Dermal-epidermal Junction Research Articles

Management of Linear IgA through Ayurveda -A Case study

Linear IgA bullous dermatosis (LABD)/ Chronic bullous disease of childhood (CBDC) is a rare autoimmune blistering disease characterized by the linear deposition of IgA at the dermoepidermal junction. The disease has an incidence of 0.2 to 2.3 per 1000000 million per year showing diverse clinical manifestations and etiologies1.In this case, a 6-year-old child presented with erythematous vesiculobullous lesions on her perianal region, groins, thighs and in both upper and lower limbs since 1 month.The symptoms were gradually progressive in nature.A biopsy was performed, which was consistent with linear immunoglobulin A bullous dermatosis and later confirmed by direct immunofluorescence studies.The child was advised to consume samana aushadhis (internal medicines)for 6 months, which led to remarkable changes in the lakshanas and the patient had no such symptoms till date.This case shows the significant role of ayurvedic intervention in the management of such diseases.

Pressure-Insensitive Epidermal Thickness of Fingertip Skin for Optical Image Encryption.

In this study, an internal fingerprint-guided epidermal thickness of fingertip skin is proposed for optical image encryption based on optical coherence tomography (OCT) combined with U-Net architecture of a convolutional neural network (CNN). The epidermal thickness of fingertip skin is calculated by the distance between the upper and lower boundaries of the epidermal layer in cross-sectional optical coherence tomography (OCT) images, which is segmented using CNN, and the internal fingerprint at the epidermis-dermis junction (DEJ) is extracted based on the maximum intensity projection (MIP) algorithm. The experimental results indicate that the internal fingerprint-guided epidermal thickness is insensitive to pressure due to normal correlation coefficients and the encryption process between epidermal thickness maps of fingertip skin under different pressures. In addition, the result of the numerical simulation demonstrates the feasibility and security of the encryption scheme by structural similarity index matrix (SSIM) analysis between the original image and the recovered image with the correct and error keys decryption, respectively. The robustness is analyzed based on the SSIM value in three aspects: different pressures, noise attacks, and data loss. Key randomness is valid by the gray histograms, and the average correlation coefficients of adjacent pixelated values in three directions and the average entropy were calculated. This study suggests that the epidermal thickness of fingertip skin could be seen as important biometric information for information encryption.

Diagnostic Utility of Direct Immunofluorescence on Paraffin-Embedded Skin Biopsy Samples for the Diagnosis of Autoimmune Vesiculobullous Lesions.

Background Autoimmune vesiculobullous diseases (AIBDs) are a group of diseases characterized by blisters of the skin/mucosa due to the presence of circulating autoantibodies against antigens in the epidermis or the dermo-epidermal junction. Direct immunofluorescence (DIF) for immunoglobulin (Ig)G, IgC3, and IgA on fresh-frozen tissue is the gold standard diagnostic test for AIBDs. However, DIF in the absence of frozen tissue is challenging for the diagnosis of AIBDs. This study aimed to analyze the practical utility of DIF using paraffin-embedded skin biopsy rather than fresh frozen tissue for the diagnosis of AIBDs. Methodology This cross-sectional comparative study included 30 cases of AIBDs. DIF for IgG and IgA was performed on paraffin-embedded tissue (PE-DIF) after proteinase digestion on histopathologically confirmed 15 pemphigus vulgaris (PV), three pemphigus foliaceous (PF), four bullous pemphigoid (BP), three dermatitis herpetiformis (DH), three subcorneal pustular dermatosis (SCPD), and one case each of linear IgA disease and pemphigoid gestationis (PG). PE-DIF staining pattern was compared with the DIF on fresh frozen tissue (FF-DIF). Results All cases of PV and PF showed an intercellular IgG chicken wire staining pattern similar to FF-DIF. However, background staining was more intense in PV cases while less intense in PF cases. Three BP cases showed linear IgG staining in PE-DIF. DH, SCPD, linear IgA disease, and PG cases did not show IgG positivity. Out of three DH cases, two cases showed granular IgA positivity while linear IgA positivity along the basement membrane was seen in a single case of linear IgA disease. Negative IgG staining was observed in SCPD. Immunofluorescence in PE-DIF was rapidly deteriorating than in FF-DIF. Conclusions DIF done on paraffin-embedded tissue can be used as a supplement and salvage technique with histopathology for the diagnosis of AIBDs,particularly when a cryostat facility for frozen tissue is not available and the patient is unable to undergo a second biopsy procedure.

