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Background: Despite considerable advancements in cancer immunotherapy, advanced melanoma still presents a substantial clinical challenge. In an effort to explore treatment options, we examined the immunotherapeutic potential of effector Vγ9Vδ2-T cells in vitro in a three-dimensional (3D) human organotypic melanoma-in-skin (Mel-RhS) model. Materials and methods: Vγ9Vδ2-T cells were introduced into Mel-RhS via intradermal injection and cultured within the tissue microenvironment for up to 3 days. Results: Vγ9Vδ2-T cells remained viable for up to 3 days and were in close proximity to or within tumor nests. Upon Mel-RhS dissociation, a fraction was shown to be decorated by melanoma-associated chondroitin sulfate proteoglycan (MCSP), demonstrating their ability to actively navigate the tumor microenvironment and trogocytose cancer cells. Investigation into the apparent trogocytosis revealed an enhanced activated state of MCSP-decorated Vγ9Vδ2-T cells, evidenced by increased expression levels of 4-1BB, NKp44, programmed cell death protein-1 (PD-1), and programmed death-ligand 1 (PD-L1), compared with their MCSP- counterpart. These findings suggest that Vγ9Vδ2-T cells, upon successfully contacting melanoma cells, actively recognize and acquire MCSP from these malignant cells. Evidence of actual tumor cell elimination, although not significant, was only obtained after preincubation of Mel-RhS with pamidronate, a phosphoantigen-inducing agent, indicating the need for additional T cell receptor-mediated signaling for Vγ9Vδ2-T cells to reach their full oncolytic potential. Conclusions: This study highlights the viability and persistence of Vγ9Vδ2-T cells within the 3D microenvironment, their migratory and antitumor functionality, and the suitability of the model for testing T cell-based therapies, contributing both to the understanding of Vγ9Vδ2-T cell biology and their application in cancer immunotherapy.

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Staphylococcus aureus acts both as a colonizing commensal bacterium and invasive pathogen. Nasal colonization is associated with an increased risk of infection caused by the identical strain. In patients with atopic dermatitis (AD), the degree of S. aureus colonization is associated with the severity of the disease. Here, we comparatively analyzed the in vivo transcriptional profile of S. aureus colonizing the nose and non-diseased skin (non-lesional skin) as opposed to the diseased skin (lesional skin-defined here as infection) of 12 patients with AD. The transcriptional profile during the asymptomatic colonization of the nose closely resembled that of the lesional skin samples for many of the genes studied, with an elevated expression of the genes encoding adhesion-related proteins and proteases. In addition, the genes that modify and remodel the cell wall and encode proteins that facilitate immune evasion showed increased transcriptional activity. Notably, in a subgroup of patients, the global virulence regulator Agr (accessory gene regulator) and downstream target genes were inactive during nasal colonization but upregulated in the lesional and non-lesional skin samples. Taken together, our results demonstrate a colonization-like transcriptional profile on diseased skin and suggest a role for the peptide quorum sensing system Agr during the transition from asymptomatic nasal colonization to skin colonization/infection.

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Photobiomodulation (PBM) using 830 nm light-emitting diode (LED) benefits tissue regeneration, wound healing and neural stimulation. However, there is not much exploration of its effect on melanocytes and ex vivo skin model. This study aims to investigate the mechanism behind the anti-melanogenic activity of 830 nm LED and provides evidence for its activity in human ex vivo skin model. Our results showed that 830 nm LED at fluences ranging from 5 to 20 J/cm2 inhibited melanosome maturation and reduced melanin content, tyrosinase activity and melanogenesis-related proteins. 830 nm LED inhibited the phosphorylation of AKT and its downstream FOXO3a, leading to nuclear translocation of FOXO3a. Furthermore, FOXO3a knockdown and AKT activator like SC79 could reverse the melanogenesis inhibition phenotype induced by 830 nm LED. In human ex vivo skin model, Fontana-Masson staining revealed a decrease in epidermal basal pigmentation after 830 nm LED irradiation. Taken together, 830 nm LED demonstrated the anti-melanogenic activity via FOXO3a.

