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To investigate the characteristics of multilineage-differentiating stress-enduring (Muse) cells labeled with chloromethyl dialkylcarbocyanine (CM-Dil) in culture and in skin wounds of rats. Normal human dermal fibroblasts (NHDFs) were obtained from foreskins and were confirmed by immunocytochemistry with vimentin. Muse cells were derived from NHDFs using long-term trypsinization (LTT), were confirmed using immunocytochemistry with antibodies against stage specific embryonic antigen-3 (SSEA-3) and CD105 and were expanded in suspension cultures. The Muse cells were labeled with CM-Dil and were further evaluated with respect to their biological properties using CCK-8 assays and scratch tests. One hundred µl CM-Dil-labeled Muse cells at a concentration of 5 × 103/µl were injected subcutaneously at the edges of skin wounds in adult male SD rats. At weeks 1, 3 and 5 after the injection, the distribution of CM-Dil-labeled Muse cells in skin tissues was observed using immunofluorescence microscopy. Muse cells were double-positive for CD105 and SSEA-3. ALP staining of the M-clusters were positive and they displayed orange-red fluorescence after labelling with CM-Dil, which had no adverse effects on their viability, migration or differentiation capacity. One week after the subcutaneous injection of CM-Dil-labeled Muse cells, many cells with orange-red fluorescence were observed at the edges of the skin injuries; those fluorescent spots gradually decreased over time, and only a few Muse cells with fluorescence could be detected by week 5. CM-Dil can be used to label Muse cells without affecting their proliferation, migration or differentiation, and can be used for short-term tracking of Muse cells for the treatment of skin wounds in a rat model.
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Alprostadil , Ratas , Masculino , Humanos , Animales , Alprostadil/farmacología , Ratas Sprague-Dawley , Diferenciación Celular , Carbocianinas/farmacologíaRESUMEN
OBJECTIVE: To observe the morphological characteristics of clusters of Muse cells from normal human dermal fibroblasts (NHDFs) under different culture conditions. METHODS: Muse cells were sorted by magnetic activated cell sorting (MACS) from NHDFs, and were evaluated by flow cytometry. Muse cells were cultured in suspension and in adherent conditions to obtain Muse cell clusters (M-clusters), which were further characterized by alkaline phosphatase (AP) staining, immunofluorescence (IF) staining and transmission electron microscopy (TEM). The M-clusters were further cultured on Lando artificial dermal regeneration matrix (LADRM) for analysis by scanning electron microscopy (SEM) and IF staining of frozen sections. RESULTS: The proportion of SSEA3 and CD105 double-positive cells obtained by MACS was 87.4%. The sorted cells rapidly formed M-clusters after suspension culture, and showed internal characteristics of stem cells under TEM. After adherent culture, M-clusters stained positively for AP, SSEA-3 and OCT-4. Each M-cluster on the surface of the LADRM displayed an outer membrane of amorphous materials under SEM. Frozen sections and fluorescence staining of LADRM loaded with M-clusters showed an uneven fluorescence intensity of SSEA-3 within the clusters. CONCLUSIONS: Muse cells sorted by MACS from NHDFs could generate M-clusters, which included cells of different stemness and are wrapped in membrane-like structures.
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Alprostadil , Fibroblastos , Humanos , Diferenciación Celular , Células Cultivadas , Alprostadil/metabolismo , PielRESUMEN
The aim of this study is to characterize the molecular properties of multilineage differentiating stress-enduring (Muse) cells compared with dermal fibroblasts (FBs) and to characterize differences in their transcriptomes and open chromatin regions that are involved in cellular plasticity. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) analyses was then performed on FBs and Muse cells. Subsequently, cell type-selective gene regulatory regions were identified by coalition analysis. Expression patterns of transcription factors (TFs) and signaling pathways intermediates were verified using quantitative real-time polymerase chain reaction and Western blot analyses. RNA-seq identified 2355 significantly differentially expressed genes (DEGs) that regulate the transcriptome, including 1222 upregulated and 1133 downregulated DEGs. The general panorama of RNA-seq and ATAC-seq analyses confirmed the differences in TFs and open chromatin regions between FBs and Muse cells. ATAC-seq analysis showed that Muse cells had more reproducible and meaningful peaks than FBs, and the peak signals were concentrated near promoter-transcription start site areas. In genomic regions that can be preferentially accessed in FBs and Muse cells, more than 200 TFs had binding motif sequences. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and coalition analyses identified differences in factors involved in the cell cycle and the protein kinase B (AKT) signaling pathway of FBs and Muse cells. The results of RNA-seq and ATAC-seq analyses clarified the genetic basis of the different biological properties of Muse cells and FBs. These results suggest that the cell cycle transition and the AKT signaling pathway may affect the morphology and biological characteristics of Muse cells.
