Implication of regulatory T cells' telomere shortening in pathogenesis of generalized vitiligo.

Generalized vitiligo(GV) is a skin depigmenting condition due to loss of melanocytes. Regulatory T cells(Tregs), responsible for peripheral tolerance, show altered numbers and functions in GV patients, likely influenced by the aging process. Therefore, the present study was focused on measuring the relative telomere length of Tregs in 96 GV patients and 90 controls by qPCR, along with correlation of relative telomere length with in vitro Treg suppressive capacity. Interestingly, we found significantly decreased relative telomere length in Tregs of GV patients as compared to controls(p = 0.0001). Additionally, age based-analysis suggested significant decrease in relative telomere length in elderly GV patients(>40 years) in comparison to young GV patients(0-20 years; p = 0.0027). Furthermore, age of onset analysis suggested for reduced relative telomere length in early onset GV patients (0-20 years) in comparison to late onset GV patients(>40 years; p = 0.0036). The correlation analysis suggested positive correlation for relative telomere length with in vitro Tregs suppressive capacity(r = 0.68 & r = 0.45; p < 0.0001). Additionally, the in vitro Tregs suppressive capacity was significantly reduced in elderly GV patients(p = 0.003) and early onset GV patients(p = 0.0074). Overall, our study for the first time demonstrated that, the Tregs ageing due to telomere shortening may be responsible for altered Treg functions and number.

HLAB27 may confer protection to COVID-19 in generalized vitiligo patients from South Gujarat population.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), while generalized vitiligo(GV) is an autoimmune disease that causes the loss of functional melanocytes, resulting in white patches all over the body. Human Leukocyte Antigen (HLA) plays a crucial role in immune response to pathogens. Studies assessing the link between GV and COVID-19 are lacking; therefore, our current study was aimed to establish the association between GV and HLAB27 by genotyping the HLAB27 allele in 150 GV patients and 150 controls from South Gujarat population through polymerase chain reaction-sequence-specific primers (PCR-SSP) method. Additionally, we assessed the correlation of GV with COVID-19 and the influence of HLAB27 on COVID-19 development. Interestingly, our study suggested that the HLAB27 allele was prevalent in GV patients as compared to controls (52% vs 35.33%; p = 0.0051). Moreover, the occurrence of COVID-19 was significantly lower in GV patients than in controls (10% vs 32.66%; p < 0.0001). Disease activity-based analysis suggested that COVID-19 occurrence was significantly lower in active vitiligo (AV) patients as compared to stable vitiligo (SV) patients(6.87% vs 31.57%; p = 0.0045). Furthermore, COVID-19 development was significantly reduced in HLAB27 positive individuals as compared to HLAB27 negative individuals (p = 0.0025). Overall, our study suggests, for the first time, that HLAB27 allele might be a genetic risk factor for GV susceptibility, and an ongoing immune response in GV patients, more specifically in AV patients, might protect against COVID-19 infection in South Gujarat population. Additionally, our study highlighted the likely role of HLAB27 in protection against COVID-19 development.

Altered regulatory T cell-mediated Natural Killer cells suppression may lead to generalized vitiligo.

Generalized vitiligo (GV) is characterized by white patches due to autoimmune loss of melanocytes. Regulatory T cells (Tregs) maintain immune homeostasis, while NK cells eliminate pathogens and tumors. Increased NK cell frequency and reduced Treg frequency and suppressive capacity are observed in vitiligo patients. However, studies assessing Treg-mediated suppression of NK cell functions in GV are lacking. Therefore, our study aimed to assess in vitro Treg-mediated suppression of NK cells function over K562 and SK-Mel-28 cells in 31 GV patients and 30 controls using the BrdU-cell proliferation assay. We found decreased Treg-mediated suppression of NK cell function in GV patients (p = 0.0289). Moreover, increased NK cell-mediated K562 and SK-Mel-28 cells' suppression was observed in GV patients (p = 0.0207,p = 0.0419). Disease activity-based analysis, suggested reduced Treg-mediated suppression of NK cell function and increased NK cell function in active vitiligo patients (p = 0.03,p = 0.0436). Interestingly, age-based analysis suggested decreased Treg-mediated suppression of NK cell function in 1-20 and 21-40 years age groups compared to 41-60 years age group of GV patients (p = 0.005,p = 0.0380). Overall, our study, for the first time, suggests that decreased Treg-mediated suppression of NK cells may lead to increased destruction of melanocytes in GV, and this knowledge may help in developing effective therapeutics based on Tregs and NK cells for GV.

Compromised melanocyte survival due to decreased suppression of CD4+ & CD8+ resident memory T cells by impaired TRM-regulatory T cells in generalized vitiligo patients.

