Impacts of exposure to topical calcineurin inhibitors on metabolism in vitiligo infants.

BACKGROUND: Tacrolimus ointment is a recently developed topical immunomodulator that has been approved for use in patients with vitiligo older than 2 years. Concern regarding potential systemic toxic effects has limited treatment options for children younger than 2 years. We wanted to determine whether topical tacrolimus therapy is safe and effective in patients with vitiligo younger than 2 years. METHODS: The present 6-month clinical trial was conducted to evaluate the efficacy and safety of 0.03% tacrolimus in the treatment of vitiligo in children under 2 years of age. Meanwhile, serum and urine samples were collected, and liquid chromatography-mass spectrometry was performed to generate the serum and urine metabolic profile data of patients and healthy controls. RESULTS: The overall response rate at the sixth month, which was defined by the degree of re-pigmentation, was 100%. As revealed by blood monitoring and metabolite detection 6 months later, there was no difference between the treatment group and the control group. There is no evidence that long-term topical application of 0.03% tacrolimus ointment will cause metabolite or other physical changes in the body. CONCLUSIONS: Tacrolimus ointment appears to be effective and safe in the treatment of vitiligo in children younger than 2 year. TRIAL REGISTRATION: http://www.chictr.org.cn identifier: ChiCTR 2100045920. IMPACT: We first reported the efficacy and safety of topical application of 0.03% tacrolimus ointment in infants with vitiligo characterized by the metabolites. There is no evidence that long-term topical application of 0.03% tacrolimus ointment will cause metabolite or other physical changes in the body. This study provide evidence for the TCI treatment of infants with vitiligo.

Altered circulating memory T cells in vitiligo cases followed NB-UVB therapy.

BACKGROUND: Vitiligo represents a commonly diagnosed autoimmune disease caused by the depletion of epidermal melanocytes. Many subsets of T cells contribute to vitiligo pathogenesis, including resident and circulating memory T cells. OBJECTIVES: To analyze the amounts of CD4+ and CD8+ memory T-cell subsets in peripheral blood specimens from vitiligo patients and alterations caused by narrowband ultraviolet B (NB-UVB) phototherapy. METHODS: Circulating CD4+ and CD8+ central memory T (TCM ) and effector memory T (TEM ) cell frequencies in 33 patients with non-segmental vitiligo and 16 healthy donors were evaluated by flow cytometry. Related chemokine levels were also detected. RESULTS: Peripheral blood CD4+ TCM and CD8+ TCM counts were markedly reduced in vitiligo cases while they were higher in active vitiligo compared with stable vitiligo cases. Circulating CD8+ TCM frequency in vitiligo was closely related to disease duration. Interestingly, CD4+ TCM and CD8+ TCM frequencies, alongside CXCL9 and CXCL10 amounts in peripheral blood of patients with vitiligo, were significantly decreased after NB-UVB phototherapy. CONCLUSIONS: Decreased frequencies of circulating CD4+ TCM and CD8+ TCM by NB-UVB suggest a possible immunosuppressive effect of phototherapy. The chemokines CXCL9 and CXCL10 are the bridge between circulating and skin resident memory T cells. NB-UVB blocks the homing of circulating memory T cells into vitiligo lesions by down-regulating CXCL9 and CXCL10. Targeting the above proteins could provide novel, durable treatment options to cure and prevent flares of this disease.

Repigmentation in two patients with vitiligo on AcEGCG 0.5% cream.

Per-acetylated epigallocatechin-3-gallate (AcEGCG), a fully acetylated derivative of EGCG, a more potent agent for protection of melanocytes from oxidative damage. We present two patients with vitiligo treated with AcEGCG 0.5% cream, who demonstrated skin repigmentation and control of depigmentation progression.

Topical epigallocatechin-3-gallate in the treatment of vitiligo.