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Secuenciación de Inmunoprecipitación de Cromatina , Proteínas Proto-Oncogénicas c-akt , Alprostadil/metabolismo , Cromatina/metabolismo , Fibroblastos/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas Proto-Oncogénicas c-akt/metabolismo , RNA-Seq , Análisis de Secuencia de ARNRESUMEN
Human epidermal melanocytes can be induced to form melanocyte spheroids and revert to immature characteristics by long-term trypsinization (LTT). To further explore the biological characteristics of melanocytes after LTT and to study the underlying mechanism. Melanocytes were subjected to long-term (2 h) trypsinization in this study, after which their viability, proliferation and autophagy were characterized. The expression of melanocyte markers [human melanoma black45 (HMB45), tyrosinase (TYR) and Nestin] was detected and relative expression levels of mRNAs encoding TYR, Nestin, c-Kit and microphthalmia-associated transcription factor (MITF) were determined. After LTT, more short spindle-shaped melanocytes appeared and viability assays demonstrated that most melanocytes survived that treatment but had decreased proliferation rates compared to the untreated controls. There was a significant increase in autophagy of melanocytes after LTT and the expression of TYR was decreased compared with untreated control melanocytes. There were no significant differences in the expression of HMB45 or Nestin between the two groups. Compared with untreated melanocytes, levels of message ribonucleic acid (mRNAs) encoding TYR, c-Kit and MITF were decreased after LTT, while Nestin mRNA levels were increased. These results clarified that Long-term treatment with trypsin causes the dedifferentiation of mature epidermal melanocytes in vitro.
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Epidermis , Melanocitos , Humanos , Monofenol Monooxigenasa , Proteínas Proto-Oncogénicas c-kit/genética , ARN Mensajero/genéticaRESUMEN
Majocchi's granuloma is an uncommon fungal infection of the dermis and subcutaneous tissue. The most frequently identified cause of Majocchi's granuloma is anthropophilic Trichophyton rubrum, and it is most commonly located on the anterior aspect of the lower limbs in women. Here, we report a case of Majocchi's granuloma on the forearm, a site that is rarely involved, in a 62-year-old woman who had been bitten by a dog. Histological examination revealed a dense dermal infiltrate composed of lymphoplasmacytic cells and neutrophils, with hyphae in the dermis. The presence of the fungus, Trichophyton tonsurans, was confirmed by mycological examination and molecular methods. Therefore, histological and mycological examination confirmed the diagnosis of Majocchi's granuloma. The patient was treated with local moxibustion and itraconazole, 200 mg/day, for 60 days, which facilitated a complete resolution of the lesions.
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Mordeduras y Picaduras/complicaciones , Granuloma/diagnóstico , Granuloma/microbiología , Tiña/diagnóstico , Tiña/microbiología , Animales , Antifúngicos/uso terapéutico , Arthrodermataceae/aislamiento & purificación , Perros , Femenino , Granuloma/tratamiento farmacológico , Humanos , Persona de Mediana Edad , Tiña/tratamiento farmacológicoRESUMEN
To characterize the tolerance of different types of human epidermal cells to trypsinization in vitro and develop a new method to separate and purify melanocytes according to their tolerance to trypsinization. Epidermal cells were obtained by separating the epidermis from human foreskins. Some of those cells were used for routine culture, and then were subjected to differential trypsin digestion. The remaining epidermal cells were resuspended in a 0.25% trypsin solution and then were neutralized by the addition of bovine serum at different time points. Immunofluorescence staining of HMB45, K15 and vimentin was used to identify melanocytes, keratinocytes and fibroblasts, respectively. We found that Keratinocytes, melanocytes and fibroblasts are primary cells obtained from conventional cultures of human skin. Purified keratinocytes and melanocytes can be obtained by conventional differential trypsin digestion, but fibroblasts in the melanocyte population quickly gain a survival advantage after passage. With longer trypsin digestion times, the number of adherent cells decreased, the time required for cell attachment increased, and the proportion of melanocytes increased. There were no obvious keratinocytes in cell populations obtained after 12 h of trypsinization of epidermal cells, and more short spindle-shaped melanocytes appeared, all of which were HMB45-positive. In conclusion, the tolerance of human epidermal melanocytes to trypsinization in vitro was better than epidermal keratinocytes, and that property can be used to purify melanocytes and was better than traditional differential trypsin digestion. The morphology of cells that survived the long-term trypsin digestion changed and they had good proliferative activity, but seemed to be more immature.