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BACKGROUND: The field of drug development is witnessing a remarkable surge in the development of innovative strategies. There is a need to develop technological platforms capable of generating human data prior to progressing to clinical trials. METHODS: Here we introduce a new flexible solution designed for the comprehensive monitoring of the natural human skin ecosystem's response to immunogenic drugs over time. Based on unique bioengineering to preserve surgical resections in a long survival state, it allows for the first time a comprehensive analysis of resident immune cells response at both organ and single-cell levels. RESULTS: Upon injection of the mRNA-1273 COVID-19 vaccine, we characterized precise sequential molecular events triggered upon detection of the exogenous substance. The vaccine consistently targets DC/macrophages and mast cells, regardless of the administration route, while promoting specific cell-cell communications in surrounding immune cell subsets. CONCLUSION: Given its direct translational relevance, this approach provides a multiscale vision of genuine human tissue immunity that could pave the way toward the development of new vaccination and drug development strategies.

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On the human face, the lips are one of the most important anatomical elements, both morphologically and functionally. Morphologically, they have a significant impact on aesthetics, and abnormal lip morphology causes sociopsychological problems. Functionally, they play a crucial role in breathing, articulation, feeding, and swallowing. An apparatus that can accurately and easily measure the elastic modulus of perioral tissues in clinical tests was developed, and its measurement sensitivity was evaluated. The apparatus is basically a uniaxial compression apparatus consisting of a force sensor and a displacement sensor. The displacement sensor works by enhancing the restoring force due to the deformation of soft materials. Using the apparatus, the force and the displacement were measured for polyurethane elastomers with various levels of softness, which are a model material of human tissues. The stress measured by the developed apparatus increased in proportion to Young's modulus, and was measured by the compression apparatus at the whole region of Young's modulus, indicating that the relation can be used for calibration. Clinical tests using the developed apparatus revealed that Young's moduli for upper lip, left cheek, and right cheek were evaluated to be 45, 4.0, and 9.9 kPa, respectively. In this paper, the advantages of this apparatus and the interpretation of the data obtained are discussed from the perspective of orthodontics.

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BACKGROUND: Oxidative stress plays an important role in the skin aging process. Rapamycin has been shown to have anti-aging effects, but its role in oxidative senescence of skin cells remains unclear. The aim of this study was to explore the effect of rapamycin on oxidative stress-induced skin cell senescence and to illustrate the mechanism. METHODS: Primary human skin fibroblasts (HSFs) were extracted and a model of H2O2-induced oxidative senescence was constructed, and the effects of rapamycin on their value-added and migratory capacities were detected by CCK-8 and scratch assays. SA-ß-gal was utilized to detect senescence, oxidatively closely related factors were also assessed. Gene and protein expressions of senescence, oxidative, and autophagy were detected by western blotting and quantitative-PCR. The data were analyzed by one-way analysis of variance. RESULTS: Rapamycin (0.1 nmol/L for 48 h) promoted the proliferative and migration of H2O2-treated HSFs (p < 0.05), decreased senescent phenotypes SA-ß-gal staining and the expression of P53, and MMP-1 proteins, and increased the expression level of COL1A-1 (p < 0.001). Rapamycin also enhanced the activities of SOD and HO-1, and effectively removed intracellular ROS, MDA levels (p < 0.05), in addition, autophagy-related proteins and genes were significantly elevated after rapamycin pretreatment (p < 0.001). Rapamycin upregulated the autophagy pathway to exert its protective effects. CONCLUSION: Our findings indicate that rapamycin shields HSFs from H2O2-induced oxidative damage, the mechanism is related to the reduction of intracellular peroxidation and upregulation of autophagy pathway. Therefore, rapamycin has the potential to be useful in the investigation and prevention of signs of aging and oxidative stress.

