Skin in the game: a review of single-cell and spatial transcriptomics in dermatological research.

Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) are two emerging research technologies that uniquely characterize gene expression microenvironments on a cellular or subcellular level. The skin, a clinically accessible tissue composed of diverse, essential cell populations, serves as an ideal target for these high-resolution investigative approaches. Using these tools, researchers are assembling a compendium of data and discoveries in healthy skin as well as a range of dermatologic pathophysiologies, including atopic dermatitis, psoriasis, and cutaneous malignancies. The ongoing advancement of single-cell approaches, coupled with anticipated decreases in cost with increased adoption, will reshape dermatologic research, profoundly influencing disease characterization, prognosis, and ultimately clinical practice.

Orofacial Anomalies in Kindler Epidermolysis Bullosa.

Importance: Kindler epidermolysis bullosa is a genetic skin-blistering disease associated with recessive inherited pathogenic variants in FERMT1, which encodes kindlin-1. Severe orofacial manifestations of Kindler epidermolysis bullosa, including early oral squamous cell carcinoma, have been reported. Objective: To determine whether hypoplastic pitted amelogenesis imperfecta is a feature of Kindler epidermolysis bullosa. Design, Settings, and Participants: This longitudinal, 2-center cohort study was performed from 2003 to 2023 at the Epidermolysis Bullosa Centre, University of Freiburg, Germany, and the Special Care Dentistry Clinic, University of Chile in association with DEBRA Chile. Participants included a convenience sampling of all patients with a diagnosis of Kindler epidermolysis bullosa. Main Outcomes and Measures: The primary outcomes were the presence of hypoplastic pitted amelogenesis imperfecta, intraoral wounds, gingivitis and periodontal disease, gingival hyperplasia, vestibular obliteration, cheilitis, angular cheilitis, chronic lip wounds, microstomia, and oral squamous cell carcinoma. Results: The cohort consisted of 36 patients (15 female [42%] and 21 male [58%]; mean age at first examination, 23 years [range, 2 weeks to 70 years]) with Kindler epidermolysis bullosa. The follow-up ranged from 1 to 24 years. The enamel structure was assessed in 11 patients, all of whom presented with enamel structure abnormalities. The severity of hypoplastic pitted amelogenesis imperfecta varied from generalized to localized pitting. Additional orofacial features observed include gingivitis and periodontal disease, which was present in 90% (27 of 30 patients) of those assessed, followed by intraoral lesions (16 of 22 patients [73%]), angular cheilitis (24 of 33 patients [73%]), cheilitis (22 of 34 patients [65%]), gingival overgrowth (17 of 26 patients [65%]), microstomia (14 of 25 patients [56%]), and vestibular obliteration (8 of 16 patients [50%]). Other features included chronic lip ulcers (2 patients) and oral squamous cell carcinoma with lethal outcome (2 patients). Conclusions and Relevance: These findings suggest that hypoplastic pitted amelogenesis imperfecta is a feature of Kindler epidermolysis bullosa and underscore the extent and severity of oral manifestations in Kindler epidermolysis bullosa and the need for early and sustained dental care.

Antiviral drugs prolong survival in murine recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin disease characterized by defects in type VII collagen leading to a range of fibrotic pathologies resulting from skin fragility, aberrant wound healing, and altered dermal fibroblast physiology. Using a novel in vitro model of fibrosis based on endogenously produced extracellular matrix, we screened an FDA-approved compound library and identified antivirals as a class of drug not previously associated with anti-fibrotic action. Preclinical validation of our lead hit, daclatasvir, in a mouse model of RDEB demonstrated significant improvement in fibrosis as well as overall quality of life with increased survival, weight gain and activity, and a decrease in pruritus-induced hair loss. Immunohistochemical assessment of daclatasvir-treated RDEB mouse skin showed a reduction in fibrotic markers, which was supported by in vitro data demonstrating TGFß pathway targeting and a reduction of total collagen retained in the extracellular matrix. Our data support the clinical development of antivirals for the treatment of patients with RDEB and potentially other fibrotic diseases.

Maintenance of chronicity signatures in fibroblasts isolated from recessive dystrophic epidermolysis bullosa chronic wound dressings under culture conditions.