OBJECTIVE: To preliminarily assess the efficacy and safety of the topical application of Epigallocatechin-3-gallate (EGCG) in treating vitiligo, a 6-month clinical trial was carried out. METHOD: Patients were randomly given topical application of EGCG on the assigned lesions, with pimecrolimus being used as the control for twice a day over a 6-month treatment period. Responses to treatment were assessed based on the changes in VASI score for percentage reduction in body surface area and the PGA scores. RESULTS: According to our results, both drugs were discovered to be markedly effective on repigmentation. The VASI of lesion had diminished from 1.19 ± 0.42 to 0.63 ± 0.38, in the EGCG-treated lesions, while from 1.18 ± 0.43 to 0.61 ± 0.36 in the pimecrolimus-treated lesions, and there was no statistically significant difference in VASI score between the EGCG-treated lesions and pimecrolimus-treated lesions (P = 0.755). Meanwhile, the mean PGA score on the EGCG applied side was 4.39 ± 2.23, while that was 4.43 ± 2.02 on the pimecrolimus applied side (P = 0.886). Furthermore, difference in the improvement degree between pimecrolimus side and EGCG side was not statistically significant (P = 0.845). Notably, no serious side effects were observed throughout the study. CONCLUSION: Findings of the study indicate that topical EGCG can be effective on treating vitiligo.

Caffeic acid derivative WSY6 protects melanocytes from oxidative stress by reducing ROS production and MAPK activation.

Purpose: Vitiligo is a chronic depigmentation disease caused by a loss of functioning melanocytes and melanin from the epidermis. Oxidative stress-induced damage to melanocytes is key in the pathogenesis of vitiligo. WSY6 is a caffeic acid derivative synthesized from epigallocatechin-3-gallate (EGCG). This study is to investigate whether the new chemical WSY6 protected melanocytes from H2O2-induced cell damage and to elucidate the underlying molecular mechanism. Patients and methods: The present study compared the antioxidative potential of WSY6 with EGCG in hydrogen peroxide (H2O2)-treated PIG1 cells. Western blotting was used to study the protein expression of cyto-c, cleaved-caspase3, cleaved-caspase9, and the activation of MAPK family members, including p38, ERK1/2, JNK and their phosphorylation in melanocytes. ROS assay kit to detect intracellular reactive oxygen species production; CCK8 and lactate dehydrogenase leak assay to detect cytotoxicity. Results: EGCG and WSY6 ameliorated H2O2-induced oxidative stress damage in PIG1 cells in a does-dependent manner, while WSY6 was much more effective. WSY6 reduced cellular ROS production, cytochrome c release, downregulated caspase-3 and caspase-9 activation. MAPK pathway signaling including phosphorylated p38, ERK and JNK were observed under oxidative stress and can be much protected by pre-treatment of WSY6. Conclusion: These results indicated that WSY6 could be a more powerful antioxidant than EGCG and protect melanocytes against oxidative cytotoxicity.

Target area treatment ratio of varied lesions in the cultured pure melanocyte transplantation repigmentation of vitiligo: A retrospective study.

Autologous cultured pure melanocyte transplantation (CMT) can be utilized to treat stable vitiligo cases, but clinical data are insufficient to improve its efficacy. To evaluate the influence of various factors on the therapeutic effect of CMT, this single-center retrospective study enrolled stable vitiligo patients who underwent CMT between 2009 and 2020. Univariate and multivariable analysis were used to determine the factors affecting the outcome of repigmentation. The study included 491 patients with long-term follow-up data (6-120 months). It was found that 69.7% of patients achieved an excellent re-color effect and 18.4% achieved a good re-color effect. There were statistically significant differences in pigmentation between patients with stable disease course, vitiligo type, and lesion site. Overall, a significant positive correlation between the target area treatment ratio of varied lesions and the percentage of repigmentation was found. CMT is effective and well tolerated in the treatment of stable vitiligo. Various factors, especially the target area treatment ratio of varied lesions, should be carefully assessed before using CMT. As the target area treatment ratio of varied lesions could further improve the post-operative repigmentation other than type of vitiligo. This clinic trial was approved by Hangzhou Third People's Hospital (number 2023KA015, national clinical record number MR-33-23-034502).

The association between declining lung function and stroke risk: insights from an observational study and Mendelian randomization.

