Weisong Hong

A Randomized Controlled Study of the Effects of Different Modalities of Narrow‐Band Ultraviolet B Therapy on the Outcome of Cultured Autologous Melanocytes Transplantation in Treating Vitiligo

BACKGROUND Vitiligo is an acquired skin disorder with great social impact. It can be successfully treated using cultured autologous melanocytes transplantation. OBJECTIVE To evaluate the effect of different modalities of narrow‐band ultraviolet B (NB‐UVB) therapy on the outcome of cultured autologous melanocyte transplantation in treating vitiligo. METHODS Patients undergoing cultured autologous melanocyte transplantation were randomly assigned to four different study groups. Group 1 underwent 20 sessions of NB‐UVB treatment before transplantation; Group 2 underwent 30 sessions of NB‐UVB treatment after transplantation; Group 3 underwent 20 sessions of NB‐UVB treatment before transplantation and 30 sessions after transplantation; Group 4 underwent only transplantation. RESULTS Four hundred thirty‐seven patients were enrolled. Group 3 responded best, more than 90% repigmentation was achieved in 81.3% of patients, and 94.8% patients experienced 50% or greater repigmentation. Statistical analysis showed that there was a highly significant difference between the four groups (χ2 = 35.56, p < .001). Homogeneous skin color was obtained on the repigmentation areas, and no scarring or other serious side effects were observed. CONCLUSIONS Cultured autologous melanocyte transplantation is an effective treatment for stable vitiligo. Combination of NB‐UVB therapy with melanocyte transplantation can accelerate repigmentation of transplanted vitiliginous areas, especially if NB‐UVB is given before and after transplantation.

Interferon-γ Induces Senescence in Normal Human Melanocytes

Background Interferon-γ (IFN-γ) plays an important role in the proceedings of vitiligo through recruiting lymphocytes to the lesional skin. However, the potential effects of IFN-γ on skin melanocytes and the subsequent contribution to the vitiligo pathogenesis are still unclear. Objective To investigate the effects of IFN-γ on viability and cellular functions of melanocytes. Methods Primary human melanocytes were treated with IFN-γ. Cell viability, apoptosis, cell cycle melanin content and intracellular reactive oxygen species (ROS) level were measured. mRNA expression was examined by real-time PCR. The release of interleukin 6 (IL-6) and heat shock protein 70 (HSP-70) was monitored by ELISA. β-galactosidase staining was utilized to evaluate melanocyte senescence. Results Persistent IFN-γ treatment induced viability loss, apoptosis, cell cycle arrest and senescence in melanocytes. Melanocyte senescence was characterized as the changes in pigmentation and morphology, as well as the increase of β-galactosidase activity. Increase of p21Cip1/Waf1 protein was evident in melanocytes after IFN-γ treatment. IFN-γ induction of senescence was attenuated by siRNAs against p21, Janus kinase 2 (JAK2) or signal transducer and activator of transcription 1 (STAT1), but not by JAK1 siRNA nor by p53 inhibitor pifithrin-α. IFN-γ treatment increased the accumulation of intracellular ROS in melanocytes, while ROS scavenger N-acetyl cysteine (NAC) effectively inhibited IFN-γ induced p21 expression and melanocyte senescence. IL-6 and HSP-70 release was significantly induced by IFN-γ treatment, which was largely inhibited by NAC. The increase of IL-6 and HSP-70 release could also be observed in senescent melanocytes. Conclusion IFN-γ can induce senescence in melanocytes and consequently enhance their immuno-competency, leading to a vitiligo-prone milieu.

Quercetin attenuates the effects of H2O2 on endoplasmic reticulum morphology and tyrosinase export from the endoplasmic reticulum in melanocytes

Swollen endoplasmic reticulum (ER) is commonly observed in the melanocytes of vitiligo patients; however, the cause and proteins involved in this remain to be elucidated. Oxidative stress has been reported to be involved in the pathogenesis of vitiligo and previous studies have demonstrated that hydrogen peroxide (H2O2) induced melanocyte apoptosis, whereas quercetin exhibited cytoprotective activities against the effects of H2O2. The aim of the present study was to further investigate the role of H2O2 in the ER of melanocytes as well as its role in the export of tyrosinase from ER; in addition, the present study aimed to determine the mechanism by which quercetin protects against the effects of H2O2. The results demonstrated that melanocyte cells treated with H2O2 presented with swollen ER; however, a normal ER configuration was observed in untreated cells as well as quercetin/H2O2‑treated cells. Furthermore, H2O2 inhibited tyrosinase export from the ER and decreased expression levels of tyrosinase; however, quercetin was found to attenuate the effects induced by H2O2. In conclusion, the results of the present study confirmed the hypothesis that H2O2 induced ER dilation and hindered functional tyrosinase export from the ER of melanocytes. It was also found that quercetin significantly weakened these effects mediated by H2O2, therefore it may have the potential for use in the treatment of vitiligo.