Abstract 4906: Biopsy free virtual histology to discriminate benign from malignant squamous neoplasms

Abstract Reflectance confocal microscopy (RCM) is a noninvasive optical imaging modality that allows for cellular-level resolution, in vivo images of skin without performing a traditional skin biopsy. RCM image interpretation currently requires specialized training to interpret the grayscale output images that are difficult to correlate with tissue pathology. Here, we use a deep learning-based framework that uses a convolutional neural network to transform grayscale output images into virtually-stained hematoxylin and eosin (H&E)-like images allowing for the visualization of various skin layers, including the epidermis, dermal-epidermal junction, and superficial dermis layers. To train the deep-learning framework, a stack of a minimum of 7 time-lapsed, successive RCM images of excised tissue were obtained from epidermis to dermis 1.52 microns apart to a depth of 60.96 microns using the Vivascope 3000. The tissue was embedded in agarose tissue and a curette was used to create a tunnel through which drops of 50% acetic acid was used to stain cell nuclei. These acetic acid-stained images were used as “ground truth” to train a deep convolutional neural network using a conditional generative adversarial network (GAN)-based machine learning algorithm to digitally convert the images into GAN-based H&E-stained digital images. We used the already trained machine learning algorithm and retrained the algorithm with new samples to include squamous neoplasms. Through further training and refinement of the algorithm, high-resolution, histological quality images can be obtained to aid in earlier diagnosis and treatment of cutaneous neoplasms. The overall goal of obtaining biopsy-free virtual histology images with this technology can be used to provide real-time outputs of virtually-stained H&E skin lesions, thus decreasing the need for invasive diagnostic procedures and enabling greater uptake of the technology by the medical community. Citation Format: Nelly Kokikian, Rachel Wahhab, Jingxi Li, Stephanie Martin, David P. Beynet, Aydogan Ozcan, Phillip Scumpia. Biopsy free virtual histology to discriminate benign from malignant squamous neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4906.

Testing the Reliability of Optical Coherence Tomography to Measure Epidermal Thickness and Distinguish Volar and Nonvolar Skin

In persons with limb loss, prosthetic devices cause skin breakdown, largely because residual limb skin (nonvolar) is not intended to bear weight such as palmoplantar (volar) skin. Before evaluation of treatment efficacy to improve skin resiliency, efforts are needed to establish normative data and assess outcome metric reliability. The purpose of this study was to use optical coherence tomography to (i) characterize volar and nonvolar skin epidermal thickness and (ii) examine the reliability of optical coherence tomography. Four orientations of optical coherence tomography images were collected on 33 volunteers (6 with limb loss) at 2 time points, and the epidermis was traced to quantify thickness by 3 evaluators. Epidermal thickness was greater (P < .01) for volar skin (palm) (265.1 ± 50.9 μm, n= 33) than for both nonvolar locations: posterior thigh (89.8 ± 18.1 μm, n= 27) or residual limb (93.4 ± 27.4 μm, n= 6). The inter-rater intraclass correlation coefficient was high for volar skin (0.887-0.956) but low for nonvolar skin (thigh: 0.292-0.391, residual limb: 0.211-0.580). Correlation improved when comparing only 2 evaluators who used the same display technique (palm: 0.827-0.940, thigh: 0.633-0.877, residual limb: 0.213-0.952). Despite poor inter-rater agreement for nonvolar skin, perhaps due to challenges in identifying the dermal-epidermal junction, this study helps to support the utility of optical coherence tomography to distinguish volar from nonvolar skin.