Regulatory T cells (Tregs) are involved in the suppression of activated T cells in generalized vitiligo (GV). The study was aimed to investigate resident memory (TRM)-Tregs and antigen-specific Tregs' numbers and functional defects in 25 GV patients and 20 controls. CD4+ & CD8+ TRM cell proliferation was assessed by BrDU assay; production of IL-10, TGF-ß, IFN-γ, perforin and granzyme B were assessed by ELISA and enumeration of TRM cells was done by flowcytometry. GV patients showed significantly increased frequency and absolute count of CD4+ & CD8+ TRM cells in lesional (L), perilesional (PL) and non-lesional (NL) skin compared to controls (p = 0.0003, p = 0.0029 & p = 0.0115, respectively & p = 0.0003, p = 0.003 & p = 0.086, respectively). Whereas, TRM-Treg (p < 0.0001 & p = 0.0015) and antigen-specific Tregs (p = 0.0014 & p = 0.003) exhibited significantly decreased frequency and absolute counts in L & PL skin. GV patients showed reduced suppression of CD8+ & CD4+ TRM cells (with increased IFN-γ, perforin & granzyme B) and decreased TRM-Tregs and antigen-specific Tregs (with decreased IL-10 & TGF-ß production) and reduced proliferation of SK-Mel-28 cells in co-culture systems. Immunohistochemistry revealed increased expression of TRM stimulating cytokines: IL-15 & IL-17A and reduced expression of TGF-ß & IL-10 in L, PL, NL skins compared to controls. These results for the first time suggest that decreased and impaired TRM-Tregs and antigen-specific Tregs are unable to suppress CD4+ & CD8+ TRMs' cytotoxic function and their proliferation due to decrease production of immunosuppressive cytokines (IL-10 & TGF-ß) and increased production of TRM based IFN-γ, perforin and granzyme B production, thus compromising the melanocyte survival in GV.

Harmine and Kaempferol treatment enhances NFATC1 and FOXP3 mediated regulatory T-cells' suppressive capacity in generalized vitiligo.

BACKGROUND: Generalized vitiligo (GV) is an autoimmune disease characterized by the progressive loss of melanocytes. OBJECTIVES: Current study was undertaken to assess in-vitro therapeutic potential of Harmine and Kaempferol for GV. METHODS: Calcium, calcineurin, NFATC1 levels, cell proliferation were assessed by various kits and ORAI1, PEIZO1, Calcineurin, GSK3B, DYRK1A transcripts and IFN-γ,IL-10,TGF-ß protein levels were assessed by qPCR and ELISA in blood and skin biopsy samples from Tregs of 52 patients and 50 controls. RESULTS: Harmine and Kaempferol treatment enhances Treg suppressive capacity, NFATs and FOXP3 expression in blood and skin Tregs of GV patients (p < 0.05). Furthermore, Harmine and Kaempferol treatment in Tregs increased calcineurin and NFATC1 activity and decreased DYRK1A transcripts in blood and skin Tregs of GV patients(p < 0.05). In-silico analysis revealed that Harmine and Kaempferol might boost Treg suppressive capacity by increasing calcineurin dephosphorylation activity leading to increase NFATs activation and also increase nuclear retention of NFATs by inhibiting DYRK1a phosphorylation activity. Moreover, calcineurin and NFATC1 activity in Tregs were positively correlated with Treg suppressive capacity, NFATC1 and FOXP3 expression (p < 0.05), whereas, DYRK1A transcripts were negatively correlated with Treg suppressive capacity, NFATC1 and FOXP3 expression (p < 0.05). These compounds significantly increased melanocytes' survival and proliferation in Treg:CD4+/CD8+:SK-Mel-28 cell line co-culture system from GV patients (p < 0.0001). CONCLUSIONS: For the first time the study suggests that Harmine and Kaempferol treated Tregs could control the CD8+ and CD4+T-cells' proliferation and IFN-γ production, leading to melanocytes' survival and proliferation. These compounds may serve as novel Treg-based therapeutics for GV; however, in vivo studies are warranted to assess the safety and efficacy of these compounds.

Genetic association of nuclear factor of activated T cells' 3'UTR and structural polymorphisms with susceptibility to generalized vitiligo in Gujarat population.