Background: Stroke, prevalent globally, particularly impacts low- and middle-income countries. Decreased lung function is one of the risk factors for stroke, and there is a lack of sufficient research on the association between the two, especially based on evidence from representative large samples. We aimed to explore the association between lung function and stroke incidence. Methods: We collected data from 13,371 participants from the 2007-2012 U.S. national cross-sectional study and 11,192 participants from the Chinese national cohort study during the 2011-2018 follow-up period. Multivariate logistic regression and Cox proportional hazards regression were used to assess cross-sectional and longitudinal associations of peak expiratory flow with stroke risks. Additionally, we used publicly available GWAS data from a European population to conduct Mendelian randomization analysis, further exploring the potential causal relationship. Results: The results of the cross-sectional study suggest that a decline in peak expiratory flow may be associated with an increased risk of stroke. The cohort study revealed that, compared to the first tertile group, the risk of stroke incidence in the second and third tertile groups of PEF decreased by 19% (hazard ratio (HR) = 0.810, 95%CI = 0.684-0.960) and 21.4% (HR = 0.786, 95%CI = 0.647-0.956), respectively. Mendelian randomization analysis clarified that higher PEF levels are significantly associated with a reduced risk of stroke (OR = 0.852, 95%CI = 0.727-0.997). Conclusion: Decreased lung function is a risk factor for stroke. As a simple and accurate indicator of lung function, PEF can be used to monitor lung function in community populations and patients for primary stroke prevention.

[Impact on tyrosinase expression and export from endoplasmic reticulum by inhibition of 26S proteasome].

OBJECTIVE: To determine the impact on tyrosinase expression and export from endoplasmic reticulum by inhibition of 26S proteasome. METHODS: Western blot was used to detect 26S proteasome from 8 vitiligo patients and 4 healthy controls. Melanocytes were incubated with proteasome inhibitor (lactacystin) and further detected as follows: cell survival by MTT assay, proteasome activity with fluorescence, ultrastructure observation with electron microscope, co-localization of tyrosinase and calreticulin (endoplasmic reticulum marker) by confocal laser scanning microscopy and 26S proteasome and tyrosinase with Western blot. RESULTS: The 26S proteasome expression level from lesions of vitiligo (1.05 ± 0.40) was significantly lower than the donor sites (1.82 ± 0.88) and the healthy controls (1.88 ± 0.16) (P < 0.05). But no significant difference existed between the latter two groups (P > 0.05). Compared to the untreated group, a 12-h incubation of 10 µmol/L lactacystin showed inhibitory effects on melanocytes (0.999 ± 0.110 vs 1.372 ± 0.127, P < 0.05) and significantly decreased proteasome activity (0.234 ± 0.019 vs 1, P < 0.01). Expansion rate of endoplasmic reticulum in the lactacystin group (1.91 ± 0.17) was significantly higher than that of the untreated cells (1.17 ± 0.11) (P < 0.05). More tyrosinase co-localized with calreticulin in endoplasmic reticulum in lactacystin-treated cells was observed than that of the untreated group. Compared with the untreated group, significantly decreased levels of tyrosinase (146 ± 10 vs 269 ± 8, P < 0.01) and tyrosinase activity (0.159 ± 0.017 vs 0.221 ± 0.019, P < 0.01) were shown in the lactacystin group (P < 0.05). CONCLUSIONS: Significantly decrease of 26S proteasome is found in lesions of vitiligo patients. Inhibition of 26S proteasome may lead to expansion of endoplasmic reticulum of melanocytes, impact export of tyrosinase from melanocyte endoplasmic reticulum and expression of tyrosinase.

Generalized Vitiligo After Stem Cell Transplantation: A Case Report.

Graft versus host disease (GVHD) is a complex immune-mediated pathophysiological process, which is caused by allogenic immune reactions between donors and recipients. No matter ac-ute or chronic GVHD, skin involvement is the most common, severe skin damage can lead to permanent disfigurement, which seriously affects the long-term quality of life of patients. We herein report a patient with generalized vitiligo after allogeneic peripheral hematopoietic stem cell transplantation (allo-HSCT) for aplastic anemia.

EGR1 Mediated Reduction of Fibroblast Secreted-TGF-ß1 Exacerbated CD8+ T Cell Inflammation and Migration in Vitiligo.

