Primary Skin Research Articles

Generation of skin-derived iPSCs from an Osteogenesis imperfecta patient carrying WNT1c.677C>T mutation

To obtain skin-derived induced pluripotent stem cells (iPSCs) from an Osteogenesis imperfecta (OI) patient carrying WNT1c.677C>T mutation in order to provide a new cell model for investigating the underlying molecular mechanism and stem cell therapy for OI. The pathogenic variant of the patient was identified by Sanger sequencing. With informed consent from the patient, skin tissue was biopsied, and primary skin fibroblasts were cultured. Skin fibroblasts were induced into iPSCs using Sendai virus-mediated non-genomic integration reprogramming method. The iPSC cell lines were characterized for pluripotency, differentiation capacity, and karyotyping assay. The patient was found to carry homozygous missense c.677C>T (p.Ser226Leu) mutation of the WNT1 gene. The established iPSC lines possessed self-renewal and capacity for in vitro differentiation. It also has a diploid karyotype (46,XX). A patient-specific WNT1 gene mutation (WNT1c.677C>T) iPSC line was established, which can provide a cell model for the study of OI caused by the mutation.

The prognostic role of PD-L1 expression and the presence of polyomavirus in Merkel cell carcinoma cases

BackgroundMerkel cell carcinoma (MCC) comprises a rare malignant primary skin tumor presenting neuroendocrine differentiation. Recently, agents blocking the programmed cell death protein 1 and programmed cell death protein ligand 1 pathway (PD-1/PD-L1) have demonstrated objective and durable tumor regressions in patients presenting advanced MCC. This study aimed to describe the sociodemographic, clinical, and histopathological characteristics of MCC patients, also assessing the prevalence of PD-L1 expression and Merkel cell Polyomavirus (MCPyV), as well as their prognostic roles.MethodsData from patients diagnosed with MCC between 1996 and 2019 at a reference cancer center in Rio de Janeiro, southeastern Brazil, were evaluated in a retrospective study. Tumor samples were tested for MCPyV and PD-L1 employing immunohistochemistry. Survival analyses were carried out employing the Kaplan–Meier method and curves were compared using the log-rank test. A multiple semiparametric Cox model was used. Values p < 0.05 were considered significant.ResultsA total of 65 patients were included in the study, with a mean age at diagnosis of 72 (standard deviation 13.9). A total of 56.9% (37/65) of the patients were male, 86.2% (56/65) were white, and 56.9% (37/64) were illiterate or with incomplete elementary school. MCPyV immunohistochemistry was positive in 29 cases (44.6%) and PD-L1 positivity was ≥ 1% in 42 cases (64.6%). Significant associations between MCPyV and PD-L1 expression ≥ 1% (p = 0.003) and PD-L1 expression ≥ 5% (p = 0.005) were noted. Concerning the multivariate analysis, only education level and advanced MCC stage indicated statistically significant worse progression-free survival. Regarding overall survival (OS), being male, education level and advanced stage comprised risk factors. The estimated OS at 60 months for stages I to III was of 48.9% and for stage IV, 8.9%.ConclusionsThis is the first large Brazilian cohort to assess the prevalence of MCPyV in MCC tumors, as well as PD-L1 expression and their associations. No correlations were noted between MCPyV infection or PD-L1 expression and survival rates.

Effect of Cells Derived from Periodontal Ligament Tissue on Bone Formation.

Recent reports indicate that sclerostin is secreted by periodontal ligament tissue-derived (PDL) cells during orthodontic force loading and that the secreted sclerostin contributes to bone metabolism. However, the detailed mechanism is poorly understood. The aim of this study was to determine how PDL cells affect bone formation. Rat periodontal ligament tissue was immunohistochemically stained for sclerostin. Cultured primary PDL cells, osteoblasts, and skin fibroblasts (Sfbs) isolated from rat periodontal ligament tissue, calvaria, and skin, respectively, were examined. Osteoblasts were cultured with control conditioned medium (Cont-CDM) and PDL cell culture conditioned medium (PDL-CDM) for up to 21 days. Cultured osteoblasts were then stained with alkaline phosphatase and von Kossa stain. Osteoblasts cultured in each conditioned medium were analyzed by real-time quantitative PCR for bone Gla protein (Bgp), Axin2, and Ki67 expression. PDL cells used to obtain conditioned medium were analyzed for Sost, Ectodin and Wnt1 expression and compared with expression in Sfbs. Expression of sclerostin was observed in periodontal ligament tissue by immunohistochemical staining. The formation of mineralization nodules was inhibited in PDL-CDM compared with Cont-CDM in osteoblast culture. In PDL-CDM, the expression levels of Bgp and Axin2 in osteoblasts were decreased compared with Cont-CDM. In PDL cells, expression levels of Sost and Ectodin were much higher than in Sfbs; however, expression of Wnt1 was lower in PDL cells compared with Sfbs. PDL cells secrete various proteins, including sclerostin and suppress osteogenesis in osteoblasts through the canonical Wnt pathway.