Generalized vitiligo (GV) is an autoimmune skin depigmenting disease characterized by loss of functional melanocytes. Nuclear factor of activated T cells (NFATs) play a key role in regulatory T cells' (Tregs) activation and function. Our previous studies have highlighted the role of reduced NFATs expression and activity in impaired Tregs suppressive capacity, leading to GV pathogenesis. 3'UTR region and structural single nucleotide polymorphisms(SNPs) could lead to reduced NFAT expression and activity. Therefore, we studied the association of NFATs 3'UTR [NFATC2 rs4811198 (T > G) & NFATC4 rs11848279 (A > G)] and structural [NFATC1 rs754093 (T > G) & NFATC2 rs12479626 (T > C)] SNPs in 427 GV patients and 415 controls from Gujarat population by Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). Additionally, we carried out genotype-phenotype correlation and in silico analysis to assess the effect of NFATs SNPs on NFATs expression and structure. NFATC2 rs4811198 (T > G) 3' UTR & NFATC2 rs12479626 (T > C) structural SNPs were significantly associated with GV (p < 0.0001). Interestingly, for NFATC2 rs4811198 (T > G) SNP, there was a significant difference in the TT vs GG genotypes' frequencies (p = 0.0034; Table 2), and for NFATC2 rs12479626 (T > C) SNP there was a significant difference between TT vs TC and CC genotypes' frequencies (p < 0.0001 & p = 0.0002) between GV patients and controls. Furthermore, Odds ratio suggested that the susceptible alleles for NFATC2 rs4811198 (T > G) & NFATC2 rs12479626 (T > C) SNPs increased the risk of GV by 1.38 & 3.04 fold. However, the NFAT 3' UTR [NFATC2 rs4811198 (T > G)] and structural [NFATC1 rs754093 (T > G)] SNPs were not significantly associated with GV. Interestingly, the genotype-phenotype correlation suggested that the susceptible 'G' allele of NFATC2 rs4811198 (T > G) & NFATC4 rs11848279 (A > G) 3' UTR SNPs lead to reduced NFATC2 and NFATC4 expression (p < 0.0001). Furthermore, in silico analysis suggested that hsa-miR-3183 & hsa-miR-6720-3p miRNAs specifically bound to 'G' allele of NFATC2 rs4811198 SNP and has-miR-4652-3p miRNA specifically bound to 'G' allele of NFATC4 rs11848279 SNP. Overall, our study suggests that NFATC2 rs4811198 (T > G) 3' UTR & NFATC2 rs12479626 (T > C) structural SNPs may be associated with GV susceptibility in Gujarat population. Moreover, the susceptible alleles for the 3' UTR SNPs could lead to reduced NFATs levels, which may further possibly, affect the Treg suppressive function leading to GV.

Differential Expression and in Silico Functional Analysis of Plasma MicroRNAs in the Pathogenesis of Non-segmental Vitiligo.

Vitiligo is a disease characterized by acquired depigmentation, white macules, and patches on the skin due to the dysfunction of epidermal melanocytes. In this study, we attempt to profile the microRNA (miRNA) expression patterns and predict the potential targets, assessing the biological functions of differentially expressed miRNAs in the blood of generalized vitiligo patients. Peripheral blood samples were taken from all participants, and the expression levels of 89 identified miRNAs were analyzed with real-time quantitative polymerase chain reaction (PCR). The results indicated significant upregulation of six miRNAs and downregulation of 19 miRNAs in the plasma of vitiligo patients. The top three upregulated miRNAs were hsa-miR-451a, hsa-miR-25-3p, and hsa-miR-19a-3p, and the top three downregulated miRNAs were hsa-miR-146a-5p, hsa-miR-940, and hsa-miR-142-3p. Moreover, the miRNA expression profiles of patients with Type 3 and Type 4 phototypes were substantially different in such a way that the patients with Type 3 phototype would be more prone to the emergence of melanoma and cancer. While significant variations in the expression patterns of miRNAs in male and female vitiligo patients were demonstrated, miR-let-7i-5p, miR-19a-3p, miR-25-3p, and miR-451a were commonly upregulated, and miR-142-3p and miR-146a-5p were commonly repressed in both sexes. This study may shed light on the roles of differentially expressed miRNAs in vitiligo patients by examining the miRNA expression patterns and the combined effects of miRNA and their predicted targets.

Dendritic cells and their associated pro-inflammatory cytokines augment to the inflammatory milieu in vitiligo skin.

BACKGROUND AND AIM: Vitiligo is a progressive, autoimmune, hypomelanotic acquired disorder of skin which is characterized by depigmentation. The initial immunological events of this diseases are still at enigma that includes breach of immune tolerance, and defect in antigen presentation. Hence, we aimed to explore role of Dendritic cells (DCs) and its associated cytokines in the pathogenesis of generalized vitiligo (GV) patients. METHODOLOGY: For this case-control study, 20 active patients and controls were enrolled. Phenotypic characterization of myeloid and plasmacytoid DCs (mDCs, pDCs) were done by flow-cytometry. Primary culture of DCs was done by monocyte differentiation supplemented with rIL-4 and rGM-CSF. Functional analysis DCs related cytokines and co-stimulatory molecules (CD80, CD40) was done by ELISA and qPCR respectively. Tissue localization of DCs was evaluated by immunohistochemistry. RESULT: The frequency of mDCs (0.3715% v/s 0.188%) and pDCs (0.2331% v/s 0.1156%) were elevated in patients as compared to controls. Circulatory level of IL-12, TNF-α were significantly higher whereas IFN-α was decreased in patients than controls. Similar results were obtained in the culture supernatants of patients. Relative mRNA expression profiling of co-stimulatory molecules (CD80, CD40) were found to be up regulated in patient's skin. Tissue localization of Langerhans cells (Langerin, CD1a+) were found to be significantly higher in patients. CONCLUSION: Elevated frequency of mDCs and pDCs along with elevated levels of IL-12, TNF-α and CD80, CD40 may contribute in defective antigen presentation of DCs. Altered pro-inflammatory and anti-inflammatory cytokines along with tissue localization of Langerhans cells might be involved in the pathogenesis of GV. These DCs associated cytokines can be explored as a therapeutic target in future.