Deficient skin proteins rescue of expression in patients with epidermolysis bullosa: efficacy of gentamicin

Epidermolysis bullosa is a group of rare hereditary skin diseases based on mutations in the genes of structural proteins of the epidermis and the dermal-epidermal junction. Clinically, epidermolysis bullosa is characterized by the appearance of erosions and blisters on the skin and mucous membranes in response to any minor impact. Currently, the treatment of epidermolysis bullosa is only symptomatic. Pathogenetic methods of the epidermolysis bullosa therapy are under development. One of the new possible pathogenetic directions in the treatment of epidermolysis bullosa is aminoglycoside antibiotics (gentamicin, geneticin, paromomycin). A number of studies have shown the ability of gentamicin to promote readthrough terminating codon and resume the synthesis of type VII collagen in keratinocytes and fibroblasts in patients with epidermolysis bullosa with nonsense mutations in the COL7A1. The review presented the possibilities of gentamicin therapy for patients with epidermolysis bullosa, describes the mechanism of its action, summarizes data from clinical trials.

Calpain Inhibition Protects against UVB-Induced Degradation of Dermal–Epidermal Junction–Associated Proteins

Calpain Inhibition Protects against UVB-Induced Degradation of Dermal–Epidermal Junction–Associated Proteins

Evolution of genome diagnostics in epidermolysis bullosa: Unveiling the power of next-generation sequencing.

Genome diagnostics is considered gold standard diagnostics for epidermolysis bullosa (EB), a phenotypically and genetically heterogeneous group of rare disorders characterized by blistering and wounding of mucocutaneous tissues. EB is caused by pathogenic variants in genes encoding proteins of the dermo-epidermal junction. Accurate genetic diagnosis of EB is crucial for prognostication, counselling and precision-medicine. Genome diagnostics for EB started in 1991 with the introduction of Sanger sequencing (SS), analysing one gene at a time. In 2013, SS was superseded by next-generation sequencing (NGS), that allow for high-throughput sequencing of multiple genes in parallel. Several studies have shown a beneficial role for NGS in EB diagnostics, but its true benefit has not been quantified. To determine the benefit of NGS in EB by systematically evaluating the performance of different genome diagnostics used over time based on robust data from the Dutch EB Registry. The diagnostic performances of SS and NGS were systematically evaluated in a retrospective observational study including all index cases with a clinical diagnosis of EB in whom genome diagnostics was performed between 01 January 1994 and 01 January 2022 (n = 308), registered at the Dutch EB Expertise Centre. Over time, a genetic diagnosis was made in 289/308 (94%) EB cases. The diagnostic yield increased from 89% (SS) to 95% (NGS). Most importantly, NGS significantly reduced diagnostic turnaround time (39 days vs. 211 days, p < 0.001). The likelihood of detecting variants of uncertain significance and additional findings increased from 5% and 1% (SS) to 22% and 13% (NGS) respectively. Our study quantifies the benefit of NGS-based methods and demonstrate they have had a major impact on EB diagnostics through an increased diagnostic yield and a dramatically decreased turnaround time (39 days). Although our diagnostic yield is high (95%), further improvement of genome diagnostics is urgently needed to provide a genetic diagnosis in all EB patients.