Blister roof grafting, cultured melanocytes transplantation and non-cultured epidermal cell suspension transplantation in treating stable vitiligo: A mutual self-control study

Abstract Objectives: To compare the efficacy of blister roof grafting (BG), cultured melanocytes transplantation (CMT) and non-cultured epidermal cell suspension transplantation (NCES) in the treatment of stable vitiligo. Methods: In each person of 83 vitiligo patients one vitiligo macule was selected and divided in three areas for separate treatment with BG, CMT and NCES in the same session. The results were evaluated 12-month post-surgery for the extent of repigmentation and color match. Results: A satisfactory result (>50% repigmentation) was achieved in 92%, 82% and 81% of the 83 patients with the BG, CMT and NCES methods, respectively. Significant differences between the BG and CMT groups (p = 0.038), and between BG and NCES groups (p = 0.017) were observed, but not between the CMT and NCES groups (p = 0.986). The extent of repigmentation on the head neck and trunk was superior to that of the extremities by all the three methods. A difference in the time of onset of repigmentation was observed, with repigmentation first appearing after 10 days, 20–30 days and >30 days in the BG, CMT and NCES groups, respectively. Conclusions: All the three methods are safe and effective to treat vitiligo. Future studies with larger groups are warranted to confirm our results.

Niacin protects against UVB radiation-induced apoptosis in cultured human skin keratinocytes.

Niacin and its related derivatives have been shown to have effects on cellular activities. However, the molecular mechanism of its reduced immunosuppressive effects and photoprotective effects remains unclear. In this study, we investigated the molecular mechanism of the photoprotective effect of niacin in ultraviolet (UV)-irradiated human skin keratinocytes (HaCaT cells). We found that niacin effectively suppressed the UV-induced cell death and cell apoptosis of HaCaT cells. Existing data have shown that AKT activation is involved in the cell survival process. Yet, the potential mechanism of niacin in protection against UV-induced skin damage has thus far not fully been eluvidated. We observed that niacin pretreatment enhances UV induced activation of AKT (Ser473 phosphorylation) as well as that of the downstream signal mTOR (S6 and 4E-BP1 phosphorylation). The PI3K/AKT inhibitor, LY294002, and the mTOR inhibitor, rapamycin, largely neutralized the protective effects of niacin, suggesting that AKT and downstream signaling mTOR/S6 activation are necessary for the niacin-induced protective effects against UV-induced cell death and cell apoptosis. Collectively, our data suggest that niacin may be utilized to prevent UV-induced skin damage and provide a novel mechanism of its photoprotective effects against the UV radiation of sunlight by modulating both AKT and downstream mTOR signaling pathways.

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.

Immunodetection of the MCHR1 antibody in vitiligo patient sera

Human vitiligo is an acquired depigmenting skin disorder characterized by milk-white skin macules resulting from a chronic and progressive loss of melanocytes. It has been suggested that autoimmune mechanisms are involved in this disorder. We undertook this project to obtain an MCHR1-C linear peptide containing four main MCHR1 B-cell epitopes in an attempt to detect the IgG antibody against MCHR1 in vitiligo. The target gene encoding the MCHR1-C peptide was cloned into a pGEX-4T-2 expression vector, expressed in Escherichia coli BL21, and purified using a GST column. The molecular weight was analyzed by SDS-PAGE and western blotting. The IgG antibody response to MCHR1 was detected by ELISA with MCHR1-C as a coated antigen, and the absorption experiment was used to assess the binding ability of the Ab detected by MCHR1-C with a membrane protein in human epidermal melanocytes. The purified peptide MCHR1-C with a molecular weight of 33 kDa was obtained, and could bind to the MCHR1 antibody in human sera. Of the vitiligo patient sera examined, 24 of 145 (16.55%) tested positive for the MCHR1 antibody, and the frequency in the vitiligo patient group was significantly greater compared to the normal control. However, no significant difference between gender, disease duration, clinical subtype or family history between the two groups was observed. In addition, the Ab detected by MCHR1-C in sera was specifically absorbed by the membrane protein obtained from human epidermal melanocytes. Collectively, our data suggest that the MCHR1-C peptide can be used to detect the MCHR1 antibody in vitiligo patients as a coated antigen instead of MCHR1 protein. We conclude that the level of MCHR1 antibody in active vitiligo patients is significantly higher than that in healthy individuals, while gender, disease duration, clinical subtype and family history have no association with the level of MCHR1 antibody in vitiligo patients.