YAP promotes the healing of ischemic wounds by reducing ferroptosis in skin fibroblasts through inhibition of ferritinophagy

The impaired healing of chronic wounds is often attributed to the ischemic and hypoxic microenvironment, leading to increased cell death. Ferroptosis, a novel form of cell death unveiled in recent years, could potentially be linked with the process of wound healing. In this study, we explored the significance and mechanism of ferroptosis in ischemic wounds. Using transmission electron microscopy, Western blot, flow cytometry, immunofluorescence, and glutathione (GSH) assay, we observed that the death of primary mouse skin fibroblasts induced by oxygen and glucose deprivation (OGD) was associated with ferroptosis. Specifically, we observed elevated intracellular Fe2+ and lipid peroxidation levels and decreased GSH levels in vitro, indicative of ferroptosis. Importantly, we found that ferroptosis in OGD-treated skin fibroblasts was dependent on autophagy, as the autophagy inhibitor chloroquine phosphate (CHQ) significantly reduced ferroptosis induced by OGD. Moreover, our study revealed that NCOA4-mediated ferritinophagy significantly contributed to the occurrence of ferroptosis induced by OGD in skin fibroblasts. Additionally, we identified the involvement of YAP in the regulation of ferritinophagy, with YAP suppressing NCOA4 expression in OGD-treated skin fibroblasts, thereby reducing ferroptosis. Furthermore, in ischemic wound models in mice, both inhibitors of ferroptosis and autophagy promoted wound healing, while a YAP inhibitor, verteporfin, delayed wound healing. In conclusion, these findings indicate that ferroptosis, regulated by YAP through ferritinophagy inhibition, presents a novel mechanism responsible for the delayed healing of ischemic wounds. Understanding this process could offer promising therapeutic targets to improve wound healing in ischemic conditions.

Quantification of Autophagosomes in Human Fibroblasts Using Cyto-ID® Staining and Cytation Imaging.

As an essential process for the maintenance of cellular homeostasis and function, autophagy is responsible for the lysosome-mediated degradation of damaged proteins and organelles; therefore, dysregulation of autophagy in humans can lead to a variety of diseases. The link between impaired autophagy and disease highlights the need to investigate possible interventions to address dysregulations. One possible intervention is hyperthermia, which is described in this protocol. To investigate these interventions, a method for absolute quantification of autophagosomal compartments is required that allows comparison of autophagosomal activity under different conditions. Existing methods such as western blotting and immunohistochemistry for analysing the location and relative abundance of intracellular proteins associated with autophagy, or transmission electron microscopy (TEM), which are either very time-consuming, expensive, or both, are less suitable for this purpose. The method described in this protocol allows the absolute quantification of autophagosomes per cell in human fibroblasts using the CYTO-ID® Autophagy Detection Kit after heat therapy compared to a control. The Cyto-ID® assay is based on the use of a specific dye that selectively stains autophagic compartments, combined with an additional Hoechst 33342 dye for nuclear staining. The subsequent recognition of these stained compartments by the Cytation Imager enables the software to determine the number of autophagosomes per nucleus in living cells. Additionally, this absolute quantification uses an image-based method, and the protocol is easy to use and not time-consuming. Furthermore, the method is not only suitable for heat therapy but can also be adapted to any other desired therapy or substance. Key features • Absolute quantification of autophagic compartments in living cells. • Optimised protocol for the determination of autophagy in primary human skin fibroblasts. • Allows the testing of active substances and treatments concerning autophagy. • Imaging-based method for the determination of autophagy.

Angiomatoid fibrous histiocytoma: primary intracranial lesion with thoracic spine metastasis and a malignant course. Illustrative case.

