Effects of Photodynamic Therapy Using 5 -Aminolevulinic Acid (ALA) Loaded Acrylic Nanoparticles (ANPs) on HaCaT Cells.

Objective: ALA-PDT (5-aminolevulinic acid photodynamic therapy) is a central modality in the treatment of skin diseases. Increasing the bioavailability of ALA remains a critical issue. With this in mind, our study explores a novel route of ALA delivery by loading acrylic nanoparticles (ANPs). Methods: ALA-ANPs were synthesized by emulsion polymerisation and characterised by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The effects of ALA-ANPs on HaCaT cell line were evaluated, including characteristics, morphological changes, protoporphyrin IX (PpIX) fluorescence kinetics, reactive oxygen species (ROS) levels, mitochondrial membrane potential and ki67 expression in these cells. Results: The ANPs had uniform sizes, smooth surfaces and excellent light transmittance, with diameters of 150-200 nm. In contrast, the ALA - ANPs had uneven surfaces and poor light transmittance, with diameters of 220-250 nm. During 12 hours of co-incubation of HaCaT cells with ALA, the intracellular accumulation of PpIX increased over time. Notably, after 6 hours of incubation, PpIX levels induced by 1.81 mg/mL ALA-ANPs exceeded those induced by 1.0 mM ALA (p < 0.01). CCK-8 results showed a positive correlation between PDT-induced inhibition of HaCaT cell proliferation and ALA concentration when ALA concentration remained below 2.0 mM. Compared to the 1.0 mM ALA group, the 1.81 mg/mL ALA-ANPs group showed decreased mitochondrial membrane potential, ki67 immunofluorescence intensity and cell proliferation. In contrast, ROS levels were significantly increased in the 1.81 mg/mL ALA-ANPs group (p < 0.01). Conclusion: Loading ANPs provide improved stability and potency for ALA. The ALA-ANPs-PDT approach has superior inhibitory effects on HaCaT proliferation in vitro.

Bioinformatic Analysis of Genes Associated with Autophagy in Vitiligo.

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.

Clinical Observation on the Therapeutic Effect of Port-Wine Stains with Intravenous Injection of Hematoporphyrin Monomethyl Ether (HMME).

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.

Misdiagnosis of cutaneous facial sporotrichosis: An analysis of five cases.

BACKGROUND: Facial cutaneous sporotrichosis presents with diverse clinical manifestations, often leading to misdiagnosis. OBJECTIVE: This study aims to present the clinical characteristics of five misdiagnosed cases of facial cutaneous sporotrichosis, aiming to enhance understanding of this disease and prevent misdiagnosis and mistreatment. METHODS: Clinical data, histopathology, and fungal culture results of these five cases were comprehensively analyzed. RESULTS: Among these five patients, three presented with lymphocutaneous sporotrichosis, while two had the fixed cutaneous type. Due to misdiagnosis, initial treatments were ineffective for all patients. Upon histopathological examination and fungal culture confirming sporotrichosis, treatment with itraconazole for 3 months led to complete resolution of lesions. While one patient experienced a relapse due to noncompliance with the prescribed medication. CONCLUSION: Facial sporotrichosis, with its diverse clinical manifestations and obscure trauma history, is prone to misdiagnosis. Timely and thorough examinations are crucial for precise diagnosis and management. Itraconazole treatment demonstrated notable efficacy, and patient compliance is also essential for favorable outcomes.

In Vitro Antibacterial and Anti-Inflammatory Properties of Imidazolium Poly(ionic liquids) Microspheres Loaded in GelMA-PEG Hydrogels.

Repairing damaged tissue caused by bacterial infection poses a significant challenge. Traditional antibacterial hydrogels typically incorporate various components such as metal antimicrobials, inorganic antimicrobials, organic antimicrobials, and more. However, drawbacks such as the emergence of multi-drug resistance to antibiotics, the low antibacterial efficacy of natural agents, and the potential cytotoxicity associated with metal antibacterial nanoparticles in hydrogels hindered their broader clinical application. In this study, we successfully developed imidazolium poly(ionic liquids) (PILs) polymer microspheres (APMs) through emulsion polymerization. These APMs exhibited notable antibacterial effectiveness and demonstrated minimal cell toxicity. Subsequently, we integrated the APMs into a gelatin methacryloyl (GelMA)-polyethylene glycol (PEG) hydrogel. This composite hydrogel not only showcased strong antibacterial and anti-inflammatory properties but also facilitated the migration of human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) and promoted osteogenic differentiation in vitro.