BACKGROUND: Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare inherited skin disease caused by variants in the COL7A1 gene, coding for type VII collagen (C7), an important component of anchoring fibrils in the basement membrane of the epidermis. RDEB patients suffer from skin fragility starting with blister formation and evolving into chronic wounds, inflammation and skin fibrosis, with a high risk of developing aggressive skin carcinomas. Restricted therapeutic options are limited by the lack of in vitro models of defective wound healing in RDEB patients. RESULTS: In order to explore a more efficient, non-invasive in vitro model for RDEB studies, we obtained patient fibroblasts derived from discarded dressings) and examined their phenotypic features compared with fibroblasts derived from non-injured skin of RDEB and healthy-donor skin biopsies. Our results demonstrate that fibroblasts derived from RDEB chronic wounds (RDEB-CW) displayed characteristics of senescent cells, increased myofibroblast differentiation, and augmented levels of TGF-ß1 signaling components compared to fibroblasts derived from RDEB acute wounds and unaffected RDEB skin as well as skin from healthy-donors. Furthermore, RDEB-CW fibroblasts exhibited an increased pattern of inflammatory cytokine secretion (IL-1ß and IL-6) when compared with RDEB and control fibroblasts. Interestingly, these aberrant patterns were found specifically in RDEB-CW fibroblasts independent of the culturing method, since fibroblasts obtained from dressing of acute wounds displayed a phenotype more similar to fibroblasts obtained from RDEB normal skin biopsies. CONCLUSIONS: Our results show that in vitro cultured RDEB-CW fibroblasts maintain distinctive cellular and molecular characteristics resembling the inflammatory and fibrotic microenvironment observed in RDEB patients' chronic wounds. This work describes a novel, non-invasive and painless strategy to obtain human fibroblasts chronically subjected to an inflammatory and fibrotic environment, supporting their use as an accessible model for in vitro studies of RDEB wound healing pathogenesis. As such, this approach is well suited to testing new therapeutic strategies under controlled laboratory conditions.

Longitudinal study of wound healing status and bacterial colonisation of Staphylococcus aureus and Corynebacterium diphtheriae in epidermolysis bullosa patients.

Epidermolysis bullosa (EB) is an inherited disorder characterised by skin fragility and the appearance of blisters and wounds. Patient wounds are often colonised or infected with bacteria, leading to impaired healing, pain and high risk of death by sepsis. Little is known about the impact of bacterial composition and susceptibility in wound resolution, and there is a need for longitudinal studies to understand healing outcomes with different types of bacterial colonisation. A prospective longitudinal study of 70 wounds from 15 severe EB patients (Junctional and Recessive Dystrophic EB) from Chile. Wounds were selected independently of their infected status. Wound cultures, including bacterial species identification, composition and Staphylococcus aureus (SA) antibiotic susceptibility were registered. Wounds were separated into categories according to their healing capacity, recognising chronic, and healing wounds. Hundred-one of the 102 wound cultures were positive for bacterial growth. From these, 100 were SA-positive; 31 were resistant to Ciprofloxacin (31%) and only seven were methicillin-resistant SA (7%). Ciprofloxacin-resistant SA was found significantly predominant in chronic wounds (**P < .01). Interestingly, atoxigenic Corynebacterium diphtheriae (CD) was identified and found to be the second most abundant recovered bacteria (31/101), present almost always in combination with SA (30/31). CD was only found in Recessive Dystrophic EB patients and not related to wound chronicity. Other less frequent bacterial species found included Pseudomonas aeruginosa, Streptococus spp. and Proteus spp. Infection was negatively associated with the healing status of wounds.