Angiomatoid fibrous histiocytoma (AFH) is an exceptionally rare soft tissue neoplasm. This tumor primarily presents as a benign soft tissue lesion in children with an average age of 14 years. The standard treatment regimen is wide local excision with interval follow-up. However, newer reports have demonstrated malignant potential with the possibility of intracranial metastasis. A 45-year-old male with no soft tissue primary tumor presented with a primary intracranial lesion and thoracic spine metastasis refractory to chemotherapy and radiation treatment. This report illustrates the potential for a highly malignant nature of metastatic AFH. In addition, the authors demonstrate an incidence of AFH in a middle-aged male without a primary soft tissue or skin lesion. This report highlights the importance of prompt treatment and excision for AFH, as there is still little understanding of successful options for systemic therapy.

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Melanoma is a malignant tumor that most often affects the skin, but it can also occur in many other human organs. In recent decades, the frequency of melanoma cases continues to grow and it has now become a relatively common malignant neoplasm, which is a serious socio-economic problem. The vast majority of melanomas have a known primary site of their development, but approximately 3.2 % of all melanomas are first detected in distant locations without a known primary tumor. Melanoma is considered as a multi-factorial disease, which arises as a result of the interaction of the genetic sensitivity of the human body and the influence of the environment. The most important and potentially modifying environmental risk factor of malignant melanoma is exposure to UV rays due to their genotoxic effect. The most important human body risk factors are the number of melanocytic nevi, family history and genetic predisposition. Melanomas without a primary skin lesion have been poorly studied due to their rarity, although in recent years more information has become available regarding the clinical, molecular, and genetic characteristics of such tumors. The article presents the actual observation of the so-called primary melanoma of the lung with a detailed description of known clinical data, performed surgical intervention and pathomorphological examination. The obtained data were compared with the available information on similar cases, demonstrating high grade of agreement. However, the authors hesitate to draw a conclusion regarding the treatment of primary lung melanoma, because recently, in a high-tech study of a number of similar cases of lung melanoma, objective molecular-genetic evidence has been provided for the existence of primary hidden melanoma of the skin.

Effect of Jaw Tracking During Volumetric Modulated Arc Therapy for Facial Non-melanoma Skin Cancer.

This study aimed to analyze the dosimetric effects of jaw tracking during Volumetric Modulated Arc Therapy (VMAT) planning for facial non-melanoma skin cancer (NMSC). This study included 50 patients with facial NMSC who underwent VMAT planning with or without jaw tracking. The target volume (TV) included the primary skin lesion with a 1-cm margin around the surface and a depth of 4 mm. A total of 55 Gy in 20 fractions was prescribed, and the plans were considered acceptable if the TV was covered by 95-105% of the isodose curve. A dosimetric comparison was performed for the volumes of the low-dose regions, which were defined as <50% of the prescription dose (V10-50%). Target coverage was evaluated using the homogeneity index (HI) and conformity index (CI). The patients' mean TV was 5.137 cc (range=1.03-15.89 cc). Jaw tracking resulted in mean volume reduction rates of 3.9%, 6.6% 10.6% and 13.8% for V40%, V30%, V20%, and V10%, respectively (all p<0.001). The volume change in V50% between the two groups was 2.7% (p=0.006). No significant differences were observed in HI (p=0.449) or CI (p=0.127). The application of jaw tracking during VMAT for facial NMSC is associated with a significant reduction in the volume of low dose delivered in the radiation field (V10-50%), while maintaining target coverage. Future analyses should assess whether this volume difference affects treatment-related cosmetic outcomes.

In Vitro Evaluation of Wound Healing, Stemness Potentiation, Antioxidant Activity, and Phytochemical Profile of Cucurbita moschata Duchesne Fruit Pulp Ethanolic Extract.