Vitiligo is a T cell-mediated depigment skin disease caused by the complex interplay between melanocyte dysfunction, environmental stimulation, and dysregulated immune signals. Transforming growth factor-ß1 (TGF-ß1), which typically derives from regulatory T cells, has long been identified at low levels in the peripheral system of vitiligo patients. Here, through RNA-sequencing and transcription factor enrichment, we revealed that in response to CD8+ T cell-secreted interferon-gamma (IFN-γ), stromal fibroblast downregulates early growth response 1 (EGR1) activity, leading to TGF-ß1 deficiency. The defective immune regulation loop further exacerbated local CD8+ T cell inflammation and promoted inflammatory cell migration in vitiligo. Thus, fibroblast-derived TGF-ß1 plays an important stromal signal in vitiligo pathogenesis.

Pathogenic Th2 Cytokine Profile Skewing by IFN-γ-Responding Vitiligo Fibroblasts via CCL2/CCL8.

PURPOSE: Vitiligo is a T cell-mediated skin depigmentation disease. Though treatments arresting disease progression and inducing repigmentation are available, the efficacy of these options is often limited and poorly sustained. How stromal signals contribute to the interferon-γ-dominant skin niches is unclear. This study aims to determine how fibroblasts participate in the IFN-γ-dominant vitiligo niche. PATIENTS AND METHODS: Mouse vitiligo models were established. Fibroblasts from control and vitiligo mice were extracted for RNA sequencing. In vitro IFN-γ stimulation was performed to verify the JAK-STAT pathway by qPCR and Western blot. T cell polarization with chemokines was measured by flow cytometry. Protein levels in tissues were also examined by IHC. RESULTS: The vitiligo mouse model recapitulates the human CD8-IFN-γ pathway. RNA sequencing revealed elevated chemokine CCL2 and CCL8 in vitiligo fibroblast, which may be regulated by the JAK-STAT signaling. Such phenomenon is verified by JAK inhibitor peficitinib in vitro. Moreover, CCL2 addition into the naïve T polarization system promoted type 2 cytokines secretion, which represents a hallmark of vitiligo lesions. CONCLUSION: Dermal fibroblasts, a principal constituent of skin structure, respond to IFN-γ by skewing T cells towards a type 2 cytokine profile via CCL2 and CCL8, which can be abrogated by JAK inhibitor peficitinib.

Narrowband Ultraviolet B Combined with Topical Simvastatin Solution in the Treatment of Vitiligo: A Case Report.

Background: Phototherapy is one of the treatments for vitiligo. To be specific, the combination of narrowband ultraviolet B (NB-UVB) with topical preparations has currently become the most common therapeutic modality. Moreover, the research on new topical drug has been a hot issue in the field of vitiligo. Objective: At present, simvastatin has been considered as a potential therapeutic agent for the treatment of vitiligo. To the best of our knowledge, this is the first case report concerning the successful application of NB-UVB combined with topical simvastatin in the treatment of vitiligo. Methods: In this article, a clinical case report was presented, where the patient was not responsive to NB-UVB but was markedly responsive to the treatment of UVB combined with topical simvastatin. Results: A 34-year-old Chinese female patient with vitiligo was cured by NB-UVB combined with topical simvastatin solution. Conclusions: NB-UVB combined with topical simvastatin may be a potential treatment against vitiligo. This research was approved by the Medical Ethics Committee of Third People's Hospital of Hangzhou.

[Corrigendum] The role of VIT1/FBXO11 in the regulation of apoptosis and tyrosinase export from endoplasmic reticulum in cultured melanocytes.

Following the publication of the above article, an interested reader drew to the authors' attention that the Transwell cell migration assay data shown in Fig. 4A appeared to be partly overlapping with data presented for experiments performed under different experimental conditions in Figs. 4D and E. The authors independently examined the figure and realized that inadvertent errors had been made during the assembly of Fig. 4; furthermore, owing to the time that has elapsed since this paper was published, the authors no longer had access to the original data. Accordingly, to further verify the conclusions reported in the study, the authors repeated these experiments, and the results obtained were found to be consistent with the original findings. The new version of Fig. 4 is shown below. The authors are grateful to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 26: 57-65, 2010; DOI: 10.3892/ijmm_00000435].

CXCL9 as a key biomarker of vitiligo activity and prediction of the success of cultured melanocyte transplantation.