Maintenance of chronicity signatures in fibroblasts isolated from recessive dystrophic epidermolysis bullosa chronic wound dressings under culture conditions

BACKGROUND: Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare inherited skin disease caused by variants in the COL7A1 gene, coding for type VII collagen (C7), an important component of anchoring fibrils in the basement membrane of the epidermis. RDEB patients suffer from skin fragility starting with blister formation and evolving into chronic wounds, inflammation and skin fibrosis, with a high risk of developing aggressive skin carcinomas. Restricted therapeutic options are limited by the lack of in vitro models of defective wound healing in RDEB patients. RESULTS: In order to explore a more efficient, non-invasive in vitro model for RDEB studies, we obtained patient fibroblasts derived from discarded dressings) and examined their phenotypic features compared with fibroblasts derived from non-injured skin of RDEB and healthy-donor skin biopsies. Our results demonstrate that fibroblasts derived from RDEB chronic wounds (RDEB-CW) displayed characteristics of senescent cells, increased myofibroblast differentiation, and augmented levels of TGF-ß1 signaling components compared to fibroblasts derived from RDEB acute wounds and unaffected RDEB skin as well as skin from healthy-donors. Furthermore, RDEB-CW fibroblasts exhibited an increased pattern of inflammatory cytokine secretion (IL-1ß and IL-6) when compared with RDEB and control fibroblasts. Interestingly, these aberrant patterns were found specifically in RDEB-CW fibroblasts independent of the culturing method, since fibroblasts obtained from dressing of acute wounds displayed a phenotype more similar to fibroblasts obtained from RDEB normal skin biopsies. CONCLUSIONS: Our results show that in vitro cultured RDEB-CW fibroblasts maintain distinctive cellular and molecular characteristics resembling the inflammatory and fibrotic microenvironment observed in RDEB patients' chronic wounds. This work describes a novel, non-invasive and painless strategy to obtain human fibroblasts chronically subjected to an inflammatory and fibrotic environment, supporting their use as an accessible model for in vitro studies of RDEB wound healing pathogenesis. As such, this approach is well suited to testing new therapeutic strategies under controlled laboratory conditions.

Viroscope: Plant viral diagnosis from high-throughput sequencing data using biologically-informed genome assembly coverage.

High-throughput sequencing (HTS) methods are transforming our capacity to detect pathogens and perform disease diagnosis. Although sequencing advances have enabled accessible and point-of-care HTS, data analysis pipelines have yet to provide robust tools for precise and certain diagnosis, particularly in cases of low sequencing coverage. Lack of standardized metrics and harmonized detection thresholds confound the problem further, impeding the adoption and implementation of these solutions in real-world applications. In this work, we tackle these issues and propose biologically-informed viral genome assembly coverage as a method to improve diagnostic certainty. We use the identification of viral replicases, an essential function of viral life cycles, to define genome coverage thresholds in which biological functions can be described. We validate the analysis pipeline, Viroscope, using field samples, synthetic and published datasets, and demonstrate that it provides sensitive and specific viral detection. Furthermore, we developed Viroscope.io a web-service to provide on-demand HTS data viral diagnosis to facilitate adoption and implementation by phytosanitary agencies to enable precise viral diagnosis.

Collagen VII maintains proteostasis in dermal fibroblasts by scaffolding TANGO1 cargo.

Lack of type VII collagen (C7) disrupts cellular proteostasis yet the mechanism remains undescribed. By studying the relationship between C7 and the extracellular matrix (ECM)-associated proteins thrombospondin-1 (TSP1), type XII collagen (C12) and tissue transglutaminase (TGM2) in primary human dermal fibroblasts from multiple donors with or without the genetic disease recessive dystrophic epidermolysis bullosa (RDEB) (n=31), we demonstrate that secretion of each of these proteins is increased in the presence of C7. In dermal fibroblasts isolated from patients with RDEB, where C7 is absent or defective, association with the COPII outer coat protein SEC31 and ultimately secretion of each of these ECM-associated proteins is reduced and intracellular levels are increased. In RDEB fibroblasts, overall collagen secretion (as determined by the levels of hydroxyproline in the media) is unchanged while traffic from the ER to Golgi of TSP1, C12 and TGM2 occurs in a type I collagen (C1) dependent manner. In normal fibroblasts association of TSP1, C12 and TGM2 with the ER exit site transmembrane protein Transport ANd Golgi Organization-1 (TANGO1) as determined by proximity ligation assays, requires C7. In the absence of wild-type C7, or when ECM-associated proteins are overexpressed, C1 proximity and intracellular levels increase resulting in elevated cellular stress responses and elevated TGFß signaling. Collectively, these data demonstrate a role for C7 in loading COPII vesicle cargo and provides a mechanism for disrupted proteostasis, elevated cellular stress and increased TGFß signaling in patients with RDEB. Furthermore, our data point to a threshold of cargo loading that can be exceeded with increased protein levels leading to pathological outcomes in otherwise normal cells.

