Effects of keratinocyte-derived and fibroblast-derived exosomes on human epidermal melanocytes.

BACKGROUND: Exosomes have been demonstrated to carry proteins, membrane lipids, mRNAs and microRNAs which can be transferred to surrounding cells and regulate the functions of those recipient cells. OBJECTIVES: The objective of the study was to investigate the effects of exosomes released by keratinocytes and fibroblasts on the proliferation, tyrosinase activity and melanogenesis of melanocytes. METHODS: Melanocytes, keratinocytes and fibroblasts obtained from human foreskin were cultured and exosomes secreted by keratinocytes and fibroblasts were harvested from the culture supernatants by ultracentrifugation. Each exosome fraction was divided into two parts; one part was subjected to high-throughput sequencing using an Illumina HiSeq sequencer to characterize the microRNA expression profiles, while the other part was labeled with the fluorescent dye PKH67 and was then co-cultivated with epidermal melanocytes. RESULTS: High-throughput sequencing analysis showed 168 differentially expressed microRNA within exosomes derived from keratinocytes and from fibroblasts, 97 of those being up-regulated with the other 71 down-regulated. Gene ontology analysis showed that the target genes responsible for these differentially expressed microRNAs were mainly enriched in the protein-binding region of molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that target genes regulated by differentially expressed microRNA were mainly involved in mitogen-activated protein kinase (MAPK) signaling pathway, Ras signaling pathway, cAMP signaling pathway and Wnt signaling pathway. Keratinocyte-derived exosomes were taken up by melanocytes co-cultured with them and promoted the proliferation, tyrosinase activity and melanin synthesis of those melanocytes. However, fibroblast-derived exosomes had no similar effects on melanocytes. CONCLUSION: Keratinocyte-derived exosomes but not fibroblast-derived exosomes were taken up by melanocytes in co-culture and significantly stimulated their proliferation, tyrosinase activity and melanin synthesis. Those different effects may be mainly due to the differential expression of microRNAs in exosomes derived from the different types of cells. LIMITATIONS: Electron microscopy of the obtained exosomes and in-depth study of apparently differentially expressed microRNAs were not performed.

Experimental study and clinical observations of autologous hair follicle cell transplants to treat stable vitiligo.

BACKGROUND: Vitiligo is characterized by the loss and/or dysfunction of melanocytes in the skin and has a profound impact on the social interactions of patients. Although there are many treatment options for vitiligo, the outcome is frequently unsatisfactory, especially for patients with stable vitiligo. OBJECTIVES: To study the biological properties of melanocytes derived from human hair follicles and to observe the efficacy of using transplants of autologous hair follicle cells to treat patients with stable vitiligo. METHODS: From February 2014 to March 2017, 26 patients with stable vitiligo, who were refractory to all current routine therapy, underwent treatment with transplanted autologous hair follicle cells. The skin graft from each patient's occipital region was trimmed to remove excess adipose tissue and some of the upper part of the dermis. The remaining tissue, including hair follicles and dermal papillae, was cut into pieces and incubated in collagenase type IV and then in trypsin-ethylenediaminetetraacetic acid solutions. The cells were recovered, resuspended in the patient's own serum and then applied to the recipient area. Clinical observations continued for 6 months to 1 year. Laboratory experiments were also performed during this time on scalp specimens obtained from normal human volunteers. Cells migrating from the outer root sheath and the dermal papillae at various times of culture were observed using a microscope. RESULTS: Most of the repigmentation in the vitiligo areas appeared within 8 weeks of transplantation of autologous hair follicle cells. Early skin repigmentation was not uniform and appeared more repigmented than the surrounding normal skin. As time went by, the repigmentation became more obvious and matched the color of the skin around the lesion. Most of the pigmentation presented as a diffuse pattern and was not localized around the hair follicles. Among the 26 patients, 9 (34.6%) achieved excellent repigmentation, while 13 (50.0%) had good, 3 (11.5%) fair and 1 (3.9%) poor repigmentation. During the follow-up visit at 1 year, no excess hair growth was observed in the recipient areas and there was no scarring or ulcer formation in the donor or recipient areas. In the experimental part of the study, many keratinocytes, melanocytes and fibroblasts migrated from the adherent outer root sheath. In later subcultures using a specialized medium, pure melanocytes were obtained that had a strong proliferative capacity and had bipolar or poly-dendritic shapes. On the other hand, cells from the dermal papillae grew radially in primary culture and were almost fibroblast-like. However, a few bipolar melanocytes appeared in the later stage of culture. CONCLUSIONS: The results of our study show that transplantation of autologous hair follicle cells is a simple and effective method to treat patients with stable vitiligo. Hair follicles (especially the outer root sheath) harbor many melanocytes with potential proliferative ability. LIMITATIONS: There are a few limitations of the present study: a small sample size, a short follow-up period, no cell counting or viability testing.

