The Treatment of Refractory Vitiligo With Autologous Cultured Epithelium Grafting: A Real-World Retrospective Cohort Study.

BACKGROUND: Surgical intervention is the main therapy for refractory vitiligo. We developed a modified autologous cultured epithelial grafting (ACEG) technique for vitiligo treatment. Between January 2015 and June 2019, a total of 726 patients with vitiligo underwent ACEG in China, with patient characteristics and clinical factors being meticulously documented. Using a generalized linear mixed model, we were able to assess the association between these characteristics and the repigmentation rate. RESULTS: ACEG demonstrated a total efficacy rate of 82.81% (1754/2118) in treating 726 patients, with a higher repigmentation rate of 64.87% compared to conventional surgery at 52.69%. Notably, ACEG showed a better response in treating segmental vitiligo, lesions on lower limbs, age ≤ 18, and stable period > 3 years. A keratinocyte:melanocyte ratio below 25 was found to be advantageous too. Single-cell RNA sequencing analysis revealed an increase in melanocyte count and 2 subclusters of keratinocytes after ACEG, which remained higher in repigmented sites even after 1 year. CONCLUSIONS: ACEG is a promising therapy for refractory vitiligo. Patient age, clinical type, lesion site, and stability before surgery influence repigmentation in ACEG. The mechanism of repigmentation after ACEG treatment is likely not confined to the restoration of melanocyte populations. It may also involve an increase in the number of keratinocytes that support melanocyte function within the affected area. These keratinocytes may aid the post-transplant survival and function of melanocytes by secreting cytokines and extracellular matrix components. TRIAL REGISTRATION: registered with Chictr.org.cn (ChiCTR2100051405).

Single-cell sequencing reveals increased LAMB3-positive basal keratinocytes and ZNF90-positive fibroblasts in autologous cultured epithelium.

Autologous cultured epithelium grafting (ACEG) presents a promising treatment for refractory vitiligo, yet concerns regarding infections and immunological reactions hinder its surgical use due to serum and feeder dependencies. Addressing this, we culture autologous epithelium under serum- and feeder-free (SFF) conditions, comparing its safety and efficacy with serum- and feeder-dependent (SFD) conditions in stable vitiligo patients, and we discover no significant differences in repigmentation between the SFF and SFD grafts. Single-cell RNA transcriptomics on SFF- and SFD-cultured epithelium alongside healthy skin reveal increased populations of LAMB3+ basal keratinocytes and ZNF90+ fibroblasts in the SFF sheets. Functional analyses showcase active cellular metabolism in LAMB3+ basal keratinocytes, vital in extracellular matrix homeostasis, while ZNF90+ fibroblasts demonstrate increased differentiation, essential in collagen formation for cell adhesion. Importantly, these cell populations in SFF sheets exhibit enhanced interactions with melanocytes compared to SFD sheets. Further, knockdown experiments of LAMB3 in keratinocytes and ZNF90 in fibroblasts lead to a downregulation in melanocyte ligand-receptor-related genes. Overall, SFF sheets demonstrate comparable efficacy to SFD sheets, offering superior safety. LAMB3+ basal keratinocytes and ZNF90+ fibroblasts act as potential drivers behind repigmentation in ACEG under SFF conditions. This study provides translational insights into ACEG repigmentation and potential therapeutic targets for vitiligo.

The complete chloroplast genome of a karst-dwelling plant Primulina tenuituba (Gesneriaceae).

Primulina tenuituba is a species in the Gesneriaceae family that is widely distributed in China. It is a karst-dwelling species with an enormous tolerance for extreme drought and high temperatures. The species is also used in traditional Chinese medicine. In this study, the complete chloroplast genome of P. tenuituba was assembled and characterized for the first time. The complete chloroplast genome exhibited a typical quadripartite cycle of 153,236 bp in length, including a pair of inverted repeats (IRs) of 25,494 bp, which were separated by a large single-copy (LSC) region of 84,364 bp and a small single copy (SSC) region of 17,884 bp. The GC content was 37.6%. The complete chloroplast genome of P. tenuituba contains 114 genes, including 80 protein-coding genes, 30 tRNAs genes, and four rRNAs. The phylogenetic analysis showed that P. tenuituba is closely related to P. eburnea. The newly reported chloroplast genome of P. tenuituba would provide valuable data for further studies on its evolution and adaptation mechanism.