This study aimed to investigate the potential biomarkers of vitiligo by evaluating the disease activity and curative effect of autologous cultured pure melanocyte transplantation (CMT) on patients. Altogether, 36patients with stable vitiligo were treated with CMT. Blister fluid samples were collected from patients with stable vitiligo. Patients with active vitiligo were matched with healthy controls. The chemokine levels in the serum and blister fluid samples were measured using Luminex. The curative effect on patients with stable vitiligo was evaluated 6 months after treatment. Treatment responses were defined according to the extent of repigmentation as effective (if 50% or more repigmentation was achieved) or ineffective (if less than 50% or worse repigmentation was achieved). Patients received re-transplantation if the initial treatment was ineffective. The levels of C-X-C motif chemokine ligand (CXCL)9 and CXCL10 in blister fluid samples were significantly lower in stable patients than in active participants. Receiver operating characteristic analysis revealed that the levels of CXCL9 and CXCL10 were sensitive and specific in diagnosing active vitiligo. Further, 65.6% (21/32) of patients who received CMT had effective treatment responses. The high CXCL9 level in the blister fluid was a significant predictor of ineffective treatment responses. The treatment response was significantly enhanced after treatment. Four patients with ineffective treatment responses received anti-inflammatory treatment and re-transplantation. The CXCL9 and CXCL10 levels in the blister fluid were related to the presence of active vitiligo. Also, the CXCL9 level was a predictor of the effectiveness of CMT in treating vitiligo.

Inhibition of Fam114A1 protects melanocytes from apoptosis through higher RACK1 expression.

Fam114A1 is a gene closely related to the development of nerve cells, melanocytes, and nerve cells that originate from the neural crest of the embryonic ectoderm. Recent studies showed that Fam114A1 has a role in the occurrence of ankylosing myelitis spondylitis and autoimmune enteritis; still, its cellular function remains poorly understood. In this study, we investigated the effect of Fam114A1 on the biological activity of melanocytes. We found that the expression of Fam114A1 in vitiligo melanocytes (MCV-L, MCV-N, PI3V) was higher than that in normal melanocytes, and the biological function of melanocytes was significantly affected when the Fam114A1 gene was silenced. Inhibition of Fam114A1 increased proliferation, migration, and melanin synthesis proteins, decreased apoptosis, while its overexpression reversed this process. Mechanistically, we discovered that RACK1 is a target protein of Fam114A1 and that RACK1 can be negatively regulated by Fam114A1. Further study showed that Fam114A1 inhibition could not protect melanocytes from apoptosis once the expression of RACK1 protein was silenced. In summary, Fam114A1 is an effective regulatory protein for regulating the function of melanocytes. Inhibition Fam114A1 protects melanocytes from apoptosis through increasing RACK1.

[Expression of InnVit/FBXO11 in vitiligo and its role in tyrosinase export from endoplasmic reticulum].

OBJECTIVE: To investigate the roles of InnVit (FBX011) gene in melanocytes by detecting the expression of InnVit gene in vitiligo and analyzing the impact of InnVit gene on morphology of endoplasmic reticulum (ER) and the tyrosinase export from ER. METHODS: The lesion tissues and the donor tissues were collected from 10 vitiligo patients to examine the InnVit gene expression by immunohistochemistry. Synthesized specific siRNA and constructed plasmid P3XF-P120 were separately transfected into cells for the silence and over-expression of InnVit gene with lipofectamine(TM) 2000. The untreated cells were used as control. Morphology of ER of cells from the above three groups was observed under electron microscope. Co-localization of tyrosinase and calreticulin was identified by confocal laser scanning microscopy. InnVit, tyrosinase and calreticulin were examined by Western blot. RESULTS: In vitiligo patients, the expression of InnVit gene in the lesions was markedly lower than that in the donor tissues. The normal morphology of ER was found in the untreated and the plasmid groups whereas inflated ER was shown in siRNA group. And the relative inflation rate in siRNA group (1.97 +/- 0.48) was higher than that in the untreated group (1.28 +/- 0.09) and plasmid group (1.24 +/- 0.13) (both P = 0.001). In the untreated and the plasmid groups, tyrosinase was expressed beyond the scope marked by ER marker protein calreticulin partly, but co-localized with calreticulin in ER in the siRNA group. Western blot showed that, contrast to the untreated group (0.320 +/- 0.020), a lower expression level of InnVit in the siRNA group (0.030 +/- 0.004, P = 0.001) and a higher expression of InnVit in the plasmid group were shown (0.710 +/- 0.040, P = 0.001). No significant difference about the expression level of calreticulin was observed among the three groups (P > 0.05). As compared with the untreated group (0.350 +/- 0.030), a higher tyrosinase level in the siRNA group (1.040 +/- 0.060, P = 0.001) and in the plasmid group (0.720 +/- 0.030, P = 0.001) was found. And the former was higher than the latter (P = 0.001). CONCLUSION: A lower expression of InnVit is observed in the lesion tissues than in the donor tissues from vitiligo patients. The InnVit gene can have an impact on the morphology of ER and tyrosinase export from ER. And it may further affect the function of melanocytes.