Prevalence and associated factors of intra-articular lesions in acute ankle fractures evaluated by arthroscopy and clinical outcomes with minimum 24-month follow-up.

BACKGROUND: Acute ankle fractures can lead to high rate of concomitant intra-articular lesions which may compromise clinical results. The purpose of this study was to evaluate the incidence of concomitant intra-articular lesions in acute ankle fractures with arthroscopy. We also sought to analyze the relationship between intra-articular lesions and the fracture type, as well as the severity of the fracture. METHODS: It was a retrospective cohort study. From April 2014 to December 2015, we have chosen arthroscopy-assisted open reduction and internal fixation (AORIF) for the treatment of unstable acute ankle fractures. All concomitant intra-articular lesions were assessed and documented carefully and prospectively, such as ligament injuries, osteochondral lesions, and tibiofibular syndesmosis injuries. All fractures were classified according to the Lauge-Hansen classification system. The American Orthopedic Foot and Ankle Society's (AOFAS) ankle-hindfoot scale was used to assess post-operative function. Statistical comparisons between the intra-articular lesions, the fracture type, and the severity of the presenting fracture were performed using a Chi-squared analysis. RESULTS: Data of 36 patients were analyzed in the study, including 23 supination-type fractures and 13 pronation-type fractures. The incidence of tibiofibular syndesmosis injuries, chondral lesions, and loose bodies were 92%, 72%, and 39%, respectively. Avulsion fractures of the anterior tibiofibular syndesmosis were more commonly found in supination-type fractures than pronation-type fracture (45% vs. 15%, χ = 5.78, P = 0.02), which would cause mechanical blocking in the anterior portion of the ankle. On the contrary, chondral lesions were more commonly found in the more severe fractures than mild fractures (86% vs. 53%, χ = 4.57, P = 0.03). A mean 41.7 months (range, 33.0-51.0 months) of follow-up was achieved. A mean AOFAS's ankle-hindfoot scale was 96.9, and 97.2% of the patients were satisfied with the procedure. CONCLUSIONS: Acute ankle fractures have a high incidence of concomitant intra-articular lesions. Avulsion fractures of the anterior tibiofibular syndesmosis are more commonly found in supination-type fractures. Chondral lesions are related to the severity of the fractures, but not with the classification of the fractures. AORIF can be one reliable solution in dealing with the associated injuries seen with acute ankle fractures.

Melanocyte spheroids are formed by repetitive long-term trypsinization.

BACKGROUND: Autologous melanocyte transplantation plays an important role in the treatment of vitiligo. OBJECTIVE: Previous studies have indicated that, compared with melanocytes growing in monolayers, melanocyte spheroids have a better survival in growth factor- and serum-deprived conditions. METHODS: Melanocyte spheroids were obtained from human epidermis by repetitive long-term trypsinization and maintained an aggregated morphology for a short period in certain conditions. RESULTS: Melanocyte spheroids were capable of growing into normal dendritic melanocytes in monolayer when they were harvested and reinoculated in 24-well plates. Immunohistochemical analysis of the melanocyte spheroids revealed that they were positive for HMB45, a melanosome-specific marker. No melanomas occurred when melanocyte spheroids were transplanted into mice. CONCLUSION: Our study provides a promising approach for melanocyte transplantation to treat vitiligo.

[Development of external medicine and dermatology of TCM in the Republican period].

The inheritance of the theory in external medicine in the Republican period was mainly derived from Wai ke zheng zong (Orthodox Manual of External Medicine) and Yi zong jin jian (Golden Mirror of Medicine) and the sorting out of medical plasters, pills, and powders from Wai ke shi san fang (Thirteen Recipes of External Medicine). Zhang Shanlei's Yang ke gang yao (Guidelines for Ulcerations), based on the five-year experience of Zhu's family, offers new experience and ideas for syndrome differentiation; Ding Ganren's diagnosis for external medicine is exquisite with many well known formulae prescribed by himself; Monographs on leprosy and syphilis were published by famous physicians. Departments of external medicine and anal diseases, and classes on external medicine and dermatology and venereal diseases were set up in TCM schools in Guangdong and Zhejiang provinces with teaching materials compiled. All these mark the development of external medicine in modern age.