Deverji's disease after COVID-19: Case report

Deverji's disease is a rare idiopathic skin disease characterized by keratinization disorder and manifested in follicular hyperkeratosis, orange-red peeling plaques, palmar-plantar keratodermia with the possible development of erythroderma. The etiopathogenesis of this dermatosis is still unknown. There are such possible trigger factors as traumatization, ultraviolet radiation, taking certain medications, autoimmune and oncological diseases, bacterial or viral infection, vaccination. The presence of familial cases is due to a mutation in the CARD14 gene. Diagnosis of the disease is based on characteristic clinical symptoms. The histological picture has no pathognomonic features; however, a biopsy is necessary for differential diagnosis with other papulosquamous dermatoses. Treatment of Deverji's disease remains a difficult task, since the disease pathogenesis has not been fully studied.&#x0D; The article describes a clinical case of Deverji's disease manifestation in a 64-year-old woman who had suffered COVID-19 infection twice. She was admitted to the Department of Dermatology and Venerology (Sechenov University) with complaints of skin rashes on her face, trunk, upper and lower extremities, accompanied by severe itching. The absence of any distinctive clinical and histological changes, the torpidity of the skin process and resistance to the therapy made it difficult to make a diagnosis. After the emergence of characteristic clinical symptoms (palmar-plantar keratodermia, salmon-tinged rashes with islands of healthy skin), as well as the results of repeated histological examination (alternating areas of ortho- and parakeratosis; uneven granular layer; vacuolization of basal cells; uneven broad acantholytic strands; loosened dermo-epidermal junction; small perivascular lymph-macrophage infiltrates) Deverji's disease was diagnosed.&#x0D; The use of standard therapies (systemic glucocorticosteroid therapy, methotrexate, topical therapy) did not give any results, and therefore it was decided to initiate the netakimab. After 5 injections, the first positive results were obtained in the form of the color paling and a decrease in the number of rashes, palmar-plantar keratodermia regression and improvement of the patient psychoemotional state. After 11 injections, almost complete remission was achieved, and treatment was continued until all symptoms disappeared completely.&#x0D; The article provides a literature review of the etiopathogenesis, clinical manifestations and treatment methods of Deverji's disease. The described clinical case is the fifth example in the world of the Deverji's disease manifestation after a COVID-19 infection, and is also the first case of the IL-17 inhibitor netakimab successful use for the disease treatment.

Stop codon readthrough as a treatment option for epidermolysis bullosa-Where we are and where we are going.

In the context of rare genetic diseases caused by nonsense mutations, the concept of induced stop codon readthrough (SCR) represents an attractive avenue in the ongoing search for improved treatment options. Epidermolysis bullosa (EB)-exemplary for this group of diseases-describes a diverse group of rare, blistering genodermatoses. Characterized by extreme skin fragility upon minor mechanical trauma, the most severe forms often result from nonsense mutations that lead to premature translation termination and loss of function of essential proteins at the dermo-epidermal junction. Since no curative interventions are currently available, medical care is mainly limited to alleviating symptoms and preventing complications. Complementary to attempts of gene, cell and protein therapy in EB, SCR represents a promising medical alternative. While gentamicin has already been examined in several clinical trials involving EB, other potent SCR inducers, such as ataluren, may also show promise in treating the hitherto non-curative disease. In addition to the extensively studied aminoglycosides and their derivatives, several other substance classes-non-aminoglycoside antibiotics and non-aminoglycoside compounds-are currently under investigation. The extensive data gathered in numerous invitro experiments and the perspectives they reveal in the clinical setting will be discussed in this review.

Molecular Insights Into the Effects of PLLA-SCA on Gene Expression and Collagen Synthesis in Human 3D Skin Models Containing Macrophages.

