with segmental vitiligo, the serum levels of all the cytokines tested were not significantly different from those of the normal controls. The GM-CSF levels of both focal type and generalized type, and the IL-6 level of the generalized type in progressive stage were significantly higher than those in stable state. CONCLUSION: It is speculated that IL-6 and GM-CSF may be involved in the autoimmune mechanism of non-segmental vitiligo. However, more evidence is required before a definite conclusion can be drawn.     

Increased micronucleus frequency in phytohaemagglutinin-stimulated blood cells of patients with vitiligo

Background Vitiligo is a relatively common, acquired pigmentary disorder characterized by areas of depigmented skin resulting from loss of melanocytes in the epidermis. Although several hypotheses have been proposed for the aetiology and pathogenesis of vitiligo, the cause of vitiligo remains unclear. Objective To evaluate spontaneous micronucleus (MN) frequency using the cytokinesis block MN assay to determine damages at the DNA or chromosome level in phytohaemagglutinin (PHA)‐stimulated blood cells of patients with vitiligo and healthy control subjects. Methods Peripheral blood samples were obtained and cultured from 21 patients with vitiligo (mean age: 21.48 ± 9.78 years) and 21 age‐ and sex‐matched healthy control subjects (mean age: 21.52 ± 9.80 years). MN values were scored in binucleated cells obtained from whole‐blood cultures of patients and control subjects. Results MN frequencies (mean ± SD) in PHA‐stimulated blood cells of patients with vitiligo and control subjects were 0.94 ± 0.58 and 0.58 ± 0.32, respectively. Compared with control subjects, MN frequencies of patients with vitiligo were found significantly higher than those of the control subjects (P = 0.012). Conclusion Our results indicate unexpectedly some chromosomal/DNA damage in whole‐blood cultures of patients with vitiligo. We do not know, however, if these chromosome/DNA instabilities observed in the cells of vitiligo patients resulted from the cause or from the consequences of the disorder.     

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.     

β-Endorphin serum levels in patients with vitiligo

Aim The aim of this study is to correlate the β-endorphin levels at the early and more chronic stages of the disease in an attempt to find or confirm an etiological factor of vitiligo. Background The exact pathogenesis of vitiligo is still unclear. The most important theories are the self destruction, the autoimmune and the neural theories. Methods Patients with vitiligo (n= 28) were divided into two groups according to the duration of their disease. A group of 15 members of medical staff was the control group. β-endorphin levels were determined with a radioimmunoassay (¹²⁵I-β-endorphin IncstarCo). Results The mean β-endorphin levels (11.88 ± 2.25 pmol/l) in patients at the early years of the disease (Group A) were statistically elevated compared to those of patients with ‘chronic’ vitiligo (9.27 ± 2.73 pmol/l) and to those of controls (8.53 ± 2.53) pmol/l). Conclusion We suggest that high β-endorphin levels play a role in the pathogenesis of vitiligo as well as in the prognosis of the disease.     

白癜风患者血浆IL-17水平的检测

【摘要】 目的 探讨白癜风与IL-17的相关性。 方法 酶联免疫吸附测定法对32例白癜风患者和30例健康对照组外周血血浆IL-17含量进行检测,分析IL-17水平与白癜风患者不同分期、面积、病程的关系。 结果 进展期和稳定期白癜风患者血浆IL-17含量与健康对照组相比明显升高（均P < 0.05）,且进展期明显高于稳定期（P < 0.05）;IL-17水平与皮损面积呈正相关（r = 0.456,P < 0.05）,与病程无相关（r = 0.239, P > 0.05）。 结论 IL-17在白癜风的发生发展过程中起到一定作用。     

Low catalase levels in the epidermis of patients with vitiligo.

Suction blister roofs taken from the involved and uninvolved epidermis of patients with vitiligo showed a consistent reduction in levels of catalase compared to normal healthy controls of matched photo-skin types (Fitzpatrick classification). A decrease in catalase activity is expected to increase the concentration of hydrogen peroxide in the epidermis of these patients. Hydrogen peroxide functions as a reversible inhibitor of human tyrosinase with a KI of 8 X 10(-6) M. Also, hydrogen peroxide undergoes photochemical reduction yielding highly reactive hydroxyl radicals (OH.) and hydroxyl ions (OH-) mainly by the Haber-Weiss reaction. Hydroxyl radicals are capable of bleaching constitutional melanin and cause membrane lysis through lipid peroxidation reactions. Hydroxyl ions increase the pH in the epidermis, and as a consequence glutathione reductase activity is increased in patients with vitiligo compared to controls. Based on these new results, together with the previously reported calcium transport defect, a new hypothesis has been formulated for the pathogenesis of vitiligo.     