Effects of calcium cyanamide on soil microbial communities and Fusarium oxysporum f. sp. cucumberinum.

Calcium cyanamide (CaCN(2)) has been one of the potential candidates as soil disinfectant since the restriction of methyl bromide in soil fumigation due to its ecological risk. However, little information is available on effects of CaCN(2) on soil microbial community. In this study, the soil microbial communities and the fate of pathogen Fusarium oxysporum (Schlechtend, Fr) f. sp. cucumberinum (Owen) Snyder and Hansen (F.O. f. sp. cucumberinum) in response to CaCN(2) treatment was evaluated. F.O. f. sp. cucumberinum population in soil treated with CaCN(2) at rates of 80 and 200 gm(-2) was suppressed by 88.7 and 92.2% after 15 d of CaCN(2) application. Bacterial, fungal, and actinomycete populations were also greatly decreased after 3 d of CaCN(2) application, but they recovered to the control level by 15 d. The variation in functional diversity of soil microbes characterized by principal component analysis, diversity and evenness indices based on Biolog data followed a similar trend. Meanwhile, the band number from the DGGE of soil 16S rDNA fragments increased from 9 for the non-CaCN(2)-treated soil to 10 or 12 after different rates of CaCN(2) application at 15 d, indicating the increase of abundant rDNA types in the community. The results suggest that CaCN(2) application had only a short-term and transitory impact on the indigenous soil microbial community in contrast to the long-term suppression of the F.O. f. sp. cucumberinum population. It is feasible to reduce Fusarium wilt without significant impact on microbial community by application of CaCN(2) at reasonable doses.

[Research on QSPR for n-octanol-water partition coefficients of organic compounds based on genetic algorithms-support vector machine and genetic algorithms-radial basis function neural networks].

A modified method to develop quantitative structure-property relationship (QSPR) models of organic compounds was proposed based on genetic algorithm (GA) and support vector machine (SVM) (GA-SVM). GA was used to perform the variable selection, and SVM was used to construct QSPR models. GA-SVM was applied to develop the QSPR models for n-octanol-water partition coefficients ( Kow) of 38 typical organic compounds in food industry. 5 descriptors (molecular weights, Hansen polarity, boiling point, percent oxygen and percent hydrogen) were selected in the QSPR model. The coefficient of multiple determination (R2), the sum of squares due to error (SSE) and the root mean squared error (RMSE) values between the measured values and predicted values of the model developed by GA-SVM are 0.999, 0.048 and 0.036, respectively, indicating good predictive capability for lgKow values of these organic compounds. Based on leave-one-out cross validation, the QSPR model constructed by GA-SVM showed good robustness (SSE = 0.295, RMSE = 0.089, R2 = 0.995). Moreover, the models developed by GA-SVM were compared with the models constructed by genetic algorithm-radial basis function neural network (GA-RBFNN) and linear method. The models constructed by GA-SVM show the optimal predictive capability and robustness in the comparison, which illustrates GA-SVM is the optimal method for developing QSPR models for lgKow values of these organic compounds.

HLA linked with leprosy in Southern China; HLA-linked resistance alleles to leprosy

According to the World Health Organization recommended multidrug therapy (WHO/MDT), we have carried out this study to investigate the presence of HLA-linked susceptibility or resistance to leprosy in a southern Chinese population. Sixty-nine leprosy patients and 112 healthy controls participated in the study. HLA-DR2 subtypes, HLA-B and MHC Class I chain-related A (MICA) alleles were typed at the DNA level using the polymerase chain reaction-single strand conformation polymorphism method. The frequencies of HLA-DR2-DRB1 alleles did not show any significant differences between the patient and the control groups, suggesting that the disease susceptibility was not associated with the DR2 subtypes in this southern Chinese population. On the other hand, in the multibacillary (MB) patients significantly decreased allele frequencies of HLA-B46 (0.040 in MB patients vs 0.129 in controls) and MICA-A5 (0.200 vs 0.380) were observed compared with the healthy controls. The calculated relative risk (RR) for B46 was 0.28; for MICA-A5, 0.52. In addition, on haplotype analysis the frequency of the HLA-B46/MICA-A5 haplotype was significantly decreased in the MB patients compared to controls (0.060 vs 0.233, RR = 0.22, p < 0.01). These results suggest that an HLA-linked disease-resistant gene to MB leprosy in southern China is in strong linkage disequilibrium with the HLA-B46/MICA-A5 haplotype. In other words, the resistant gene may be located near the HLA-B/MICA region and not in the HLA-DR locus.