Injectable poly-L-lactic acid (PLLA-SCA) is used for the correction of shallow to deep nasolabial fold contour deficiencies, cheek wrinkles, and other facial wrinkles. In contrast to hyaluronan (HA) fillers, PLLA-SCA has a biostimulatory effect by activating resident fibroblasts to produce collagen, but the mechanisms are not known in detail at the molecular level. Therefore, our aim was to investigate the molecular effects of PLLA-SCA in a comprehensive in vitro study. Since PLLA-SCA-dependent collagen production in fibroblasts depends on the interaction with macrophages, we generated novel macrophage-containing 3D skin models. According to the clinical application, PLLA-SCA was injected once into the dermal equivalent of the 3D skin model. Histological analysis showed a significant increase in epidermal thickness in these models after 5 and 14 days. Gene expression profiling revealed an upregulation of integrins and laminins (e.g., LAMA3, ITGA6), which are essential components of the dermal-epidermal junction. In addition, we found an upregulation of cytokines and chemokines (TGFB2, CXCL6, IL1B) at day 14 after PLLA-SCA injection. Interestingly, immunohistochemical analyses exhibited a significantly stimulated collagen I production in our models. These effects might be attributed, at least in part, to the upregulation of IL1B and subsequently CXCL6, which stimulates collagen I synthesis in human dermal fibroblasts as we could demonstrate. Taken together, our data provide for the first time molecular insights into the biostimulatory effects of PLLA-SCA on collagen I production in novel human 3D skin models comprising macrophages. J Drugs Dermatol. 2024;23(4):7791.&nbsp; &nbsp; doi:10.36849/JDD.7791.

Effects of a leave-on product on the strength of the dermoepidermal junction: An exploratory, intraindividual, randomized controlled trial in older adults with dry skin.

Skin aging is associated with dry skin and a decrease of the strength of the dermoepidermal adhesion, which increases the risk for lacerations (skin tears). Application of leave-on products improves dry skin and seems to reduce skin tear incidence. The aim of this study was to measure the effects of a humectant containing leave-on product on the strength of the dermoepidermal junction in older adult participants with dry skin. A randomized controlled trial using a split body design was conducted. One forearm was randomly selected and treated with a lipophilic leave-on product containing 5% urea for 8 weeks. The other forearm was the control. The parameters stratum corneum hydration (SCH), transepidermal water loss, pH, roughness, epidermal thickness and skin stiffness were measured at the baseline, Weeks 4 and 8. At Week 8, suction blisters were created and time to blistering was measured. Blister roofs and interstitial fluid were analyzed for Interleukin-1α, 6 and 8. Twelve participants were included. After 8 weeks treatment, SCH was higher (median difference 11.6 AU), and the overall dry skin score (median difference -1) and median roughness (Rz difference -12.2 µm) were lower compared to the control arms. The median group difference for Interleukin-1α was -452 fg/µg total protein (TP) in the blister roofs and -2.2 fg/µg TP in the blister fluids. The median time to blister formation was 7.7 min higher compared to the control arms. The regular application of humectant containing leave-on products improves dry skin and seems to lower inflammation and contribute to the strengthening of the dermoepidermal adhesion. This partly explains how the use of topical leave-on products helps to prevent skin tears.

Bowens disease: clinico pathological correlation with review of literature

Bowen’s disease is a type of squamous cell carcinoma in situ. It is most frequently diagnosed in the sixth to eighth decade of life. However, there have also been a few reports of pigmented Bowen’s disease. A single or multiple erythematous, round to irregular, scaly, keratotic, nodular, pigmented patches/plaques are first signs of the lesion. All cases of Bowen’s disease presented at a tertiary care centre over a period of seven years were included in this study. The patient details and clinical history were obtained from medical records. Histopathological slides of these cases were reviewed and analyzed. There were fourteen cases of Bowen’s disease with most patients belonging to 5-6th decade of life (80%). Males were more commonly affected (60%). Duration of the lesion ranged between 3-5 years in 60% of the patients. Most common site involved was lower back (80%). Most common presenting feature was hyperpigmented, erythematous plaques with scaling seen in 90% of the patients. Epidermal changes such as marked acanthosis with full thickness atypia were noted in 90 % and hyperkeratosis, parakeratosis was noted in 70% of the patients. Lichenoid infiltrates at dermo-epidermal junction was noted in 70% cases. The correct identification helps in appropriate diagnosis and treatment after biopsy confirmation. Adequate resection helps in preventing risk of invasive malignancy in these conditions.

Biofabrication of biomimetic undulating microtopography at the dermal-epidermal junction and its effects on the growth and differentiation of epidermal cells.