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The most prevalent arthropod-borne viruses, including the dengue viruses, are primarily transmitted by infected mosquitoes. However, the dynamics of dengue virus (DENV) infection and dissemination in human skin following Aedes aegypti probing remain poorly understood. We exposed human skin explants to adult female Ae. aegypti mosquitoes following their infection with DENV-2 by intrathoracic injection. Skin explants inoculated with a similar quantity of DENV-2 by a bifurcated needle were used as controls. Quantitative in situ imaging revealed that DENV replication was greatest in keratinocytes in the base of the epidermis, accounting for 50-60% of all infected cells regardless of the route of inoculation. However, DENV inoculation by Ae. aegypti probing resulted in an earlier and increased viral replication in the dermis, infecting twice as many cells at 24 h when compared to needle inoculation. Within the dermis, enhanced replication of DENV by Ae. aegypti infected mosquitoes was mediated by increased local recruitment of skin-resident macrophages, dermal dendritic cells, and epidermal Langerhans cells relative to needle inoculation. An enhanced but less pronounced influx of resident myeloid cells to the site of mosquito probing was also observed in the absence of infection. Ae. aegypti probing also increased recruitment and infection of dermal mast cells. Our findings reveal for the first time that keratinocytes are the primary targets of DENV infection following Ae. aegypti inoculation, even though most of the virus is inoculated into the dermis during probing. The data also show that mosquito probing promotes the local recruitment and infection of skin-resident myeloid cells in the absence of an intact vasculature, indicating that influx of blood-derived neutrophils is not an essential requirement for DENV spread within and out of skin.

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Exposure to mechanical stimuli such as pressure and stretching prompts the skin to undergo physiological adaptations to accommodate and distribute applied forces, a process known as mechanotransduction. Mechanotherapy, which leverages mechanotransduction, shows significant promise across various medical disciplines. Traditional methods, such as massage and compression therapy, effectively promote skin healing by utilizing this mechanism, although they require direct skin contact. This study introduces a novel contactless modality, Shear Wave Stimulation (SWS), and evaluates its efficacy compared to traditional massage in eliciting responses from human skin and fascia. Fifteen healthy volunteers received SWS, while another fifteen volunteers received massage. Tests of skin mechanical properties revealed significant enhancements in skin shear modulus for both methods, showing an increase of approximately 20%. Additionally, deformation analysis of ultrasound images showed distinct responses of the skin and fascia to the two stimuli. SWS induced extension in the dermis (∼18%), hypodermis (∼16%), and fascia (∼22%) along the X and Y axes. In contrast, massage compressed the skin layers, reducing the dermis by around 15% and the hypodermis by about 8%, while simultaneously stretching the superficial fascia by approximately 8%. The observed extension across the entire skin with SWS highlights its potential as a groundbreaking contactless approach for promoting skin healing. Furthermore, the differing responses in blood flow reaffirm the distinct stimulation modes of SWS and massage. These findings establish a foundation for future innovative skin therapy modalities.

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A number of baby wipe formulations contain 2-phenoxyethanol (PE) as a preservative and cetylpyridinium chloride (CPC) as a surfactant with antimicrobial activity. Previously, we reported the skin absorption of PE in porcine skin and human skin in vitro. In the present work, the permeation of PE from preparations with CPC and without CPC was investigated in human skin in vivo. The studies were conducted using Confocal Raman Spectroscopy (CRS) and tape stripping (TS) methods. The CRS studies showed that the area under the curve (AUC) of PE for the formulation with and without CPC were not significantly different (p > 0.05). The TS data indicated no significant difference in the amounts of PE recovered from tapes 1-6 for the preparation with and without CPC (p > 0.05). When comparing the in vitro and in vivo data, a correlation was observed between the cumulative amount of PE permeated through human skin in vitro at 24 h and the AUC as measured by CRS (r2 = 0.97). In addition, the cumulative amount of PE permeated through human skin in vitro at 24 h was found to correlate with the amount of PE recovered from tape 1 to 6 in vivo (r2 = 0.95). Both CRS and TS techniques demonstrated limitations in assessing the distribution of PE and CPC in the skin in vivo, primarily attributed to the Raman signal intensities of compounds under investigation and the variability in the amount of SC collected by TS. Despite the limitations of CRS and TS, the results from the present study add further insights to the in vitro permeation data. Additionally, the findings of the present study encourage the further development and application of CRS for non-invasive evaluation of topical skin formulations in vivo.