Eosinophils and associated parameters in different types of skin diseases related to elevated eosinophil levels.

Background: Eosinophils, basophils, white blood cells (WBC), and immunoglobulin E (IgE) play major roles in the pathogenesis of atopic dermatitis (AD), bullous pemphigoid (BP), drug reaction with eosinophilia and systemic symptoms (DRESS), and hypereosinophilic syndrome (HES). This study aimed to describe these parameters in different skin diseases and provide further information concerning the underlying pathogenesis. Methods: A cross-sectional study of blood test results, including WBC count, peripheral eosinophil count, peripheral basophil/WBC percentage, and IgE level, from 115 cases of AD, 75 cases of BP, 55 cases of DRESS, 119 cases of HES, and 621 healthy volunteers was performed in China. Data from before and after treatment and the population distribution of different diseases were compared and described. Results: All participants showed increased peripheral eosinophil counts, eosinophil/WBC ratios, IgE levels, and decreased peripheral basophil counts, with variance among the different disease groups. Peripheral eosinophil counts in HES patients and IgE level in AD patients increased the most prominently. No significant correlation existed among eosinophils, basophils, and IgE. An obvious decrease in eosinophil count was demonstrated after treatment in all 4 diseases. Conclusions: Eosinophils, basophils, and IgE exert functions in diverse skin diseases, presenting altered peripheral blood test results. In some cases, these changes are demonstrated only in the skin and not in the blood. Compared with the other parameters considered in this study, eosinophils seemed to be a better biomarker for treatment effects, regardless of the disease type.

Prevalence of vitiligo and associated comorbidities in adults in Shanghai, China: a community-based, cross-sectional survey.

BACKGROUND: The prevalence of vitiligo has been reported to range from 0.1% to 8% worldwide, and vitiligo has been linked to some autoimmune and non-autoimmune diseases. This study aimed to estimate the prevalence of vitiligo and associated comorbidities in adults in Shanghai. METHODS: A community-based cross-sectional survey was conducted among 9,114 adults (4,288 males) in a community of Shanghai between October 2009 and January 2010. Face-to-face interviews were conducted at the home of each participant, and all respondents had their skin examined by dermatologists. The risks of comorbidities associated with vitiligo were evaluated by multiple logistic regression analysis. RESULTS: The estimated prevalence of vitiligo was 0.91%, and the standardized (age-adjusted) prevalence was 0.67%. Prevalence increased with age from 0.20% in 18-30 years to 1.59% in the 71-80 years age group. The presence of vitiligo was associated with increased risks of atopic dermatitis [adjusted odds ratio (aOR) =2.49; 95% confidence interval (95% CI): 1.46-4.23], urticaria (aOR =1.83; 95% CI: 1.11-3.04). and coronary heart disease (aOR =1.88; 95% CI: 1.03-3.41), although the association with coronary heart disease was only identified in subjects who were aged ≥60 years or overweight. CONCLUSIONS: The prevalence of vitiligo in Shanghai was comparable to that seen in previous studies and increased with age. Vitiligo was associated with increased risks of atopic dermatitis, urticaria, and coronary heart disease in adults.

Predicting the dynamic distribution of Sphagnum bogs in China under climate change since the last interglacial period.

Sphagnum bogs possess irreplaceable ecological and economic value, and they are scarce in China, with a fragmented distribution. Based on 19 high-resolution bioclimatic environmental datasets and 71 bog center point locations, we employed a maximum entropy model (MaxEnt) to reconstruct and predict the spatial-temporal geographical distribution patterns of Sphagnum bogs from the last interglacial (LIG) period to two typical CO2 representative concentration pathway scenarios (RCP2.6, RCP8.5) in the future. We further computed the migratory paths of the distribution center points. Finally, a jackknife test was used to uncover the crucial environmental factors restricting the geographical distribution of the bogs. Our data indicated that the MaxEnt niche model had a high simulation precision with an area under the ROC curve value of 0.957. Spatially, the suitable bog habitats are currently centralized in northeastern China, including the Greater Khingan Mountains, the Lesser Khingan Mountains, and the Changbai Mountains, as well as peripheral areas of the Sichuan Basin. Temporally, the contours of Sphagnum bogs were similar to the present and rendered from the last glacial maximum (LMG) period, and had much more total area than the current. The total area in LIG was nearly the same as the current because of the similar climate. It was worth noting that there would be a reduction of the total area in the future. Loss of area occurred at the edges of bogs, especially under RCP8.5. The distribution center of bogs will shift to the northwest in the immediate future. The precipitation of driest month, the mean temperature of warmest quarter and the precipitation of warmest quarter were identified as crucial climatic factors affecting the distribution of Sphagnum bogs. Overall, our research provides scientific evidence for the long-term protection and effective management of these rare, precious natural resources and suggestions for in situ conservation.