The undulating microtopography located at the junction of the dermis and epidermis of the native skin is called rete ridges (RRs), which plays an important role in enhancing keratinocyte function, improving skin structure and stability, and providing three-dimensional (3D) microenvironment for skin cells. Despite some progress in recent years, most currently designed and manufactured tissue-engineered skin models still cannot replicate the RRs, resulting in a lack of biological signals in the manufactured skin models. In this study, a composite manufacturing method including electrospinning, 3D printing, and functional coating was developed to produce the epidermal models with RRs. Polycaprolactone (PCL) nanofibers were firstly electrospun to mimic the extracellular matrix environment and be responsible for cell attachment. PCL microfibers were then printed onto top of the PCL nanofibers layer by 3D printing to quickly prepare undulating microtopography and finally the entire structures were dip-coated with gelatin hydrogel to form a functional coating layer. The morphology, chemical composition, and structural properties of the fabricated models were studied. The results proved that the multi-process composite fabricated models were suitable for skin tissue engineering. Live and dead staining, cell counting kit-8 (CCK-8) as well as histology (haematoxylin and eosin (HE) methodology) and immunofluorescence (primary and secondary antibodies combination assay) were used to investigate the viability, metabolic activity, and differentiation of skin cells forin vitroculturing.In vitroresults showed that each model had high cell viability, good proliferation, and the expression of differentiation marker. It was worth noting that the sizes of the RRs affected the cell growth status of the epidermal models. In addition, the unique undulation characteristics of the epidermal-dermal junction can be reproduced in the developed epidermal models. Overall, thesein vitrohuman epidermal models can provide valuable reference for skin transplantation, screening and safety evaluation of drugs and cosmetics.

Feasibility of High-Cellular-Resolution Full-Field, Artificial-Intelligence-Assisted, Real-Time Optical Coherence Tomography in the Evaluation of Vitiligo: A Prospective Longitudinal Follow-Up Study.

Vitiligo, a psychologically distressing pigmentary disorder characterized by white depigmented patches due to melanocyte loss, necessitates non-invasive tools for early detection and treatment response monitoring. High-cellular-resolution full-field optical coherence tomography (CRFF-OCT) is emerging in pigmentary disorder assessment, but its applicability in vitiligo repigmentation after tissue grafting remains unexplored. To investigate the feasibility of CRFF-OCT for evaluating vitiligo lesions following tissue grafting, our investigation involved ten vitiligo patients who underwent suction blister epidermal grafting and laser ablation at a tertiary center between 2021 and 2022. Over a six-month period, clinical features, dermoscopy, and photography data were recorded. Utilizing CRFF-OCT along with artificial intelligence (AI) applications, repigmentation features were captured and analyzed. The CRFF-OCT analysis revealed a distinct dark band in vitiligo lesion skin, indicating melanin loss. Grafted areas exhibited melanocytes with dendrites around the epidermal-dermal junction and hair follicles. CRFF-OCT demonstrated its efficacy in the early detection of melanocyte recovery and accurate melanin quantification. This study introduces CRFF-OCT as a real-time, non-invasive, and in vivo evaluation tool for assessing vitiligo repigmentation, offering valuable insights into pigmentary disorders and treatment responses.