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Ultraviolet A (UVA) radiation causes various irreversible damages to human skin, so the research about UVA-specific sensing device is urgent. 2D black phosphorus (BP) is used in many photosensors due to its advantages of high carrier mobility and tunable bandgap, but its application for UVA-specific photosensor is not reported. Here, a MXene-BP/Zinc oxide (ZnO) hybrid structure with lamellar-spherical interfaces like finger lime fruit is prepared by the layer-by-layer assembly (LLA) method, and p-n junctions are constructed between BP and ZnO with the Ti3C2Tx electrode, showing excellent photoelectric performance. Density functional theory (DFT) calculations demonstrate that the enhanced performance is attributed to the rapid separation of photogenerated carriers in the presence of a built-in electric field at interface. Furthermore, a flexible MXene-BP/ZnO based UVA-specific photosensor is prepared, which shows a specific response to UVA as high as 7 mA W-1 and excellent mechanical stability, maintaining 98.46% response after 100 bending cycles. In particular, the integrated anti-UVA skin protection device shows excellent UVA-specific identification and wireless transmission capability, which can provide timely UVA exposure information and skin protection warning for the visually impaired. This work demonstrates a new approach for further developments of advanced photoelectric sensing technology toward improving people's skin health protection.

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We developed an automated microregistration method that enables repeated in vivo skin microscopy imaging of the same tissue microlocation and specific cells over a long period of days and weeks with unprecedented precision. Applying this method in conjunction with an in vivo multimodality multiphoton microscope, the behavior of human skin cells such as cell proliferation, melanin upward migration, blood flow dynamics, and epidermal thickness adaptation can be recorded over time, facilitating quantitative cellular dynamics analysis. We demonstrated the usefulness of this method in a skin biology study by successfully monitoring skin cellular responses for a period of two weeks following an acute exposure to ultraviolet light.

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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.

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The skin microbiome plays a pivotal role in the production of attractive cues detected by mosquitoes. Here, we leveraged recent advances in genetic engineering to significantly reduce the production of L-(+)-lactic acid as a strategy to reduce mosquito attraction to the highly prominent skin commensals Staphylococcus epidermidis and Corynebacterium amycolatum. Engraftment of these engineered bacteria onto the skin of mice reduced mosquito attraction and feeding for up to 11 uninterrupted days, which is considerably longer than the several hours of protection conferred by the leading chemical repellent N,N-diethyl-meta-toluamide. Taken together, our findings demonstrate engineering the skin microbiome to reduce attractive volatiles represents an innovative untapped strategy to reduce vector attraction, preventing bites, and pathogen transmission. These findings set the stage for new classes of long-lasting microbiome-based repellent products.