Molecular Hydrogen Protects Human Melanocytes from Oxidative Stress by Activating Nrf2 Signaling.

Oxidative stress is proven to be critical for the initiation and progression of vitiligo. Molecular hydrogen (H2) possesses potent antioxidant activity and has been shown to protect against various oxidative stress-related diseases. In this study, we first investigated the effects and mechanisms of H2 in human melanocytes damaged by hydrogen peroxide. We initially found that H2 reduced intracellular ROS accumulation and malondialdehyde levels in both vitiligo specimens and hydrogen peroxide-treated melanocytes in vitro in a concentration- and time-dependent manner, concomitant with the enhancement of antioxidant enzyme activity. Correspondingly, H2 reversed hydrogen peroxide-induced apoptosis and dysfunction in both normal and vitiligo melanocytes. H2 protected mitochondrial morphology and function in melanocytes under stress and promoted the activation of Nrf2 signaling, whereas Nrf2 deficiency abolished the protective effect of H2 against hydrogen peroxide-induced oxidative damage. Furthermore, H2 positively modulated ß-catenin in hydrogen peroxide-treated melanocytes, and the ß-catenin pathway was implicated in H2-induced Nrf2 activation. Collectively, our results indicate that H2 could be a promising therapeutic agent for vitiligo treatment via attenuating oxidative damage, and its beneficial effect in human melanocytes might involve Wnt/ß-catenin-mediated activation of Nrf2 signaling.

Suppression of FADS1 induces ROS generation, cell cycle arrest, and apoptosis in melanocytes: implications for vitiligo.

Vitiligo is a potentially serious condition characterized by loss of melanin and death of melanocytes. To identify potential therapeutic targets for vitiligo, we conducted a microarray analysis of three human vitiligo specimens and paired adjacent normal tissues. Because we found that the fatty acid desaturase 1 (FADS1) gene was downregulated in vitiligo specimens, we carried out experiments to assess its role in melanocyte replication and survival. RT-qPCR was used to verify that FADS1 expression was lower in vitiligo-affected tissues and vitiligo melanocyte PIG3V cells than in matched controls or normal human epidermal PIG1 melanocytes. In addition, CCK-8, immunofluorescence, western blot and flow cytometry assay were used to detect the proliferation and apoptosis in PIG1 cells respectively. Overexpression of FADS1 promoted proliferation of PIG3V melanocytes, while FADS1 silencing inhibited proliferation and induced cell death in PIG1 melanocytes. Increased ROS generation; induction of mitochondrial-mediated apoptosis via upregulation of Bax and active caspases 3 and 9 and downregulation of Bcl-2; and cell cycle arrest via downregulation of c-Myc and Cyclin D1 and upregulation of p21 were all enhanced after FADS1 silencing in PIG1 melanocytes. These findings implicate FADS1 downregulation in the pathogenesis of vitiligo and may open new avenues for its treatment.

Salvianolic acid B attenuates experimental pulmonary inflammation by protecting endothelial cells against oxidative stress injury.