A Clinicopathological Study on Interface Dermatitis

Background: Interface dermatitis (ID), also known as lichenoid tissue reaction, is a form of skin reaction characterized by an inflammatory infiltration that appears to cover the dermo-epidermal junction under inexpensively analysis. A wide range of inflammatory skin disorders demonstrate interface alteration with significant overlap of histological characteristics. Aim of the Study: The purpose of this study was to link interface dermatitis clinicopathologically. Methods: This cross-sectional descriptive type of study was conducted in the department of dermatology and venereology, TMSS Medical College, Bogura, Bangladesh, from January 2021 to December 2022. The study included 200 patients aged 5 to 75. Skin biopsies were obtained from clinically confirmed cases of lichenoid skin lesions and sent for histological investigation. The correlation was then performed with clinical diagnosis. All acquired data was analysed using descriptive statistics in SPSS 11.5. Results: Out of 200 cases evaluated, the most common type of ID was Lichen simplex chronicus (95, 47.5%), with Lichen planus (LP) and its variants coming in second (84, 42%). LP-like keratosis (15, 7.5%), Inflammatory verrucous epidermal nevus (ILVEN) 2 cases, Pityriasis lichenoides et varioliformisacuta (PLE-VA) 2 cases, and Prurigosimplex (PS) 2 cases were the least prevalent. Clinicopathological concordance was seen in 84 (42%) of the lichen planus patients and discordance in 116 (58%) of the cases. Conclusion: In our investigation, the most consistent histological results were basement membrane degenerations such as lymphocytic infiltrates along the dermo-epidermal interface. Interface dermatitis refers to a group of conditions that share clinical and histological characteristics. As a result, comprehensive histological studies are required to identify distinct features of various kinds of interface dermatitis.

Intravenous gentamicin therapy induces functional type VII collagen in patients with recessive dystrophic epidermolysis bullosa: an open-label clinical trial.

Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable widespread blistering skin disorder caused by mutations in the gene encoding for type VII collagen (C7), the major component of anchoring fibrils. To evaluate the efficacy and safety of intravenous (IV) gentamicin readthrough therapy in patients with RDEB harbouring nonsense mutations. The primary outcomes were increased expression of C7 in patients' skin and safety assessments (ototoxicity, nephrotoxicity, autoimmune response); secondary outcomes included measuring wound healing in target wounds and assessment by a validated Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI) scoring system. An open-label pilot trial to assess two different IV gentamicin regimens between August 2018 and March 2020 with follow-up through to 180 days post-treatment was carried out. Three patients with RDEB with confirmed nonsense mutations in COL7A1 in either one or two alleles and decreased baseline expression of C7 at the dermal-epidermal junction (DEJ) of their skin participated in the study. Three patients received gentamicin 7.5 mg kg-1 daily for 14 days and two of the three patients further received 7.5 mg kg-1 IV gentamicin twice weekly for 12 weeks. Patients who had pre-existing auditory or renal impairment, were currently using ototoxic or nephrotoxic medications, or had allergies to aminoglycosides or sulfate compounds were excluded. After gentamicin treatment, skin biopsies from all three patients (age range 18-28 years) exhibited increased C7 in their DEJ. With both regimens, the new C7 persisted for at least 6 months post-treatment. At 1 and 3 months post-treatment, 100% of the monitored wounds exhibited > 85% closure. Both IV gentamicin infusion regimens decreased EBDASI total activity scores. Of the patients assessed with the EBDASI, all exhibited decreased total activity scores 3 months post-treatment. All three patients completed the study; no adverse effects or anti-C7 antibodies were detected. IV gentamicin induced the readthrough of nonsense mutations in patients with RDEB and restored functional C7 in their skin, enhanced wound healing and improved clinical parameters. IV gentamicin may be a safe, efficacious, low-cost and readily available treatment for this population of patients with RDEB.

Skin dark spot mapping and evaluation of brightening product efficacy using Line-field Confocal Optical Coherence Tomography (LC-OCT).