Wound healing comprises an intricate process to repair damaged tissue. Research on plant extracts with properties to expedite wound healing has been of interest, particularly their ability to enhance the stemness of keratinocyte stem cells. Hence, the present study aims to determine the wound healing and stemness potentiation properties of an ethanolic extract derived from Cucurbita moschata fruit pulp (PKE). Human keratinocytes (HaCaT) and primary skin fibroblast cells were used in this study. The migration of the cells was examined by using a scratch wound healing assay, and spheroid behavior was determined by using a spheroid formation assay. The proteins related to migration and stemness were further measured by using Western blotting to explore the mechanism of action of PKE. The methods used to evaluate PKE's antioxidant properties were 2,2-diphenyl-2-picrylhydrazyl (DPPH) scavenging, ABTS radical scavenging activity, and superoxide anion radical scavenging (SOSA) assays. The phytochemistry of the PKE was investigated using phytochemical screening and high-performance liquid chromatography (HPLC) analysis. The results of this study indicate that nontoxic concentrations of PKE increase the rate of migration and spheroid formation. Mechanistically, PKE increased the expression of the migratory-related protein active FAK (phosphorylated FAK), and the subsequence increased the level of p-AKT. The expression of stem cell marker CD133, upstream protein signaling β-catenin, and self-renewal transcription factor Nanog was increased. The PKE also possessed scavenging properties against DPPH, ABTS, and SOSA. The phytochemistry analyses exhibited the presence of alkaloids, glycosides, xanthones, triterpenes, and steroids. Additionally, bioactive compounds such as ɑ-tocopherol, riboflavin, protocatechuic acid, β-carotene, and luteolin were detected. The presence of these chemicals in PKE may contribute to its antioxidant, stem cell potentiation, and wound-healing effects. The findings could be beneficial in the identification of valuable natural resources that possess the capacity to be used in the process of wound healing through the potentiation of stemness via a readily detectable molecular mechanism.

Primary mucinous skin cancer: A case report and review of the literature

Primary mucinous carcinoma of the skin (PMCS) is an extremely uncommon skin cancer originating from sweat glands. We present a case of a male patient diagnosed with PMCS who was initially misdiagnosed with benign sebaceous cysts. A 62-year-old male patient presented to a private clinic with a chief complaint of a slowly progressing nodule located under his lower lip for the past two years; The nodule was locally excised . The histopathology analysis yielded a diagnosis of mucinous carcinoma with a positive circumferential margin. Reexcison was performed, and the final histopathology result was a small nest of mucinous carcinoma with a negative surgical margin. He underwent thorough investigation, including gastroduodenoscopy, colonoscopy, and computerized tomography (CT) scans of the chest, abdomen, and pelvis, and found no signs of primary tumor. He was discharged from our hospital and educated to follow up closely for local recurrence. This paper illustrates the rarity of this disease, its vague presentation, and a brief review of diagnosis and treatment.

Development of an In Vitro secondary transfer model for testosterone gel to elucidate protocols of safe contact.

Testosterone gels applied to skin carry a risk of secondary transfer to non-patients , leading to development of symptoms such as precocious purberty. Currently, there is a lack of&amp;nbsp;in vitro&amp;nbsp;data available to inform guidance over safe contact parameters. A secondary transfer model using porcine skin to model primary (patient) skin and secondary (contact) skin was employed, with testosterone permeation and distribution analysis after 24 hours. Primary skin saw increasing amounts of unabsorbed testosterone and a reduction in delivery to the skin during at later contact times. Secondary skin saw a reduction in unabsorbed testosterone and delivery with later contact times. Permeation data mirrored the distribution data. These differences could be explained by changes in the formulation on the skin surface after application.

Skin cancer survivorship and sun protection behaviors in the United States.

Skin cancer survivors are more vulnerable to subsequent skin cancers and other malignancies, but previous studies have not examined in detail their sun protection behavior prevalence by sociodemographic factors. We aimed to understand the sociodemographic disparities in the prevalence of three important types of sun protection behaviors: using sunscreen, seeking shade, and wearing protective clothing, among skin cancer survivors and those without skin cancer history. We used the 2015 U.S. National Health Interview Survey to analyze 29,523 participants, of which 772 were skin cancer survivors and 28,751 were those without skin cancer history. We assessed overall and specific sun protection behavior prevalence based on using sunscreen, seeking shade, and wearing protective clothing. Weighted Poisson regression was used to estimate prevalence ratios. Melanoma and nonmelanoma skin cancer survivors had similar overall sun protection behavior (p > .05). Among all skin cancer survivors, 36.0% infrequently used sunscreen, 50.2% infrequently wore protective clothing, 47.8% infrequently sought shade, and 30.0% lacked frequent overall sun protection, which significantly differed from those without skin cancer history (p < .0001). The prevalence of frequent overall sun protection behavior was lower for those who were younger at survey, males, less educated, single or never married, or lived in poverty, regardless of their skin cancer history (p < .01). By identifying subpopulations with higher prevalence of infrequent sun protection among those with or without skin cancer history, our findings may encourage efforts to reduce sociodemographic disparities in sun protection behaviors and promote primary and tertiary skin cancer prevention.