Endothelial cell injury and subsequent inflammation play pivotal roles in the pathogenesis of pulmonary fibrosis, a progressive and fatal disorder. We found previously that salvianolic acid B (SAB) attenuated experimental pulmonary fibrosis. Pulmonary fibrosis is driven by inflammation, but the anti-inflammatory role and mechanism of SAB on the treatment of pulmonary fibrosis is still unknown. Here, our in vivo studies showed that SAB had a strong anti-inflammatory effect on bleomycin-instilled mice by inhibiting inflammatory cell infiltration and inflammatory cytokine production. Moreover, SAB protected endothelial cells against oxidative stress injury and inhibited endothelial cell apoptosis in bleomycin-treated mice. The in vitro studies also showed that SAB decreased the H2O2-induced overproduction of reactive oxygen species to protect EA.hy926 endothelial cells from oxidative damage, and further inhibited H2O2-induced permeability and overexpression of pro-inflammatory molecules. The next studies revealed that SAB inhibited the H2O2-induced cell apoptosis and attenuated the decrease of tight junction-related gene expression, resulting in a decrease of the endothelial permeability in injured endothelial cells. Furthermore, Western blot analysis suggested that SAB decreased endothelial cell permeability and expression of pro-inflammatory cytokines by inhibiting MAPK and NF-κB signaling pathways. Taken together, these data indicate that SAB exerted anti-inflammatory roles in pulmonary fibrosis by protection of the endothelial cells against oxidative stress injury, mediated by inhibition of endothelial permeability and expression of pro-inflammatory cytokine via the MAPK and NF-κB signaling pathways.

Vitamin D protects human melanocytes against oxidative damage by activation of Wnt/ß-catenin signaling.

Vitamin D analogs have been widely utilized for the treatment of vitiligo, but the molecular mechanism underlying their pharmacological effects (especially their antioxidant properties) has not yet been investigated. We evaluated the relationship between serum vitamin D level and oxidative damage severity in vitiligo patients, and investigated the molecular mechanism of vitamin D in protecting melanocytes against oxidative stress. Serum levels of 25-hydroxyvitamin D and malondialdehyde (MDA) were first measured in patients. A variety of in vitro experiments such as intracellular reactive oxygen species (ROS), cellular viability, migration, and apoptotic assays were then performed to detect the effects of vitamin D or ß-catenin silencing on H2O2-treated melanocytes. Expression of Wnt/ß-catenin, Nrf2, apoptotic, and MITF pathways was finally examined using quantitative real-time PCR and western blot. In this study, we initially found that vitamin D insufficiency was closely associated with the severity of oxidative stress in vitiligo patients. Using ex vivo cell models, we further showed that vitamin D positively modulated ß-catenin signaling at both translational and posttranslational levels in melanocytes under oxidative stress. Like WNT agonists, vitamin D significantly inhibited ROS accumulation and cell apoptosis in H2O2-treated melanocytes and promoted their proliferative and migratory activity, while the protective effects of vitamin D against oxidative stress were abolished by ß-catenin silencing in melanocytes. Furthermore, ß-catenin deficiency also blocked the activation of Nrf2 and MITF as well as the inhibition of apoptosis induced by vitamin D. Taken together, vitamin D insufficiency was associated with severity of oxidative stress in vitiligo patients. Our work also provides new insights into the mechanism of vitamin D against vitiligo, in which vitamin D protects melanocytes against oxidative stress by activating Wnt/ß-catenin signaling.

Hydrogen gas inhalation protects against cutaneous ischaemia/reperfusion injury in a mouse model of pressure ulcer.

Pressure ulcer formation depends on various factors among which repetitive ischaemia/reperfusion(I/R) injury plays a vital role. Molecular hydrogen (H2 ) was reported to have protective effects on I/R injuries of various internal organs. In this study, we investigated the effects of H2 inhalation on pressure ulcer and the underlying mechanisms. H2 inhalation significantly reduced wound area, 8-oxo-dG level (oxidative DNA damage) and cell apoptosis rates in skin lesions. H2 remarkably decreased ROS accumulation and enhanced antioxidant enzymes activities by up-regulating expression of Nrf2 and its downstream components in wound tissue and/or H2 O2 -treated endothelia. Meanwhile, H2 inhibited the overexpression of MCP-1, E-selectin, P-selectin and ICAM-1 in oxidant-induced endothelia and reduced inflammatory cells infiltration and proinflammatory cytokines (TNF-α, IL-1, IL-6 and IL-8) production in the wound. Furthermore, H2 promoted the expression of pro-healing factors (IL-22, TGF-ß, VEGF and IGF1) and inhibited the production of MMP9 in wound tissue in parallel with acceleration of cutaneous collagen synthesis. Taken together, these data indicated that H2 inhalation suppressed the formation of pressure ulcer in a mouse model. Molecular hydrogen has potentials as a novel and alternative therapy for severe pressure ulcer. The therapeutic effects of molecular hydrogen might be related to its antioxidant, anti-inflammatory, pro-healing actions.