Increased frequency of self-reported parasomnias in patients suffering from vitiligo

Vitiligo is a skin disorder of unknown aetiology, affecting 0.1-2% of the general population. The aim of the present study was to investigate its relationship with sleep disorders, especially parasomnias. Two hundred and sixteen individuals were examined. Among them, 116 were suffering from vitiligo, 52 from other dermatological diseases and 48 were healthy subjects, serving as a control group. An inventory including items related to sleep disorders from childhood and adolescence was used. The study was focused specifically on parasomnias. Patients suffering from vitiligo reported a significantly higher occurrence of sleepwalking, nocturnal enuresis, night illusions, sleep terrors and nightmares than that of the control group, prior to the manifestation of the disease. Patients suffering from other dermatological diseases only reported significantly more often nightmares and nocturnal enuresis compared to the control group. A relationship between parasomnias during early life and later development of vitiligo was detected. This finding supports the hypothesis that neural mechanisms involving monoaminergic systems (especially the serotoninergic one) may potentially be involved in the aetiopathology of vitiligo.     

Increased prevalence of vitiligo, but no evidence of premature ageing, in the skin of patients with bp 3243 mutation in mitochondrial DNA in the mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS)

The MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) belongs to the category of mitochondrial disorders. The most common molecular aetiology of the syndrome is a mutation at base pair (bp) 3243 in the mitochondrial genome (mtDNA). The phenotype is varied and, apart from central nervous system involvement, the patients with this mutation may present with neurosensory hearing loss, diabetes mellitus and cardiomyopathy. These phenotypes suggest that organs dependent on aerobic metabolism suffer most. We investigated the possible clinical and physiological manifestations of impaired energy metabolism in the skin of 28 patients with the bp 3243 mutation in mtDNA. Non-invasive sonography and laser Doppler flowmetry were used to measure skin thickness and the blood flow of the skin. Skin collagen synthesis was assayed from suction blister fluid. Evaporimetry was used to assess the re-epithelialization rate of suction blister wounds. Histochemistry and immunohistochemistry were used to evaluate the melanocytes and pigment in the skin. Vitiligo was found in three of the 28 patients (11%), which was markedly more than in the general population. Histological findings showed an absence of pigment, but an apparently normal distribution of melanocytes in the dermoepidermal junction. Seborrhoeic eczema and atopy were also somewhat more frequent. No features of premature ageing, such as a marked decrease in skin thickness, blood flow, collagen synthesis or re-epithelialization rate, were demonstrated.     

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     

白癜风患者病程与抗酪氨酸酶抗体的相关分析

目的 应用酪氨酸酶表达肽TYR240-479作为抗原,检测654例白癜风患者血清抗酪氨酸酶IgG抗体水平,分析白癜风患者病程与体内抗酪氨酸酶抗体水平的关联性。方法 以TYR240-479作为包被抗原,应用ELISA法检测白癜风患者血清中抗酪氨酸酶IgG抗体的滴度。不同病程患者抗体阳性检出率的差异以及病程与抗体水平的关联性分析应用卡方检验,多组率的两两比较应用Bonferroni校正。平均滴度差异比较应用one-way ANOVA,组间两两比较应用LSD－t检验。结果 各病程组抗体检测总阳性率具有显著性差异,P＝0.027,r＝0.112。其中病程1～5年（含5年）组与5～10年（含10年）组具有显著性差异,P＝0.007。病程是否超过5年与抗体水平的关联强度分析表明病程与抗体水平有关联,P＝0.025,OR＝1.473,95％CI（1.049-2.068）,但各病程组间抗体平均滴度无显著性差异。 结论 与病程超过5年的患者相比,病程5年之内的患者抗体阳性率更高,与抗体水平的关联更为紧密。白癜风病程与患者抗酪氨酸酶抗体水平有一定的关联性。