Association of FOXP3 and GAGE10 promoter polymorphisms and decreased FOXP3 expression in regulatory T cells with susceptibility to generalized vitiligo in Gujarat population.

Alterations in regulatory T (Treg) cells have been observed in generalized vitiligo (GV) patients and decreased Forkhead Box P3 (FOXP3) has been implicated in the disease pathogenesis. The present study examined FOXP3 rs3761547(A > G), rs3761548(C > A), rs2232365(A > G) and GAGE10 rs11798415(A > T) promoter single nucleotide polymorphisms (SNPs) in 419 GV patients and 429 controls from Gujarat population using PCR-RFLP and ARMS-PCR. Real-time PCR and flow cytometry were used for assessment of FOXP3 mRNA and protein levels respectively in 96 GV patients and 90 controls. The frequency of genotypes (p < 0.001) and alleles (p = 0.012 & p = 0.040) for rs3761547(A > G) and rs11798415(A > T) SNPs significantly differed between GV patients and controls. FOXP3 mRNA and protein levels were significantly decreased (p < 0.001) in GV Tregs compared to controls. Active vitiligo (AV) Tregs showed significantly reduced FOXP3 mRNA and protein levels compared to that of stable vitiligo (SV) (p = 0.02 & p = 0.039). The correlation of genotype-phenotype of FOXP3 SNPs suggested reduced FOXP3 mRNA (p = 0.019, p < 0.001 & p < 0.001) and protein (p = 0.028, p < 0.001 & p = 0.022) levels in patients with susceptible GG, AA and GG genotypes respectively. The GAGT, GCGT & ACGT haplotypes were prevalent in GV patients (p = 0.004, p = 0.004 & p = 0.016); however, GAGT & GCGT were overrepresented in patients with AV (p = 0.044 & p = 0.024). The susceptible GAGT and GCGT haplotypes in patients exhibited reduction in FOXP3 mRNA (p = 0.014 & p = 0.019) and protein (p = 0.024 & p = 0.028). DNA-protein docking analysis revealed reduced binding for transcription factor C/EBP to the susceptible allele 'G' (rs3761547) compared to A allele. For the first time, the study suggests significant association of FOXP3 rs3761547(A > G) & GAGE10 rs11798415(A > T) SNPs with susceptibility to GV in Gujarat population. In addition, the likely role of these SNPs in altered FOXP3 expression of Tregs may possibly affect Treg suppressive function in GV.

Effect of TNF-α -308G/A (rs1800629) Promoter Polymorphism on the Serum Level of TNF-α Among Iraqi Patients with Generalized Vitiligo.

BACKGROUND: Vitiligo is a chronic acquired pigmentary disorder of the skin; it results from immunological distruction of functioning melanocytes. The cytokine TNF-α plays a central role in the initiation of melanocyte apoptosis in vitiligo. Single nucleotide polymorphism (SNP) in the promoter region of the gene coding for serum TNF-α may affect its production. OBJECTIVE: The aim of this study is to assess serum TNF-α as a risk factor for generalized vitiligo among Iraqi patients and to rule out that polymorphism at the -308 position affects serum TNF-α. MATERIALS AND METHODS: This case-control study was conducted at Sulaymaniyah Dermatology Teaching Center (SDTC), Iraq. Serum concentration of TNF-α was measured using enzyme linked immunosorbent assay (ELISA) technique in 80 patients with generalized vitiligo and 40 clinically healthy controls. The amplification refractory mutation system polymerase chain reaction (ARMS-PCR) technique was used for detection of TNF-308G/A gene polymorphism. TNF-α level correlated with TNF-308G/A gene polymorphism. Serum concentration and TNF -308G/A gene polymorphism have been analyzed in correlation with demographic features and clinical characteristics of patients with generalized vitiligo. RESULTS: Statistically significant elevation of serum TNF-α seen in patients compared to a control group (p-value 0.01). Significantly higher TNF-α level (p-value 0.01) found among patients with active generalized vitiligo. Elevated serum levels of TNF-α were significantly associated with both TNFA1 (TNF-308G) allele (p-value 0.04) and TNFA2 (TNF-308A) allele (p-value 0.03). TNF-α -308GA polymorphism was not affected by demographic features and clinical characteristics of patients with generalized vitiligo. CONCLUSION: TNF-α in the serum is a risk factor for generalized vitiligo among Iraqi patients. Patients with active vitiligo have a higher serum TNF-α level. No difference was found between serum level of TNF-α with TNF-α polymorphism at position -308 (TNF -308). This involves substituting G allele for the A allele.