Formulation and Evaluation of Butenafine Hydrochloride-Incorporated Solid Lipid Nanoparticles as Novel Excipients for the Treatment of Superficial Fungal Infections.

The objective of the present study was to develop natural excipient-based solid lipid nanoparticles (SLN) of butenafine hydrochloride (BUTE) using a modified solvent emulsification technique and to evaluate the competence of aloe vera nanolipidgel in enhancing the penetration of BUTE. BUTE-SLNs were prepared using a 23 factorial design to correlate the effect of formulation components on the BUTE-SLN. Particle size, polydispersity index (PDI), zeta potential, entrapment performance, and drug loading were assessed in the formed SLNs. The fabricated BUTE-SLN was evaluated for transmission electron microscopy, fourier transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction study studies and revealed the encapsulation of BUTE in lipid in the amorphous state. BUTE-SLN-based aloe vera gel was formulated and evaluated compared with the marketed product with respect to primary skin irritation, hydration, skin permeation, and antifungal activity. The BUTE-SLN aloe vera gel, optimized for its formulation, features excellent slip properties and controlled drug release. DSC and XRD studies confirm its amorphous nature with effective drug entrapment. The gel provides enhanced skin deposition, improved antifungal activity, and reduced irritation. This makes it a cost-effective and innovative alternative to traditional dosage forms. BUTE-SLN promisingly showed no irritation, higher hydrating potential, slow and sustained release, and enhanced antifungal activity. With an aim to target deeper skin strata, minimize the side effects of drugs and symptomatic impact of fungal infection, and shorten the duration of therapy, BUTE-SLN was successfully prepared. The mean particle size and PDI were 261.25 ± 2.38 nm and 0.268 ± 0.01, respectively. BUTE-SLN gel offers improved topical delivery of BUTE with significantly higher compatibility and antifungal activity than the marketed formulation.

Performance of TMRM and Mitotrackers in mitochondrial morphofunctional analysis of primary human skin fibroblasts

Mitochondrial membrane potential (Δψ) and morphology are considered key readouts of mitochondrial functional state. This morphofunction can be studied using fluorescent dyes (“probes”) like tetramethylrhodamine methyl ester (TMRM) and Mitotrackers (MTs). Although these dyes are broadly used, information comparing their performance in mitochondrial morphology quantification and Δψ-sensitivity in the same cell model is still scarce. Here we applied epifluorescence microscopy of primary human skin fibroblasts to evaluate TMRM, Mitotracker Red CMXros (CMXros), Mitotracker Red CMH2Xros (CMH2Xros), Mitotracker Green FM (MG) and Mitotracker Deep Red FM (MDR). All probes were suited for automated quantification of mitochondrial morphology parameters when Δψ was normal, although they did not deliver quantitatively identical results. The mitochondrial localization of TMRM and MTs was differentially sensitive to carbonyl cyanide-4-phenylhydrazone (FCCP)-induced Δψ depolarization, decreasing in the order: TMRM ≫ CHM2Xros = CMXros = MDR > MG. To study the effect of reversible Δψ changes, the impact of photo-induced Δψ “flickering” was studied in cells co-stained with TMRM and MG. During a flickering event, individual mitochondria displayed subsequent TMRM release and uptake, whereas this phenomenon was not observed for MG. Spatiotemporal and computational analysis of the flickering event provided evidence that TMRM redistributes between adjacent mitochondria by a mechanism dependent on Δψ and TMRM concentration. In summary, this study demonstrates that: (1) TMRM and MTs are suited for automated mitochondrial morphology quantification, (2) numerical data obtained with different probes is not identical, and (3) all probes are sensitive to FCCP-induced Δψ depolarization, with TMRM and MG displaying the highest and lowest sensitivity, respectively. We conclude that TMRM is better suited for integrated analysis of Δψ and mitochondrial morphology than the tested MTs under conditions that Δψ is not substantially depolarized.