The effects of phototherapy and melanocytes on keratinocytes.

Phototherapy is widely used in the treatment of vitiligo. Previous studies have focused on the effects of ultraviolet (UV) radiation on melanocytes; however, the biological effects of phototherapy and melanocytes on keratinocytes remain to be elucidated. To investigate and assess the effects of clinically doses of broad band (BB)-UVA, narrow band (NB)-UVB and melanocytes on human keratinocytes in vitro, clinical doses of BB-UVA or NB-UVB radiation and human melanoma cell A375 co-culture were performed as stress divisors to HaCaT cells. Cell proliferation, expression of protease-activated receptor-2 (PAR-2) and nuclear factor E2-related factor 2 mRNA, lipid peroxidation and intracellular antioxidant level of keratinocytes were analyzed. It was demonstrated that UV radiation inhibited the proliferation of cells apart from following exposure to low dose (1 J/cm2) UVA. Medium dose (5 J/cm2) UVA radiation had no adverse effects on lipid peroxidation and increased antioxidant levels in HaCaT cells. Medium (200 mJ/cm2) and high (400 mJ/cm2) doses of UVB radiation induced cellular damage due to increased lipid peroxidation as indicated by levels of malondialdehyde. Furthermore, A375 co-culture treatment induced a similar effect on the lipid peroxidation of HaCaT as with low dose UVB radiation. Therefore, the results of the present study determined that clinical doses of BB-UVA and NB-UVB radiation had varying effects on proliferation and related protein levels in HaCaT cells. Co-culture with A375 had similar effects as those of low dose UVA and UVB radiation, in which the PAR-2 expression was significantly upregulated.

Modified Technique of Cultured Epithelial Cells Transplantation on Facial Segmental Vitiligo.

BACKGROUND: Cultured epithelial cells transplantation is a known surgical technique for vitiligo. OBJECTIVE: To evaluate the factors influencing efficacy and safety of cultured epithelial cells transplantation in 9-month follow-up. METHODS: Demographic, clinical, and repigmentation outcomes were reviewed for patients with facial segmental vitiligo who had undergone cultured epithelial cells transplantation from November 2013 to July 2015 at the clinic of the Department of Dermatology, Huashan Hospital, China. RESULTS: Twenty-eight patients who had undergone cultured epithelial cells transplantation were included. A satisfactory result (>50% repigmentation) was achieved in 79% patients with facial segmental vitiligo in 9 months. The treatment effect was significantly different in 6th month (P = 0.032), 9th month (P = 0.006) compared with 3rd month. Disease stability did significantly affect repigmentation outcome in 9th month (Z = 2.113, P = 0.035). No significant difference was observed between single segmental type versus mixed type (Z = 1.081, P = 0.280). Adverse effects were nearly absent. CONCLUSION: Cultured epithelial cells transplantation is a relatively safe and effective therapy for facial segmental stable vitiligo patients.

Low-concentration hydrogen peroxide can upregulate keratinocyte intracellular calcium and PAR-2 expression in a human keratinocyte-melanocyte co-culture system.

Hydrogen peroxide (H2O2) may have a biphasic effect on melanin synthesis and melanosome transfer. High H2O2 concentrations are involved in impaired melanosome transfer in vitiligo. However, low H2O2 concentration promotes the beneficial proliferation and migration of melanocytes. The aim of this study was to explore low H2O2 and its mechanism in melanosome transfer, protease-activated receptor-2 (PAR-2) expression and calcium balance. Melanosomes were fluorescein-labeled for clear visualization of their transfer. The expression of protease-activated receptor-2 (PAR-2) in keratinocytes was determined by western blot analysis. Flow cytometry was employed to evaluate the effects of H2O2 on calcium levels in keratinocytes. Fluorescence microscopy showed the upregulation of melanosome transfer into keratinocytes following 0.3 mM H2O2 treatment in the co-cultures rather than in the untreated control groups, which was associated with higher expression of PAR-2 protein and increased calcium concentration. The addition of a PAR-2 antagonist inhibited the positive activity of H2O2 and calcium flow in keratinocytes. When calcium flow was blocked by a calcium chelator, the addition of H2O2 did not increase the PAR-2 expression level in keratinocytes, therefore, inhibiting dendrite formation and melanosome transfer. Low H2O2 concentration promotes melanosome transfer with increased PAR-2 expression level and calcium concentration in keratinocytes. In addition, the interaction between melanocytes and keratinocytes is more beneficial to enhance calcium levels in keratinocytes which mediate melanin transfer. Moreover, low H2O2 concentration promotes dendrite formation, in which extracellular calcium and Par-2 were involved.