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Células Epidérmicas/metabolismo , Tripsina/metabolismo , Células Epidérmicas/citología , Humanos , Masculino , Melanocitos/citologíaRESUMEN
A 45-year-old Chinese man had begun to show asymmetry of the face 30 years previously. Subsequently, he developed visual extinction of the right eye, slight numbness, and weakness of the left extremities. Simultaneously, multiple atrophic brownish patches occurred on his side. He denied prior trauma or tick bites at those sites. There was no report of preceding redness, induration, or a history of trauma. The atrophic lesions extended and enlarged slowly. Ten years previously, some brownish patches with normal texture had appeared on the right side of the trunk. There was no further progression of the lesions. In November 2010, the patient consulted our department for the final diagnosis and prognosis of his disease. He did not suffer from epileptic seizures and had no history of a tick bite or Lyme disease.
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Asimetría Facial/complicaciones , Dermatosis Facial/patología , Hiperpigmentación/patología , Piel/patología , Lengua/patología , Atrofia/complicaciones , Atrofia/diagnóstico , Dorso , Extremidades , Dermatosis Facial/complicaciones , Dermatosis Facial/diagnóstico , Humanos , Hiperpigmentación/complicaciones , Masculino , Persona de Mediana Edad , Trastornos de la Visión/complicacionesRESUMEN
A 2-year-old infant boy presented with a large ulcerative lesion on his tongue. The grandmother who cared for the boy was in the habit of chewing food before giving it to the boy and had active syphilis. The infant was diagnosed with acquired early syphilis, which had been transmitted by prechewed food from his grandmother. Prechewing food is a custom in most parts of China. Prechewing an infant's food could be an avenue of disease transmission, although this is not fully recognized. No studies have been conducted to evaluate prechewed food as a disease transmission route.
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Enfermedades de la Boca/etiología , Sífilis/transmisión , Preescolar , Femenino , Humanos , Masculino , Masticación , Sífilis/patología , Lengua/patologíaRESUMEN
Genital human papillomavirus (HPV) is associated with the development of cutaneous malignant tumors, and differences in HPV subtypes are found in several cancers by histology. NF-κB is persistently activated in most cancers and confers a survival advantage to cancer cells, while A20 is a critical negative regulator of NF-κB and is an important tumor suppressor inactivated in B cell lymphomas. This study was undertaken to identify HPV types in cutaneous squamous cell carcinoma (SCC) as well as to determine whether the crosstalk of A20/NF-κB was involved in HPV-induced SCC. Overall, HPV positivity was observed to be 66.2 %, with HPV16 being most common followed by infection with HPV18. Out of 43 HPV-positive samples, 35 samples were positive for one or more high-risk HPV (HR-HPV) types, suggesting a high association of SCC with HR-HPV infection, while only five HPV infections were detected in 21 normal skin samples and low-risk HPV (LR-HPV) infection was the most common. Both A20 and NF-κB were overexpressed in HPV-positive SCC samples (56 vs 87.4 %) and were closely correlated with TNM stage and lymph node transfer, respectively. More interestingly, the expression of A20 and NF-κB was much higher in HR-HPV samples than in LR-HPV samples. These results suggest that the crosstalk of A20 and NF-κB may contribute to HR-HPV-associated tumor growth and metastasis of SCC and may be a novel therapeutic target for SCC in the future.
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Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/metabolismo , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Infecciones por Papillomavirus/complicaciones , Adulto , Anciano , Alphapapillomavirus/genética , Carcinoma de Células Escamosas/patología , Femenino , Genotipo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Tipificación Molecular , Clasificación del Tumor , Metástasis de la Neoplasia , Estadificación de Neoplasias , Infecciones por Papillomavirus/virología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
A 22-year-old woman was referred to our hospital for pigmented lesions located on her face. These had gradually increased during the past 4 years. Computed tomography (CT) of her head revealed no significant parenchymal abnormalities of temporal, maxillary and sphenoid bones or of either parietal bone. Further screening, including neurologic, ophthalmologic, orthopedic, and visceral investigations, did not reveal any abnormalities. There was no family history of abnormal cutaneous pigmentation.