Unravelling Childhood Tinea Capitis: A Multi-Dimensional Investigation Using Dermoscopy, Scanning Electron Microscopy and Mass Spectrometry.

Tinea capitis, a common cutaneous fungal infection, shows an increasing prevalence with the increasing number of pets. We present tinea capitis in a 4-year-old girl presenting without typical symptoms such as alopecia or hair breakage. After a comprehensive evaluation including dermoscopy, Wood's light, direct KOH fluorescent staining, scanning electron microscopy, fungal culture and mass spectrometry analysis, a diagnosis of tinea capitis infected Microsporum canis carried by domestic cats was made. We preliminarily explored the two modes of hair erosion by tinea capitis fungi and analyzed the possibility of the feature in this case. This case highlights the importance of accurate diagnosis and appropriate therapeutic intervention in cases of paediatric tinea capitis, particularly in households with resident pets.

Eruptive Cherry Angiomas After Nitrogen Mustard Treatment for Vitiligo.

A 43-year-old male was diagnosed with vitiligo and had been treated with topical nitrogen mustard at the age of 13. Following two years of treatment, eruptive cherry angiomas developed and presented as widely distributed red papules throughout his trunk and proximal limbs. Ceasing the use of nitrogen mustard slowed the emergence of lesions. This case highlights the potential adverse effects associated with nitrogen mustard treatment in individuals with susceptibility, as it may lead to the onset of eruptive cherry angiomas.

The Morphology and Ultrastructure of Dermal Telocytes Characterized by TEM and AFM.

This investigation delves into the structural foundation of human dermal telocytes (TCs) with the aim of elucidating their role in signal transmission. Dermal TCs were isolated from human foreskins via enzymatic digestion and flow cytometric sorting, and identified by immunohistochemical staining with an antibody against CD34. The ultrastructure of TCs was examined using transmission electron microscopy (TEM). The proliferation rates of sorted TCs and CD34-negative fibroblasts were compared using the MTS assay (Cell Proliferation Assay). Images of viable cultured TCs were analyzed using atomic force microscopy (AFM) under normal atmospheric pressure and temperature. Results demonstrated that dermal TCs were positive for CD34 and vimentin, predominantly distributed in the reticular dermis and subcutaneous tissue, forming interwoven networks. Each TC had a small body with a high nuclear-plasma ratio and two or three extremely long and thin telopodes (TPs), exhibiting a typical 'moniliform' appearance. Compared with CD34-negative fibroblasts, dermal TCs exhibited significantly lower proliferation rates. Cultured TCs displayed typical moniliform projections (namely, TPs) in the AFM images. The distal ends of TPs were enlarged, shaped like a broom, and extended multiple pseudopods to contact other cell bodies. Slender filamentary pseudopodia and thick, short cone-like structures were observed on the surfaces of the dilated segments and terminals of TPs. These structures are assumed to be evidence of the secretion and release of endosomes, such as exosomes, and the communication between cells. TCs form interstitial networks in the reticular dermis and subcutaneous tissue, providing a structural basis for contacts between cells and the secretion of signal-carrying substances, involving intercellular connections and communication.

Characterization of CM-Dil-labeled Muse cells in culture and in skin wounds in rats.

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.

The inhibition of VDAC1 oligomerization promotes pigmentation through the CaMK-CRTCs/CREB-MITF pathway.

The voltage-dependent anion channel 1 (VDAC1) forms an oligomeric structure on the mitochondrial outer membrane, which plays critical roles in many physiological processes. Research studies have demonstrated that the knockout of VDAC1 increases pigment content and up-regulates the expression of melanogenic genes. Due to its involvement in various physiological processes, the depletion of VDAC1 has significant detrimental effects on cellular functions and the inhibition of VDAC1 oligomerization has recently emerged as a promising strategy for the treatment of several diseases. In this study, we found that VDAC1 oligomerization inhibitors, VBIT-12 and NSC-15364, promote melanogenesis, dendrite formation and melanosome transport in human epidermal melanocytes (HEMCs). Mechanistically, treatment of HEMCs with an oligomerization inhibitor increased the level of cytoplasmic calcium ions, which activated calcium-calmodulin dependent protein kinase (CaMK) and led to the phosphorylation of CREB and the nuclear translocation of CREB-regulated transcription coactivators (CRTCs). Subsequently, CRTCs, p-CREB and CREB-binding protein (CBP) in the nucleus cooperatively recruit the transcription machinery to initiate the transcription of MITF thus promoting pigmentation. Importantly, our study also demonstrates that VDAC1 oligomerization inhibitors increase pigmentation in zebrafish and in human skin explants, highlighting their potential as a therapeutic strategy for skin pigmentation disorders.