Decreased suppression of CD8+ and CD4+ T cells by peripheral regulatory T cells in generalized vitiligo due to reduced NFATC1 and FOXP3 proteins.

Regulatory T cells (Tregs) are involved in the suppression of activated T cells in generalized vitiligo (GV). The study was aimed to investigate Tregs functional defects in Treg:CD8+ and Treg:CD4+ T cells' co-culture systems of 55 GV patients and 45 controls. CD8+ and CD4+ T-cell proliferation was assessed by BrdU assay; production of IL-10, TGF-ß and IFN-γ cytokines was assessed by ELISA; and FOXP3, CD25, NFATC1 and CD44 proteins were measured by flow cytometry. Generalized vitiligo patients showed reduced suppression of CD8+ and CD4+ T cells (P = .0384, P = .0084), increased IFN-γ (P < .0001, P = .0019), decreased IL-10 and TGF-ß (P < .0001) and decreased FOXP3, CD25 and NFATC1 proteins (P < .0001). Active vitiligo (AV) patients showed reduced suppression of CD8+ & CD4+ T cells (P = .006, P = .015), increased IFN-γ (P = .036, P = .045), decreased IL-10 (P = .009, P = .021), FOXP3 (P = .0244) and NFATC1 (P = .019). Severe GV (50%-75% VASI) patients showed reduced suppression of CD8+ and CD4+ T cells (P = .0003, P = .001), increased IFN-γ (P = .0029, P < .0001), decreased IL-10 (P = .0057, P = .0017), FOXP3 (P = .002) and NFATC1 (P = .0347). VASI score was positively correlated with the suppression of CD8+ and CD4+ T cells (P = .0006, P < .0001), IL-10 (P = .0096, P = .029), FOXP3 (P = .0008) and NFATC1 (P = .043), whereas it was negatively correlated with IFN-γ (P = .0029, P = .0017). Early age of onset patients' Tregs demonstrated decreased suppression of CD8+ and CD4+ T cells (P = .0156, P = .0074), decreased TGF-ß (P = .0212, P = .0083) and NFATC1 (P = .0103). NFATC1 was positively correlated with FOXP3 in Tregs (P < .0001). Our results suggest impaired Tregs suppressive function in GV patients due to decreased NFATC1, FOXP3, CD25, IL-10 and TGF-ß resulting into increased CD8+ and CD4+ T-cell proliferation and IFN-γ production. For the first time, decreased NFATC1 levels were correlated with decreased FOXP3, thereby altering Treg cell function in GV patients. Additionally, decreased Treg cell function also affected onset, activity and severity of GV.

Altered expression of nuclear factor of activated T cells, forkhead box P3, and immune-suppressive genes in regulatory T cells of generalized vitiligo patients.

The study was aimed to analyze expression of nuclear factor of activated T cells (NFATs), forkhead box P3 (FOXP3), and their associated genes (sCTLA4, flCTLA4, IL10, TGFB, IL2, IL4, CD25) in regulatory T cells (Tregs) of 48 generalized vitiligo (GV) patients and 45 unaffected controls. The transcripts of NFATC1 to NFATC4, FOXP3, IL10, flCTLA4 (p < .0001), NFAT5 (p = .0003), sCTLA4 (p = .001), and FOXP3 protein in Tregs and plasma IL-10 levels were reduced significantly (p < .0001) in GV Tregs compared to controls. The FOXP3 promoter polymorphisms [rs3761548(C > A), rs3761547(A > G), and rs2232365(A > G)] revealed significantly decreased FOXP3 protein levels in patients' Tregs with susceptible AA, GG, and GG genotypes (p < .0001, p = .028, p = .022, respectively). The active vitiligo Tregs showed reduced levels of NFATC3, NFATC4, NFAT5, FOXP3, TGFB, and flCTLA4 transcripts (p = .0005, p = .0003, p = .0002, p = .020, p < .0001, p = .006, respectively) and FOXP3 and TGF-ß proteins (p = .0394 and p = .0013) compared to stable vitiligo. Early-onset patients (1-20 years) demonstrated decreased IL-10, sCTLA-4, flCTLA-4, TGFB, and FOXP3 transcripts and FOXP3 protein as compared to late-onset patients (41-60 years) (p = .001, p = .003, p = .009, p = .005, p = .038, p = .0226, respectively). Overall, our results for the first time suggest a likely role of NFATs and FOXP3 together with Treg immune-suppressive genes in GV pathogenesis and disease progression, warranting additional investigations.