Multiple-lesion, non-familial basal cell carcinoma-An interesting oddity

Basal cell carcinoma (BCC) is the most common primary carcinoma of skin, which accounts for three-fourth of all primary skin tumours. Basal carcinoma occurs commonly as a single lesion, though occasionally it can occur as Multiple lesions, which may occur in close association with heredofamilial conditions such as nevoid basal cell carcinoma syndrome (Gorlin’s syndrome), Bazex syndrome, Rombo syndrome, and unilateral basal cell nevus syndrome. The case of multiple basal cell carcinomas being presented here is unique in its occurrence inasmuch as it is not associated with heredofamilial conditions, and despite not being associated with heredofamilial conditions there is occurrence of multiple basal carcinomas in a patient in a tertiary care hospital in a dominantly tribal region. The patient has no family history of genodermatosis that could increase the incidence of multiple basal cell carcinomas such as xeroderma pigmentosum, and no history of other predisposing conditions including actinic keratosis, Bowen’s Disease, leukoplakia, Erythroplasia of Queyrat, keratoacanthoma, radiation dermatitis and exposure to arsenicals, psoralen and other photosensitizing medications. However, the patient had history of having raised macules over the face which were diagnosed as seborrheic keratosis.

Classification of psychodermatological disorders: Proposal of a new international classification.

Several classifications of psychodermatology disorders have been proposed, with most of them based on two to four main disorder category groups. However, there is, to date, no classification that has resulted from a consensus established by psychodermatology experts. The DSM-5-TR (Diagnostic and statistical manual of mental disorders (5th ed.), Text Revision) and the ICD-11 (International classification of diseases (11th revision)) also do not provide a systematized approach of psychodermatology disorders. Taking into consideration that classifications are a key pillar for a comprehensive approach to the pathologies of each branch of medicine, the proposal of a classification in psychodermatology appeared as a central need for the recognition of psychodermatological disorders, in an attempt to improve their recognition and, in that sense, to find a common language for the development of this subspecialty that crosses dermatology and psychiatry. Previously published classifications in psychodermatology were critically reviewed and discussed by expert opinion from an international multidisciplinary panel of 16 experts in psychodermatology and a new classification system is proposed, considering classical concepts in general dermatology and psychopathology. Two main categories of disorders are presented (a main group related to primary mental health disorders and another main group related to primary skin disorders), which are subsequently subdivided into subgroups considering pathophysiological and phenomenological similarities, including key aspects of dermatological examination, namely the presence of visible skin lesions (primary and secondary skin lesions) and psychopathological correlates. This new classification aims to unify previous classifications, systematize the disorders that belong to psychodermatology and highlight their tenuous boundaries, to improve their management. It has been built and approved by the Psychodermatology Task Force of the European Academy of Dermatology and Venereology (EADV), the European Society for Dermatology and Psychiatry (ESDaP) and the Association for Psychoneurocutaneous Medicine of North America (APMNA).

Relationship between surgical rhinoplasty and Demodex infestation: a case-control study.

Human Demodex mites are parasites that live in the pilosebaceous unit and can cause demodicosis. While demodicosis may occur as a primary skin disease, it may also result from immunosuppression and topical or systemic immunosuppressive therapies. Surgical rhinoplasty is one of the most commonly performed cosmetic procedures, and it is the cause of a variety of cutaneous complications, particularly acne, as it affects the skin's adnexal structures. Thus, this study aimed to investigate whether the cutaneous changes in surgical rhinoplasty patients render them vulnerable to Demodex infestation. Individuals who had undergone rhinoplasty (patients) and age- and sex-matched healthy volunteers (controls) were included in this prospective case-control study. To determine the Demodex density, samples were collected from the malar and nasal regions of both the patients and controls using the standard superficial skin biopsy method. A total of 50 rhinoplasty patients and 50 healthy controls were enrolled in the study. The Demodex density on the nose was significantly higher in the rhinoplasty patients (p = 0.0001). Furthermore, the frequency of xerosis and pustules was significantly higher in the rhinoplasty patients compared to the control group (p = 0.046 and p = 0.001, respectively). Surgical rhinoplasty may be a risk factor for demodicosis, and patients will recover faster after surgery with proper diagnosis and treatment.

Human Probiotic Lactobacillus paracasei-Derived Extracellular Vesicles Improve Tumor Necrosis Factor-α-Induced Inflammatory Phenotypes in Human Skin.