VIT1/FBXO11 knockdown induces morphological alterations and apoptosis in B10BR mouse melanocytes.

It has been shown that the VIT1 gene is down-regulated in vitiligo melanocytes, but the expression of the VIT1 gene in melanocytes of Chinese patients with vitiligo and the potential function of VIT1 in pathogenesis of vitiligo are not as well known. In this study, we first compared VIT1 gene expression in melanocytes cultured from non-lesional skin of generalized vitiligo patients, normal melanocytes and melanomas in China. The cloned VIT1 ORF cDNA fragments were sequenced and then compared. VIT1 siRNAs were transfected into mouse B10BR melanocytes. The results from semi-quantitative RT-PCR demonstrated that the expression levels of VIT1 in non-lesional melanocytes from vitiligo patients are significantly lower than those in normal melanocytes. In contrast, the expression levels of VIT1 in melanoma are higher. The results also demonstrated that VIT1 is not a novel gene including a fragment of 81 bp flanking by consensus GT and AG sequences on the 5' and 3' ends, which is regarded as an intron. VIT1 without the extra intron is a known gene called FBXO11. Our findings revealed abnormal morphology by light and electron microscopes in FBXO11 siRNA transfected melancytes, which display large epithelioid perikaryon and stubby dendrites with occasional multidendricity as opposed to slender perikaryon and bipolar dendrites of controls. The electron microscopic observation indicated swelling mitochondria and endoplasmic reticulum (ER), and increased lysosomes and bubbles. Further, transfection of FBXO11 siRNA significantly promoted cell apoptosis. Collectively, this study provides comprehensive morphological proof of the relationship between dilation of ER and decreased levels of the FBXO11 gene in vitiligo melanocytes.

Efficacy of the Topical Calcineurin Inhibitors Tacrolimus and Pimecrolimus in the Treatment of Vitiligo in Infants Under 2 Years of Age: A Randomized, Open-Label Pilot Study.

BACKGROUND: The efficacy of topical calcineurin inhibitors (TCIs) for the treatment of infants with vitiligo aged less than 2 years remains to be fully determined. OBJECTIVE: This aim of this pilot study was to assess the efficacy and tolerability of the TCIs tacrolimus and pimecrolimus in infants with vitiligo aged under 2 years. METHODS: Infants with vitiligo aged < 2 years were randomly assigned to receive either tacrolimus ointment 0.03% or pimecrolimus cream 1% for a period of 6 months. During this period, topical treatment was applied twice daily. The proportion of body surface area of the treated lesions, locations, and possible adverse effects were recorded. In addition, the overall satisfaction scores of the patients' parents was evaluated by virtue of the visual analog scale (VAS). RESULTS: Forty-six infants with vitiligo were enrolled in this study. The overall response rate (> 0% repigmentation) was 100%, while the effective rate (> 50% repigmentation) of the tacrolimus and pimecrolimus groups was 69.6% and 65.2%, respectively. Meanwhile, the effective rates for vitiligo located on the head and neck, trunk, and extremities were 70%, 64.3% and 50%, respectively, while the response rates for non-segmental and segmental vitiligo were 74.4% and 28.6%, respectively. Only a low incidence of local adverse reactions (including mild redness and skin picking) was reported during the treatment process. CONCLUSIONS: Topical tacrolimus ointment 0.03% or pimecrolimus cream 1% have efficacy for vitiligo in infants, which serves to achieve an appropriate level of safety and tolerability during the 6-month period of applications. Thus, TCIs proved to be a therapeutic option for vitiligo in infants under 2 years of age.