Sulfasalazine-induced generalized vitiligo in a patient with dermatitis herpetiformis and celiac disease.

Vitiligo is an acquired idiopathic pigmentary skin disorder characterized by the development of white macules and patches due to the loss of functioning melanocytes. In this report, we describe a case of a patient with a longstanding history of dermatitis herpetiformis (DH) and celiac disease that developed rapidly progressing, biopsy-confirmed generalized vitiligo after 11 months of treatment with anti-inflammatory medication sulfasalazine, prescribed for the patient's DH. To the best of our knowledge, this is the first case report which has demonstrated the possible biochemical pathways, triggered by sulfasalazine, in the development of vitiligo.

Expression of nucleobindin 2-encoded satiety and fat-influencing protein-1 and interleukin-26 in the serum of patients with vitiligo and its significance / 中华皮肤科杂志

Objective To determine the expression of nucleobindin 2-encoded satiety and fatinfluencing protein-1 (nesfatin-1) and interleukin (IL)-26 in the serum of patients with vitiligo,and to explore the relationship of nesfatin-1 and IL-26 with anxiety status,disease stage and distribution pattern of vitiligo lesions.Methods From March 2017 to September 2018,123 outpatients with vitiligo,as well as 30 healthy controls (control group),were enrolled from Department of Dermatology,Affiliated Hospital of Jiangsu University.Of these patients,93 rated as anxious (vitiligo with anxiety group),and 30 as nonanxious (vitiligo without anxiety group).Another 30 anxious patients with other autoimmune diseases and background diseases like hypertension and diabetes mellitus but not vitiligo (anxiety group) were enrolled from Zhenjiang Fifth People's Hospital.Enzyme-linked immunosorbent assay (ELISA) was performed to detect levels of nesfatin-1 and IL-26 in the above groups,and state-trait anxiety inventory (STAI) was used to evaluate the anxiety status.According to the vitiligo disease activity (VIDA) score,the vitiligo patients with anxiety were divided into 3 subgroups:stable stage group,active stage group and rapid progressive stage group.According to the distribution pattern of skin lesions,the vitiligo patients with anxiety were divided into 5 subgroups:localized vitiligo group,segmental vitiligo group,generalized vitiligo group,sporadic vitiligo group and acral vitiligo group.The levels of nesfatin-1 and IL-26 were compared among different stage groups and type groups.Statistical analysis was carried out using one-way analysis of variance for comparison among several groups,multi-way analysis of variance and stratification analysis for multifactor data,paired t-test for comparison before and after treatment,and Pearson correlation analysis for analyzing correlations.Results The serum level of nesfatin-1 significantly differed among the vitiligo with anxiety group,vitiligo without anxiety group,anxiety group and control group (F =10.78,P ＜ 0.001),and was significantly higher in the vitiligo with anxiety group than in the other 3 groups (P ＜ 0.001).No significant difference in the serum level of IL-26 was found among the 4 above groups (F =1.34,P =0.26).As pearson correlation analysis showed,the serum level of nesfatin-1 was positively correlated with the STAI score in the 93 vitiligo patients with anxiety (r =0.55,P ＜ 0.001).The serum level of nesfatin-1 was significantly higher in the rapid progressive stage group than in the stable stage group (P ＜ 0.05),while there was no significant difference in the IL-26 level among different stage groups (P ＞ 0.05).The generalized vitiligo patients with anxiety showed significantly increased serum level of nesfatin-1 compared with the localized vitiligo patients with anxiety (P ＜ 0.001).After 3-month treatment,the nesfatin-1 level in the 10 vitiligo patients with anxiety significantly decreased compared with that before the treatment (paired t =4.40,P =0.02),but the STAI score did not change (P ＞ 0.05).Conclusion Nesfatin-1 as an emotioninfluencing factor may participate in the occurrence of vitiligo,especially affect patients with rapid progressive vitiligo or generalized vitiligo,while IL-26 may be irrelevant to vitiligo.

The changes of gene expression profiling between segmental vitiligo, generalized vitiligo and healthy individual.