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BACKGROUND: In tissue engineering, real-time monitoring of tumors and of the dynamics of the microenvironment within in vitro models has traditionally been hindered by the need to harvest the cultures to obtain material to analyze. Line-field confocal optical coherence tomography (LC-OCT) has proven to be useful in evaluating in vivo skin conditions, including melanoma, by capturing dynamic, three-dimensional (3D) information without the need for invasive procedures, such as biopsies. Additionally, the M-Duo Technology® developed by IMcoMET presents a unique opportunity for continuous in situ biomarker sampling, providing insights into local cellular behavior and interactions. OBJECTIVE: This study aimed to validate the non-destructive mapping capabilities of two advanced methodologies (LC-OCT by DAMAE Medical and M-Duo Technology® by IMcoMET) to investigate the living microenvironment of in vitro reconstructed human skin (RhS) and melanoma-RhS (Mel-RhS). METHODS: LC-OCT and M-Duo Technology® were compared to conventional analysis of the RhS and Mel-RhS microenvironments. RESULTS: LC-OCT successfully visualized the distinct layers of the epidermis and tumor structures within the Mel-RhS, identifying keratinocytes, melanocytes, tumor nests, and fibroblasts. The M-Duo Technology® revealed differences in in situ cytokine (IL-6) and chemokine (CCL2, CXCL10, and IL-8) secretion between Mel-RhS and the control RhS. Notably, such differences were not detected through conventional investigation of secreted proteins in culture supernatants. CONCLUSION: The combination of LC-OCT's high-resolution imaging and M-Duo Technology®'s in situ microenvironmental mapping has the potential to provide a synergistic platform for non-invasive, real-time analysis, allowing for prolonged observation of processes within Mel-RhS models without the need for culture disruption.

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Electroporation, or the use of electric pulses to facilitate the intracellular delivery of DNA, RNA, and other molecules, is a well-established technique, that has been demonstrated to significantly augment the immunogenicity of DNA/mRNA vaccines and therapeutics. However, the clinical translation of traditional electroporators has been limited due to high costs, large size, complex user operation, and poor tolerability in humans due to nerve stimulation. In prior work, we introduced ePatch: an ultra-low-cost, handheld, battery-free electroporator employing a piezoelectric pulser coupled with a microneedle electrode array that showed enhanced immunogenic responses to an intradermal SARS-CoV-2 DNA vaccine in mice. The current study shifts focus from efficacy to tolerability, hypothesizing that ePatch's microneedle array, which localizes the electric field to the superficial skin strata, will minimize nerve stimulation and improve patient comfort. We tested this hypothesis in 14 healthy adults, monitoring pain and other potential adverse effects associated with electroporation. Compared to the insertion of a traditional hypodermic needle, the ePatch was less painful. Adverse effects such as pain, tenderness, erythema and swelling at the application sites were minimal, transient, and statistically indistinguishable between the experimental and placebo ePatch application, suggesting excellent tolerability towards electroporation. In summary, ePatch has a favorable tolerability profile in humans and offers the potential for the safe use of electroporation in a variety of clinical settings, including DNA and mRNA vaccination.

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Human skin ageing is closely related to the ageing of the whole organism, and it's a continuous multisided process that is influenced not only by genetic and physiological factors but also by the cumulative impact of environmental factors. Currently, there is a scientific community need for developing skin models representing ageing processes to (i) enhance understanding on the mechanisms of ageing, (ii) discover new drugs for the treatment of age-related diseases, and (iii) develop effective dermo-cosmetics. Bioengineers worldwide are trying to reproduce skin ageing in the laboratory aiming to better comprehend and mitigate the senescence process. This review provides details on the main ageing molecular mechanisms and procedures to obtain in vitro aged skin models.

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Advanced numerical simulations of the mechanical behavior of human skin require thorough calibration of the material's constitutive models based on experimental ex vivo mechanical tests along with images of tissue microstructure for a variety of biomedical applications. In this work, a total of 14 human healthy skin samples and 4 additional scarred skin samples were experimentally analyzed to gain deep insights into the biomechanics of human skin. In particular, second harmonic generation (SHG) microscopy was used to extract detailed images of the distribution of collagen fibers, which were subsequently processed using a three-dimensional Fourier transform-based method recently proposed by the authors to quantify the distribution of fiber orientations. Mechanical tests under both biaxial and uniaxial loading were performed to calibrate the relevant mechanical parameters of two widely used constitutive models of soft fiber-reinforced biological tissues that account for non-symmetrical fiber dispersion. The calibration of the models allowed us to identify correlations between the mechanical parameters of the constitutive models considered. STATEMENT OF SIGNIFICANCE: Constitutive models for soft collagenous tissues can accurately reproduce the complex nonlinear and anisotropic mechanical behavior of skin. However, a comprehensive analysis of both microstructural and mechanical parameters is still missing for human skin. In this study, these parameters are determined by combining biaxial mechanical tests and SHG stacks of collagen fibers on ex vivo healthy human skin samples. The constitutive parameters are provided for two widely used hyperelastic models and enable accurate characterization of skin mechanical behavior for advanced numerical simulations.