Decreased SUMOylation of the retinoblastoma protein in keratinocytes during the pathogenesis of vitiligo.

The role of SUMOylation in the pathogenesis of vitiligo has not been reported previously. The present study aimed to reveal abnormalities in small ubiquitin­like modifier (SUMO) conjugation in keratinocytes from depigmented lesions of patients with vitiligo and confirm the role of SUMOylation in keratinocytes from patients with vitiligo. Skin samples used for immunohistochemistry were obtained by punch biopsy from the depigmented lesions of 6 patients. Blisters were produced by vacuum and the roofs were collected for keratinocyte culture. HaCaT cells were transduced with SUMO1 knockdown vectors. The protein expression of SUMO1, SUMO­specific peptidase 1 (SENP1), ubiquitin­conjugating enzyme E2 I (Ubc9), SUMO­activating enzyme subunit 1 (SAE1), cyclin­dependent kinase (CDK)2, CDK6, proliferating cell nuclear antigen (PCNA), retinoblastoma protein (Rb), phosphorylated Rb (pRb) and ß­actin was assessed by western blotting. The SUMOylation status of proteins was assessed by immunoprecipitation. Cell cycle analysis was performed by flow cytometry and cell proliferation rate was investigated using a Cell Counting Kit­8. The results demonstrated that the levels of SUMO1­conjugated proteins were decreased in vitiligo lesions and vitiligo keratinocytes compared with normal controls. The protein expression of Ubc9 was decreased and SENP1 was increased in vitiligo keratinocytes compared with normal keratinocytes, with no alterations in SAE1 expression. Following knockdown of SUMO1 in HaCaT cells, the proliferation of HaCaT cells was reduced and the cell cycle was arrested in G1 phase. Furthermore, the protein expression levels of PCNA, CDK2, CDK6 and pRb were reduced in SUMO1­knockdown HaCaT cells, and SUMOylated Rb was also decreased markedly in keratinocytes from lesions of patients with vitiligo compared with normal keratinocytes. In conclusion, vitiligo lesions in the present study exhibited dysregulated SUMOylation and deSUMOylation balance and dysregulation of cell cycle progression may be present in SUMO1 knockdown HaCaT cells. These results indicate that deSUMOylation of Rb of keratinocytes may serve an important role in vitiligo, providing a novel direction for the study into the mechanism of vitiligo.

Role of chemokines and the corresponding receptors in vitiligo: A pilot study.

To determine the levels and sources of chemokines in the serum and epidermis of vitiligo patients, we examined 80 active patients, 80 stable patients and 40 healthy controls. First, the serum levels of candidate chemokines were measured by Luminex assay, and levels of CCR5, CXCR1 and CXCR3 were measured in peripheral mononuclear cells (PMBC) by flow cytometry. Then, the local epidermis levels of elevated chemokines in vitiligo were tested by Luminex. Finally, the mRNA and protein expression levels of elevated chemokines in HaCaT cells stimulated with interferon (IFN)-γ or tumor necrosis factor (TNF)-α were tested by quantitative real-time polymerase chain reaction and Luminex. The serum levels of CCL5, CXCL8 and CXCL10 in active vitiligo were significantly elevated compared with those in stable vitiligo patients. Furthermore, the levels of CCL3 and CCL4 had weak and positive correlations with the Vitiligo Area Scoring Index. In the peripheral blood of active vitiligo patients, the percentages of CD3+ CD8+ CCR5+ and CD3+ CD8+ CXCR3+ T cells were significantly increased compared with those in stable vitiligo and healthy controls. In the epidermis of lesions, the expression levels of CCL5 and CXCL10 in active vitiligo were significantly increased. In addition, the mRNA and protein levels of CCL5, CXCL8 and CXCL10 were significantly elevated in HaCaT cells after stimulation with TNF-α or IFN-γ. The CCR5/CCL5 and CXCR3/CXCL10 axes may play an important role in the progression and maintenance of vitiligo. Moreover, keratinocytes stimulated with TNF-α and IFN-γ may be a primary source of CCL5 and CXCL10.