Oxidation levels differentially impact melanocytes: low versus high concentration of hydrogen peroxide promotes melanin synthesis and melanosome transfer.

BACKGROUND: UVB light can generate potentially harmful hydrogen peroxide (H(2)O(2)) in vivo, but it can also promote the beneficial proliferation and migration of melanocytes. The successful use of UVB monotherapy for treatment of vitiligo suggests that H(2)O(2) may have a biphasic effect on melanin synthesis and melanosome transfer. OBJECTIVE: To study the beneficial role of H(2)O(2) on melanogenesis and melanosome transport in living melanocytes and keratinocytes. METHODS: A co-culture system model was constructed using the primary human melanocytes and keratinocytes. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine cell proliferation, NaOH was used to determine the melanin content, and real-time PCR was used to determine tyrosinase expression. Western blot was used to determine Rab-27A and protease-activated receptor 2 (PAR-2) expression. RESULTS: This study demonstrated that tyrosinase was activated by low concentrations of H(2)O(2) (≤0.3 mM); however, this activity was downregulated by high concentrations of H(2)O(2) (>0.3 mM). Activation of high levels of melanin synthesis was induced when cells were treated with low concentrations of H(2)O(2) (0.3 mM). Further observation using an in vitro co-culture system of fluorescein (carboxyfluorescein diacetate succinimidyl ester, CFDA-SE)-labeled melanocytes and keratinocytes indicated that melanosome transfer occurred in normal human epidermal melanocytes. Fluorescence microscopy revealed increased melanosome transfer into keratinocytes treated with 0.3 mM H(2)O(2) in the co-culture compared to the control. Examination of melanosomes in the keratinocytes by flow cytometry confirmed these results. Furthermore, treatment with H(2)O(2) (0.3 mM) upregulated the expression of Rab-27A and PAR-2, significant proteins involved in melanosome transfer, according to Western blot. CONCLUSION: These results confirmed that low concentration levels of H(2)O(2) play a major role in the regulation of human pigmentation by increasing melanin synthesis and melanosome transfer.

Childhood vitiligo in China: clinical profiles and immunological findings in 620 cases.

BACKGROUND: Childhood vitiligo is a common pediatric skin disorder. The pathogenesis of vitiligo is unclear, and immunological dysfunction may play an important role. OBJECTIVES: This prospective study aimed to profile childhood vitiligo and to discuss its correlation with immunological dysfunction. METHODS: All of the 620 enrolled patients were aged younger than 14 years, and were assessed with a standard questionnaire. The levels of immunoglobulins, complement, and T-lymphocyte subsets were measured in 270 of these 620 patients. RESULTS: Of the 620 children, 302 (48.71%) were boys and 318 (51.29%) were girls, with an average disease onset age of 7.57 years. The average duration was 13.45 months. 453 (73.06%) children had head and neck involvement and 160 (25.81%) children had segmental vitiligo. 84 (13.55%) children had a family history. There was a correlation between the disease and seasons. The onset or progression usually occurred in summer and spring. Halo nevus was seen in both segmental and non-segmental vitiligo. Precipitating factors such as stress appeared more commonly in segmental vitiligo. As to the immunological findings, in segmental vitiligo, the levels of C3 and C4 were lower in the active relative to the quiescent stage (p < 0.05); and in non-segmental vitiligo, the percentages of CD3+ and CD4+ lymphocytes and the CD4+/CD8+ ratio were lower in the active relative to the quiescent stage (p < 0.01). CONCLUSIONS: Childhood vitiligo has its own clinical features. The different types of vitiligo have different characteristics. There is immunological dysfunction in children with vitiligo. Dysfunction of humoral immunity may play a role in the progression of segmental vitiligo, while non-segmental vitiligo is more related to cellular immunity.