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Lidocaine is generally recognized and preferred for local anaesthesia, but in addition, studies have described additional benefits of lidocaine in cancer therapy, inflammation reduction, and wound healing. These properties contribute to its increasing importance in dermatological applications, and not only in pain relief but also in other potential therapeutic outcomes. Therefore, the purpose of our study was to enhance lidocaine delivery through the skin. A stable nanostructured lipid carrier (NLC), as a passive permeation enhancer, was developed using a 23 full factorial design. The nanosystems were characterized by crystallinity behaviour, particle size, zeta potential, encapsulation efficiency measurements, and one of them was selected for further investigation. Then, NLC gel was formulated for dermal application and compared to a traditional dermal ointment in terms of physicochemical (rheological behaviour) and biopharmaceutical (qualitative Franz diffusion and quantitative Raman investigations) properties. The study also examined the use of 3D printed solid microneedles as active permeation enhancers for these systems, offering a minimally invasive approach to enhance transdermal drug delivery. By actively facilitating drug permeation through the skin, microneedles can complement the passive transport achieved by NLCs, thereby providing an innovative and synergistic approach to improving lidocaine delivery.

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Skin wound healing is driven by proliferation, migration and differentiation of several cell types that are controlled by the alterations in the gene expression programmes. Brahma Gene 1 (BRG1) (also known as SMARCA4) is a core ATPase in the BRG1 Associated Factors (BAF) ATP-dependent chromatin remodelling complexes that alter DNA-histone interaction in chromatin at the specific gene regulatory elements resulting in increase or decrease of the target gene transcription. Using siRNA mediated suppression of BRG1 during wound healing in a human ex vivo and in vitro (scratch assay) models, we demonstrated that BRG1 is essential for efficient skin wound healing by promoting epidermal keratinocytes migration, but not their proliferation or survival. BRG1 controls changes in the expression of genes associated with gene transcription, response to wounding, cell migration and cell signalling. Altogether, our data revealed that BRG1 play positive role in skin repair by promoting keratinocyte migration and impacting the genes expression programmes associated with cell migration and cellular signalling.

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The mineral content of thermal spring water (TSW) applied to the skin surface can directly influence the skin barrier. Indeed, our previous study showed that Avène TSW (ATSW), a low mineral content thermal spring water, protects the stratum corneum from dehydration compared to a mineral-rich TSW (MR-TSW) and maintains skin surface ultrastructure. While many TSWs have been recognized to have beneficial effects on skin, little is known about their localized and specific effects on skin barrier biomechanics at the nanometric scale. The aim of this study was to compare the effects of ATSW with a reference, MR-TSW, on the biomechanical barrier properties of the skin under homeostasis conditions using atomic force microscopy (AFM). AFM was used to obtain a precise nanomechanical mapping of the skin surface after three applications of both TSW. This provides specific information on the skin topographical profile and elasticity. The topographic profile of skin samples showed a specific compaction of the skin layers after application of MR-TSW, characterized by an increase of the total number of external skin layers, compared to non-treated samples. By contrast, ATSW did not modify the skin topographic profile. High-resolution force/volume acquisitions to capture the elastic modulus showed that it was directly correlated with skin rigidity. The elastic modulus strongly and significantly increased after MR-TSW application compared to non-treated skin. By contrast, applications of ATSW did not increase elastic modulus. These data demonstrate that applications of MR-TSW significantly modified skin barrier properties by increasing skin surface layer compaction and skin rigidity. By contrast, ATSW did not modify the topographical profile of skin explants nor induce mechanical stress at the level of the stratum corneum, indicating it does not disrupt the biophysical properties linked to skin surface integrity.

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