BACKGROUND: Vitiligo is a common acquired depigmentation skin disease characterized by loss or dysfunction of melanocytes within the skin lesion, but its pathologenesis is far from lucid. The gene expression profiling of segmental vitiligo (SV) and generalized vitiligo (GV) need further investigation. OBJECTIVE: To better understanding the common and distinct factors, especially in the view of gene expression profile, which were involved in the diseases development and maintenance of segmental vitiligo (SV) and generalized vitiligo (GV). METHODS: Peripheral bloods were collected from SV, GV and healthy individual (HI), followed by leukocytes separation and total RNA extraction. The high-throughput whole genome expression microarrays were used to assay the gene expression profiles between HI, SV and GV. Bioinformatics tools were employed to annotated the biological function of differently expressed genes. Quantitative PCR assay was used to validate the gene expression of array. RESULTS: Compared to HI, 239 over-expressed genes and 175 down-expressed genes detected in SV, 688 over-expressed genes and 560 down-expressed genes were found in GV, following the criteria of log2 (fold change)≥0.585 and P value<0.05. In these differently expressed genes, 60 over-expressed genes and 60 down-expressed genes had similar tendency in SV and GV. Compared to SV, 223 genes were up regulated and 129 genes were down regulated in GV. In the SV with HI as control, the differently expressed genes were mainly involved in the adaptive immune response, cytokine-cytokine receptor interaction, chemokine signaling, focal adhesion and sphingolipid metabolism. The differently expressed genes between GV and HI were mainly involved in the innate immune, autophagy, apoptosis, melanocyte biology, ubiquitin mediated proteolysis and tyrosine metabolism, which was different from SV. While the differently expressed genes between SV and GV were mainly involved in the metabolism pathway of purine, pyrimidine, glycolysis and sphingolipid. CONCLUSIONS: Above results suggested that they not only shared part bio-process and signal pathway, but more important, they utilized different biological mechanism in their pathogenesis and maintenance. Our results provide a comprehensive view on the gene expression profiling change between SV and GV especially in the side of leukocytes, and may facilitate the future study on their molecular mechanism and theraputic targets.

Combination therapy of orally administered glycyrrhizin and UVB improved active-stage generalized vitiligo

Glycyrrhizin has been used clinically for several years due to its beneficial effect on immunoglobulin E (IgE)-induced allergic diseases, alopecia areata and psoriasis. In this study, glycyrrhizin, ultraviolet B light (UVB) or a combination of both were used to treat active-stage generalized vitiligo. One hundred and forty-four patients between the ages of 3 and 48 years were divided into three groups: group A received oral compound glycyrrhizin (OCG); group B received UVB applications twice weekly, and group C received OCG+UVB. Follow-ups were performed at 2, 4, and 6 months after the treatment was initiated. The Vitiligo Area Scoring Index (VASI) and the Vitiligo Disease Activity (VIDA) instrument were used to assess the affected body surface, at each follow-up. Results showed that 77.1, 75.0 and 87.5% in groups A, B and C, respectively, presented repigmentation of lesions. Responsiveness to therapy seemed to be associated with lesion location and patient compliance. Adverse events were limited and transient. This study showed that, although the three treatment protocols had positive results, OCG and UVB combination therapy was the most effective and led to improvement in disease stage from active to stable.

Can systemically generated reactive oxygen species help to monitor disease activity in generalized vitiligo? A pilot study.

BACKGROUND: Generalized vitiligo is a disease with unpredictable bursts of activity, goal of treatment during the active phase being to stabilize the lesions. This emphasizes the need for a prospective marker for monitoring disease activity to help decide the duration of therapy. AIMS AND OBJECTIVES: In the present study, we examined whether reactive oxygen species (ROS) generated in erythrocytes can be translated into a marker of activity in vitiligo. MATERIALS AND METHODS: Level of intracellular ROS was measured flow cytometrically in erythrocytes from venous blood of 21 patients with generalized vitiligo and 21 healthy volunteers using the probe dichlorodihydrofluorescein diacetate. RESULTS: The levels of ROS differed significantly between patients and healthy controls, as well as between active versus stable disease groups. In the active disease group, ROS levels were significantly lower in those being treated with systemic steroids than those that were not. ROS levels poorly correlated with disease duration or body surface area involved. CONCLUSION: A long-term study based on these findings can be conducted to further validate the potential role of ROS in monitoring disease activity vitiligo.

Segmental and generalized vitiligo: both forms demonstrate inflammatory histopathological features and clinical mosaicism.