Facial dark spots remain a significant challenge for the cosmetic industry, in terms of providing effective treatment. Using Line-field Confocal Optical Coherence Tomography (LC-OCT), we investigated the internal structural features of photo-aging spot areas and evaluated the efficacy of a skin-brightening cosmetic product. Twenty-six Asian female volunteers, aged between 29 and 65 years, applied a cosmetic product on their entire face twice a day for 2 months. LC-OCT was used to evaluate the dermal-epidermal junction (DEJ) undulation and the volume density of melanin in the epidermis at D0 and D56. Skin brightening and redness were also assessed by photography (SkinCam). Using LC-OCT technology, various microscopic dark spot morphologies, spanning from minimally deformed DEJ to complex DEJ patterns, were identified. Dark spots characterized by slight deformities in the DEJ were predominantly observed in the youngest age group, while older volunteers displayed a wavier pattern. Furthermore, a total of 44 spots were monitored to evaluate the brightening product efficacy. A statistically significant reduction in melanin volumetric density of 7.3% in the spots and 12.3% in their surrounding area was observed after 56 days of product application. In line with these results, an analysis of color parameters using SkinCam reveals a significant increase in brightening and decrease in redness in both pigmented spots and the surrounding skin following application. LC-OCT proves to be a valuable tool for in-depth dark spots characterization and assessment of skin brightening products, enabling various applications in the field of dermatological sciences.

Evaluation of linear dermatoses by reflectance confocal microscopy in children.

Lichen striatus (LS), linear psoriasis (LPs), linear cutaneous lupus erythematosus (LCLE) and linear lichen planus (LLP) often have similar clinical manifestations, which makes clinical diagnosis with the naked eye difficult; therefore, they are easily misdiagnosed. The purpose of this study was to determine whether reflectance confocal microscopy (RCM) is helpful in differentiating between these four linear dermatoses in children. This retrospective study included 14 patients with LS, nine with LPs, eight with LCLE and 12 with LLP. All patients were analysed using RCM, and biopsies were collected from lesions previously imaged by RCM. For LS, the dermal papillary rings were partially absent, but when present, manifested with small, homogeneously round, bright cells and occasionally highly refractive plump cellular structures, aggregated in clusters. LPs exhibited dark cyst-like structures with small, bright, round cells aggregated at the epidermal level; at the dermal-epidermal junction, homogeneously distributed, enlarged, faint dermal papillary rings and numerous enlarged low-refractive canalicular structures were observed in the superficial dermis. LCLE and LLP exhibited similar manifestations, including epidermal disarray, almost total absence of dermal papillary rings, and various sized refractive structures densely distributed in the dermis. The key distinguishing features of LCLE were the different sized structures mainly clustered around hair follicles, while LLP demonstrated dense structures with a scattered distribution. RCM may be used to distinguish between the key features of LS, LPs, LCLE and LLP in children.

Highly efficient CRISPR/Cas9-mediated exon skipping for recessive dystrophic epidermolysis bullosa.

Gene therapy based on the CRISPR/Cas9 system has emerged as a promising strategy for treating the monogenic fragile skin disorder recessive dystrophic epidermolysis bullosa (RDEB). With this approach problematic wounds could be grafted with gene edited, patient-specificskin equivalents. Precise gene editing using homology-directed repair (HDR) is the ultimate goal, however low efficiencies have hindered progress. Reframing strategies based on highly efficient non-homologous end joining (NHEJ) repair aimed at excising dispensable, mutation-harboring exons offer a promising alternative approach for restoring the COL7A1 open reading frame. To this end, we employed an exon skipping strategy using dual single guide RNA (sgRNA)/Cas9 ribonucleoproteins (RNPs) targeted at three novel COL7A1 exons (31, 68, and 109) containing pathogenic heterozygous mutations, and achieved exon deletion rates of up to 95%. Deletion of exon 31 in both primary human RDEB keratinocytes and fibroblasts resulted in the restoration of type VII collagen (C7), leading to increased cellular adhesion in vitro and accurate C7 deposition at the dermal-epidermal junction in a 3D skin model. Taken together, we extend the list of COL7A1 exons amenable to therapeutic deletion. As an incidental finding, we find that long-read Nanopore sequencing detected large on-target structural variants comprised of deletions up to >5 kb at a frequency of ~10%. Although this frequency may be acceptable given the high rates of intended editing outcomes, our data demonstrate that standard short-read sequencing may underestimate the full range of unexpected Cas9-mediated editing events.