Lactic acid bacteria (LAB), a probiotic, provide various health benefits. We recently isolated a new Lactobacillus paracasei strain with strong anti-inflammatory effects under lipopolysaccharide-induced conditions and proposed a new mode of action-augmenting the endoplasmic reticulum stress pathway for anti-inflammatory functions in host cells. The beneficial effects of the L. paracasei strains on the skin have been described; however, the effects of L. paracasei-derived extracellular vesicles (LpEVs) on the skin are poorly understood. Herein, we investigated whether LpEVs can improve inflammation-mediated skin phenotypes by determining their effects on primary human skin cells and a three-dimensional (3D) full-thickness human skin equivalent under tumor necrosis factor (TNF)-α-challenged inflammatory conditions. LpEVs were efficiently taken up by the human skin cells and were much less cytotoxic to host cells than bacterial lysates. Furthermore, low LpEV concentrations efficiently restored TNF-α-induced cellular phenotypes, resulting in increased cell proliferation and collagen synthesis, but decreased inflammatory factor levels (matrix metalloproteinase 1, interleukin 6, and interleukin 8) in the human dermal fibroblasts, which was comparable to that of retinoic acid, a representative antiaging compound. The beneficial effects of LpEVs were validated in a 3D full-thickness human skin equivalent model. LpEV treatment remarkably restored the TNF-α-induced epidermal malformation, abnormal proliferation of keratinocytes in the basal layer, and reduction in dermal collagen synthesis. Additionally, LpEVs penetrated and reached the deepest dermal layer within 24 h when overlaid on top of a 3D full-thickness human skin equivalent. Furthermore, they possessed superior antioxidant capacity compared with the human cell-derived EVs. Taken together, the anti-inflammatory probiotic LpEVs can be attractive antiaging and antioxidant substances for improving inflammation-induced skin phenotypes and disorders.

MiR-21 Expressed by Dermal Fibroblasts Enhances Skin Wound Healing Through the Regulation of Inflammatory Cytokine Expression.

The management of skin wound healing is still a challenge. MicroRNA-21 (miR-21) has been reported to play important roles in wound repair; however, the underlying mechanism needs to be further clarified. The present study aimed to study the direct role of miR-21 in skin wound healing in miR-21 KO mice and to investigate the role of miR-21 in controlling the migration and proliferation of primary human skin cells and its underlying mechanism(s). miR-21 KO and wild-type (WT) mice were used for in vivo wound healing assays, while mouse and human primary skin cells were used for in vitro assays. miR-21 inhibitors or mimics or negative control small RNAs were transfected to either inhibit or enhance miR-21 expression in the human primary dermal fibroblasts or epidermal cells. RNA sequencing analysis was performed to identify the potential molecular pathways involved. We found that the loss of miR-21 resulted in slower wound healing in miR-21 KO mouse skin and especially delayed the healing of dermal tissue. In vitro assays demonstrated that the reduced expression of miR-21 caused by its inhibitor inhibited the migration of human primary dermal fibroblasts, which could be enhanced by increased miR-21 expression caused by miR-21 mimics. RNA-sequence analysis revealed that the inhibition of miR-21 expression downregulated the inflammatory response pathways associated with the decreased expression of inflammatory cytokines, and the addition of IL-1β into the culture medium enhanced the migration and proliferation of dermal fibroblasts in vitro. In conclusion, miR-21 in dermal fibroblasts can promote the migration and growth of epidermal and dermal cells to enhance skin wound healing through controlling the expression of inflammatory cytokines.

Quantification of skin wound tension using a newly designed wound tensiometer.

To (i) quantitatively measure wound tension in experimental skin wounds using a newly developed wound tensiometer and (ii) establish reference values for primary skin wound closure in medium- and large-breed dogs. Experimental cadaveric study. Nineteen dogs of medium to large breeds (BW 20 to 40 kg). Elliptical skin wounds of different sizes were created on the chest and abdomen. The wounds were gradually enlarged. Experienced surgeons (ECVS diplomates or professors of small animal surgery) and inexperienced surgeons (1st year after graduation) independently assessed wound tension through manual manipulation and determined whether the wound could be closed without tension-relieving measures. In addition, wound tension was objectively quantified using a newly developed wound tensiometer. The upper threshold for wound tension at which direct appositional wound closure was recommended by the experienced surgeons was 5.4 N, and the median minimal tension without recommendations for closure was 6.0 N. The data also demonstrate that wound tension and wound size do not necessarily correlate, and inexperienced surgeons need to develop a feel for wound tension. The intraoperative use of the wound tensiometer, in combination with established cut-off values, might facilitate decision-making regarding primary wound closure. The findings of this study provide evidence for the applicability of a wound tensiometer in guiding inexperienced surgeons in their choice of the skin wound closure method.