BACKGROUND: Segmental vitiligo (SV) and generalized vitiligo (GV) are perceived to evolve by different mechanisms, the former with unspecified neural mechanisms and the latter by melanocyte specific autoimmune mechanisms. However, the two diverse mechanisms are difficult to reconcile in cases of "mixed vitiligo". To test the possibility of a common pathogenesis, we reviewed clinical and histopathological features of SV and GV. MATERIALS AND METHODS: As part of an ongoing histopathological study on vitiligo and vitiligo like lesions, over a 10 year period from 2002 to 2011, biopsies were taken routinely from evolving or recently evolved lesions. 50 cases of SV with quasi-dermatomal distribution and 154 cases of GV were identified and the clinical and histopathological features were compared. RESULTS: Mild clinical inflammation was recorded in 33 of 154 GV cases but, none among 50 SV had such features. In addition to bilateral symmetrical involvement, mirror image lesions with unusual segmentation were observed in nine cases of GV. SV with a few bilateral lesions (4) and GV with quasi-dermatomal lesions (3), i.e., mixed vitiligo, were included in their corresponding groups for analytical purposes. Focal lichenoid inflammation of varying degrees around epidermal/adnexal melanocytes was identified as a common feature in evolving lesions of both SV (78%) and GV (70%). CONCLUSIONS: SV and GV demonstrated a similar inflammatory histopathological spectrum. "Segmentation/mosaicism", identified for the first time in GV is another unifying factor. Cutaneous mosaicism harboring fragile melanocyte populations, which are susceptible to external as well as auto-inflammatory mechanisms, is an attractive hypothesis to pursue in the causation of vitiligo.

Involvement of superoxide dismutase isoenzymes and their genetic variants in progression of and higher susceptibility to vitiligo.

Oxidative stress has been implicated as the initial triggering event in vitiligo pathogenesis leading to melanocyte destruction. Here, we report a significant increase in oxidative stress in vitiligo patients as evidenced by high lipid peroxidation levels suggesting an imbalance in the antioxidant enzyme system as reported in our previous studies. This study examined the role of the enzymatic antioxidant SOD, which converts the pro-oxidant superoxide into H2O2, in vitiligo pathogenesis. The activity of three isoforms of SOD, i.e., SOD1, SOD2, and SOD3, was significantly higher in vitiligo patients. To identify the underlying mechanism for the increase in activities of SOD isoforms, we explored the SOD1, SOD2, and SOD3 genes for their genetic variations and transcript levels. The SOD2 Thr58Ile (rs35289490) and Leu84Phe (rs11575993) polymorphisms were significantly associated with vitiligo patients, and the Val16Ala (rs4880) polymorphism was associated with active vitiligo patients. Interestingly, SOD2 activity was contributed by these polymorphisms along with its increase in transcript levels in patients. SOD3 activity was associated with the Arg213Gly (rs8192291) polymorphism. The SOD3 transcript levels were also increased in patients, which might contribute to the increased SOD3 activity. However, we could not establish the genotype-phenotype correlation for SOD1 as we could not detect any novel or reported SNPs in SOD1. In addition, both transcript and protein levels of SOD1 were unchanged between patients and controls, though SOD1 activity was increased in patients. Activities of SOD isoforms also correlated with progression of the disease as the activity was higher in active cases of vitiligo compared to stable cases. Here, we report that SOD2 and SOD3 polymorphisms may be genetic risk factors for susceptibility and progression of vitiligo and hence the genetic makeup of an individual may form a basis for the effective treatment of the disease. Overall, our results suggest that increased activity of SOD isoforms under the influence of genetic factors may lead to accumulation of H2O2 in cytoplasmic, mitochondrial, and extracellular compartments resulting in oxidative damage to the melanocytes.

T helper and regulatory T cell cytokine profile in active, stable and narrow band ultraviolet B treated generalized vitiligo.

BACKGROUND: Vitiligo is a T cell mediated autoimmune depigmenting disease. Altered cytokine concentrations have been suggested in the pathogenesis of vitiligo. METHODS: T helper and regulatory T cell cytokines (IL-2, IFN-γ, IL-10, IL-13, IL-17 and TGF-ß) have been estimated by ELISA and their clinical correlation was determined. The study had 3 groups: group I with 80 vitiligo patients (60 active and 20 stable), group II with 25 narrow band ultraviolet B treated vitiligo and group III with 70 healthy controls. RESULTS: Significant difference was found in the serum interleukin (IL)-10, IL-13, IL-17A and TGF-ß1 concentrations among 3 groups (P<0.05). In group I, serum IL-2, IL-17A concentrations were significantly increased and TGF-ß1 concentrations were decreased in active vitiligo compared to stable vitiligo (P<0.05). Concentrations of IL-2, IL-10 and IL-13 (rho=-0.307, rho=-0.407, rho=-0.351 and P<0.05; respectively) were negatively- and TGF-ß1 concentrations were positively-correlated (rho=0.799, P=0.001) with disease duration. Interleukin-13 concentrations were negatively- and serum TGF-ß1 concentrations were positively-correlated (rho=-0.326, rho=0.244 and P<0.05; respectively) with percentage of body surface area involvement. CONCLUSIONS: Increased concentrations of serum IL-10, IL-13, and IL-17A and decreased concentrations of TGF-ß1 suggested altered cell-mediated immunity that may facilitate the melanocyte cytotoxicity in vitiligo.