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Nevo de Ota/patología , Neoplasias Cutáneas/patología , Cara , Femenino , Humanos , Nevo de Ota/diagnóstico , Neoplasias Cutáneas/diagnóstico , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
Background: As vitiligo progresses, autophagy becomes more and more important. Objectives: To validate potential genes associated with autophagy in vitiligo through bioinformatics analysis and experimental testing. Materials and Methods: Dataset GSE75819 of mRNA expression profiles was obtained from GEO. After data normalisation, gene set enrichment analyse enrichment analysis and abundance analysis of infiltrating immune cells were performed. A list of autophagy-related differentially expressed genes (ARDEGs) associated with vitiligo was generated using R software. Protein-protein interaction (PPI) analysis, correlation analysis, and enrichment analysis on gene ontology (GO) and Kyoto encyclopaedia of genes and genome (KEGG) pathways were conducted on the ARDEG data. The microRNAs associated with hub genes were predicted using the TargetScan database. Finally, RNA expression of 10 hub genes and Western blotting (WB) of autophagy pathway factors were further verified. Results: From the lesions of 15 vitiligo patients, 44 ARDEGs were identified. PPI analysis demonstrated that these ARDEGs interacted with each other. GO and KEGG analyses of ARDEGs revealed that several enriched terms were associated with macroautophagy (biological process), vacuolar membranes (cellular components), cysteine-type peptidase activity (molecular function), and autophagy in animals, neurodegeneration-multiple disease pathways, and apoptosis. In vitiligo lesions, qRT-PCR and sequencing validation analyses showed expression levels of CCL2, RB1CC1, TP53, and ATG9A that were consistent with bioinformatic analysis of the microarray. WB results also showed that autophagy-related proteins were differentially expressed. Conclusions: Forty-four potential ARDEGs were identified in vitiligo by bioinformatic analysis. Vitiligo may be affected by autophagy regulation through CCL2, RB1CC1, TP53, and ATG9A.
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Background: Hematoporphyrin monomethyl ether (HMME) is a promising photosensitizer for photodynamic therapy (PDT) and has found wide application in the treatment of port-wine stains (PWS). Objective: This study aims to observe and analyze the clinical efficacy and safety of HMME-PDT in the treatment of PWS patients. It also aims to evaluate the usefulness of color Doppler flow imaging (CDFI), an ultrasound technique for detecting blood flow in skin lesions, in assessing clinical efficacy. Methods: Thirty-three patients with PWS underwent HMME-PDT at our dermatology outpatient clinic between January 2019 and March 2020. Data on patient demographics, lesion location, lesion type (pink, purple, nodular thickening), treatment frequency, and pre- and post-treatment images were collected and retrospectively analyzed. CDFI was performed on three patients. Results: All patients received intravenous HMME and underwent irradiation with 532 nm green LED light. Of these, 5 patients received 1 session of HMME-PDT, 14 received 2 sessions, 9 received 3 sessions and the remaining 5 patients received more than 3 sessions. Of the 33 patients, 9 were cured (27.27%), 10 showed improvement (30.30%), 11 experienced a reduction in symptoms (33.33%), and 3 showed no significant improvement (9.09%). Most patients reported local pain and oedema, and no systemic adverse effects were observed. Clinical efficacy correlated with lesion type and total number of treatment sessions. CDFI appears to be an excellent technique for assessing clinical efficacy. Conclusion: HMME-PDT is a safe and effective method for the treatment of PWS. CDFI examination appears to be a promising assessment tool. However, further validation with larger sample sizes is warranted.
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We describe a 22-year-old male with a pigmentary disorder that first appeared when he was 5 years of age. Examination revealed that these lesions were distributed in a segmental pattern on the left side of his trunk. Five shades of colors--white, tan, medium brown, dark brown, and black--were present. The patient told us there had been no preceding inflammation in the affected areas. A biopsy specimen from the inguinal region showed a complete absence of melanocytes, whereas most of the hyperpigmented areas were characterized by increased amounts of epidermal pigment and numbers of melanocytes. Based on the clinical appearance, pentachrome vitiligo in a segmental type was diagnosed.
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Melanocitos/patología , Vitíligo/patología , Adulto , Humanos , Masculino , Adulto JovenRESUMEN
This is the report of a 76-year-old male with typical lesions of acanthosis nigricans maligna (ANM), florid cutaneous papillomatosis (FCP), and tripe palms (TP) for 2 years. He did not have any gastrointestinal complaints. Pathologic findings of skin supported the diagnosis of ANM. Because gastric adenocarcinoma is the most common neoplasm associated with these paraneoplastic dermatoses, further tests were carried out. Endoscopic examination was performed and an adenocarcinoma of the esophagogastric junction was confirmed. Meanwhile, multiple small polyps in the middle and the lower thirds of the esophagus were observed. The patient was referred for further evaluation and subsequent surgical resection of the tumor.Acanthosis nigricans (AN) is a hyperkeratotic mucocutaneous eruption of heterogenous etiology, which is characterized by hyperpigmentation, velvety cutaneous thickening, intensified skin markings, and development of verrucous excrescences typically involving the intertriginous areas. AN is classified into benign and malignant forms on the basis of clinical associations. Malignant acanthosis nigricans (MAN) tends to be extensive and involves mucosal surfaces, mostly in elderly people. Florid cutaneous papillomatosis (FCP), also known as the Schwartz-Burgess syndrome, is characterized by the rapid appearance of multiple verrucous lesions that are clinically indistinguishable from common warts [1]. Tripe palms (TP) is characterized by diffuse, yellowish palmar hyperkeratosis, with enhancement of the epidermal ridges on the hands (dermatoglyphics), resembling intestinal villosities [1]. The association of these three paraneoplastic dermatoses (FCP, ANM and TP) in the same patient has been reported. Herein, we report an elderly male with three paraneoplastic dermatoses for two years. On the initial presentation, he did not report any systemic complaints; diagnostic tests confirmed the presence of a gastric adenocarcinoma.