Identification of Dopachrome Tautomerase (DCT) and Kinesin Family Member 1A (KIF1A) as Related Biomarkers and Immune Infiltration Characteristics of Vitiligo Based on Lasso-SVM Algorithms.

Objective: To identify potential diagnostic markers for vitiligo and determine the significance of immune cell infiltration in pathology. Methods: Three publicly available gene expression profiles (GSE53146, GSE75819 and GSE65127 datasets) from human vitiligo and control samples were downloaded from the GEO database. Differentially expressed genes (DEGs) were screened between 20 vitiligo and 20 control samples. Logical regression of the selection operator (LASSO) model and support vector machine recursive feature elimination (SVM-RFE) analysis were performed to identify candidate biomarkers. The area under the receiver operating characteristic curve (AUC) value was obtained and was used to evaluate the discriminatory ability. The expression level and diagnostic value of the biomarkers in vitiligo were further validated in the GSE65127 dataset (10 vitiligo patients and 10 healthy controls). Finally, the immune cell infiltration of vitiligo was evaluated by CIBERSORT, and the correlation between biomarkers and infiltrating immune cells was analyzed. The compositional patterns of the 22 types of immune cell fractions in vitiligo were estimated from the pooled cohorts using CIBERSORT. In addition, we established a mouse model of vitiligo with monobenzone and validated the screened biomarkers. Results: A total of 23 associated DEGs were identified, including 9 up-regulated and 14 down-regulated genes. Subsequently, 17 genes meeting prognostic criteria and 2 common genes (DCT and KIF1A) were obtained by SVM and Venn diagram screening. Immunodifferential analysis showed that microenvironment of vitiligo patients was altered. Finally, the different expression was verified by polymerase chain reaction (PCR). Conclusion: Biomarkers associated with vitiligo can be screened by comprehensive strategies, and immune cell infiltration plays a key role in the development of vitiligo.

Characteristics of multilineage-differentiating stress-enduring cell clusters in different culture conditions.

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.

The effects of miRNA-27a-3p on human epidermal melanocytes.

OBJECTIVE: To characterize the effects of miRNA-27a-3p on the biological properties of human epidermal melanocytes (MCs). METHODS: MCs were obtained from human foreskins and transfected with miRNA-27a-3p mimic (induces the overexpression of miRNA-27a-3p), mimic-NC (the negative control group), miRNA-27a-3p inhibitor, or inhibitor-NC. After transfection, the proliferation of MCs in each group was evaluated by cell counting kit-8 (CCK-8) at 1, 3, 5, and 7 days. Twenty-four hours later, the MCs were transferred onto a living cell imaging platform and cultured for another 12 h to detect their trajectories and velocities. On days 3, 4, and 5 after transfection, the expression of melanogenesis-related mRNAs, protein levels, and melanin contents were measured using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and NaOH solubilization, respectively. RESULTS: The RT-PCR results showed that miRNA-27a-3p was successfully transfected into MCs. The proliferation of MCs was restrained by miRNA-27a-3p. There were no significant differences in the movement trajectories of MCs in the four transfected groups, but the cell movement velocity in the mimic group was slightly lower; that is, the overexpression of miRNA-27a-3p inhibited the speed of MCs. The expression levels of melanogenesis-related mRNAs and proteins were decreased in the mimic group and were increased in the inhibitor group. Melanin content in the mimic group was lower than that in the other three groups. CONCLUSIONS: Overexpression of miRNA-27a-3p inhibits the expression of melanogenesis-related mRNAs and proteins, reduces the melanin content of human epidermal MCs, and slightly impacts their movement speed.