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Adenocarcinoma/complicaciones , Papiloma/etiología , Síndromes Paraneoplásicos/etiología , Neoplasias Cutáneas/etiología , Neoplasias Gástricas/complicaciones , Acantosis Nigricans/etiología , Adenocarcinoma/diagnóstico , Anciano , Dermatosis de la Mano/etiología , Humanos , Queratodermia Palmoplantar/etiología , Masculino , Neoplasias Gástricas/diagnósticoRESUMEN
Milia en plaque is a rare variant of miliathat occurs spontaneously on an erythematous base without identifiable causative factors. Approximately 40 cases have been recorded in the literature. Most occurred in the periauricular area, affected middle-aged patients, and showed a predilection for women. Here, we report a case of milia en plaque on the bilateral posterior helices in a 6-year-old Chinese boy.
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Pueblo Asiatico , Oído Externo , Quiste Epidérmico/patología , Eritema/patología , Niño , Quiste Epidérmico/etnología , Eritema/etnología , Humanos , MasculinoRESUMEN
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.
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Exosomas , MicroARNs , Exosomas/genética , Exosomas/metabolismo , Fibroblastos/metabolismo , Humanos , Queratinocitos/metabolismo , Melaninas/metabolismo , Melanocitos , MicroARNs/genética , MicroARNs/metabolismo , MicroARNs/farmacología , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Monofenol Monooxigenasa/farmacologíaRESUMEN
A 45-year-old male presented with painless reddish-brown plaques and nodules that had infiltrated his shoulder and back for 3 months. From the clinical manifestations and histopathological findings, the patient was diagnosed with cutaneous Rosai-Dorfman disease. Intralesional injections of betamethasone (trade name: diprospan) were recommended and the lesions improved significantly after three treatments.
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Dermatologists should be aware that the clinical manifestations of syphilis are very complex and changeable. Unilaterally distributed skin lesions and painless lip ulcers may also be the clinical manifestations of secondary syphilis.
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OBJECTIVE: In order to generate induced Pluripotent Stem Cells (iPSCs) more efficiently, it is crucial to identify somatic cells that are easily accessible and possibly require fewer factors for conversion into iPSCs. METHODS: Human epidermal melanocytes were transduced with lentiviral vectors carrying 3 transcription factors (OCT-4, KLF-4 and c-MYC, 3F) or 4 transcription factors (OCT-4, KLF-4, c-MYC and SOX-2, 4F). Once the clones had formed, assays related to stem cell pluripotency, including alkaline phosphatase staining, DNA methylation levels, expression of stem cell markers and ultrastructure analysis were carried out. The iPSCs obtained were then induced to differentiate into the cells representing the three embryonic layers in vitro. RESULTS: Seven days after the transduction of epidermal melanocytes with 3F or 4F, clones were formed that were positive for alkaline phosphatase staining. Fluorescent staining with antibodies against OCT-4 and SOX-2 was strongly positive, and the cells showed a high nucleus-cytoplasm ratio and active karyokinesis. No melanosomes were found in the cytoplasm by ultrastructural analysis. There were obvious differences in DNA methylation levels between the cloned cells and their parental cells. However, there was not a significant difference between 3F or 4F transfected clonal cells. Meanwhile, the iPSCs successfully differentiated into the three germ layer cells in vitro. CONCLUSION: Human epidermal melanocytes do not require ectopic SOX-2 expression for conversion into iPSCs, and may serve as an alternative source for deriving patient-specific iPSCs with fewer genetic elements.
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Células Madre Pluripotentes Inducidas/citología , Melanocitos/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Niño , Metilación de ADN , Terapia Genética , Humanos , Células Madre Pluripotentes Inducidas/ultraestructura , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Lentivirus/genética , Masculino , Melanocitos/ultraestructura , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Transducción GenéticaRESUMEN
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.