Characterization of the epidermal-dermal junction in hiPSC-derived skin organoids.

Human induced pluripotent stem cell (hiPSC)-derived hair-bearing skin organoids offer exciting new possibilities for modeling diseases like epidermolysis bullosa (EB). These inherited diseases affect 1 in 30,000 people worldwide and result from perturbed expression and/or structure of components of the epidermal-dermal junction (EDJ). To establish whether hiPSC-derived skin organoids might be able to capture salient features of EB, it is thus important to characterize their EDJ. Here, we report successful generation of hair-bearing skin organoids from two hiPSC lines that exhibited fully stratified interfollicular epidermis. Using immunofluorescence and electron microscopy, we showed that basal keratinocytes in organoids adhere to laminin-332 and type IV collagen-rich basement membrane via type I hemidesmosomes and integrin ß1-based adhesion complexes. Importantly, we demonstrated that EDJs in organoids are almost devoid of type VII collagen, a fibril that mediates anchorage of the epidermis to dermis. This should be considered when using skin organoids for EB modeling.

Characterisation of the pathophysiology of neuropathy and sensory dysfunction in a mouse model of recessive dystrophic epidermolysis bullosa.

ABSTRACT: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic condition in which mutations in the type VII collagen gene ( COL7A1 ) lead to decreased expression of this anchoring protein of the skin, causing the loss of stability at the dermo-epidermal junction. Most patients with RDEB experience neuropathic pain and itch due to the development of a small fibre neuropathy, characterised by decreased intraepidermal innervation and thermal hypoaesthesia. To understand the physiopathology of this neuropathy, we used a mouse model of RDEB (Col7a1 flNeo/flNeo ) and performed a detailed characterisation of the somatosensory system. Col7a1 flNeo/flNeo mice showed a decrease in heat sensitivity, an increase in spontaneous scratching, and a significant decrease in intraepidermal nerve fibre density in the hindpaw; these changes were distal because there was no significant loss of unmyelinated or myelinated fibres in the nerve trunk. Of interest, we observed a decrease in axon diameter in both myelinated and unmyelinated fibres. This axonal damage was not associated with inflammation of the dorsal root ganglion or central projection targets at the time of assessment. These results suggest that in RDEB, there is a distal degeneration of axons produced by exclusive damage of small fibres in the epidermis, and in contrast with traumatic and acute neuropathies, it does not induce sustained neuroinflammation. Thus, this animal model emphasizes the importance of a healthy cutaneous environment for maintenance of epidermal innervation and faithfully replicates the pathology in humans, offering the opportunity to use this model in the development of treatments for pain for patients with RDEB.

Generation and genetic repair of two human induced pluripotent cell lines from patients with Epidermolysis Bullosa simplex and dilated cardiomyopathy associated with a heterozygous mutation in the translation initiation codon of KLHL24.

Fibroblasts from two patients carrying a heterozygous mutation in the translation initiation codon (c.2 T > G) of the kelch-like protein 24 (KLHL24) gene were used to generate human induced pluripotent stem cells (hiPSCs), using non-integrating Sendai virus to deliver reprogramming factors. CRISPR-Cas9 editing was used for genetic correction of the mutation in the patient-hiPSCs. The top-predicted off-target sites were not altered. Patient and isogenic hiPSCs showed typical morphology, expressed pluripotency-associated markers, had the capacity for in vitro differentiation into the three germ layers and displayed a normal karyotype. These isogenic pairs will enable in vitro modelling of KLHL24-associated heart and skin conditions.

Ancestral patterns of recessive dystrophic epidermolysis bullosa mutations in Hispanic populations suggest sephardic ancestry.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genodermatosis caused by mutations in the gene coding for type VII collagen (COL7A1). More than 800 different pathogenic mutations in COL7A1 have been described to date; however, the ancestral origins of many of these mutations have not been precisely identified. In this study, 32 RDEB patient samples from the Southwestern United States, Mexico, Chile, and Colombia carrying common mutations in the COL7A1 gene were investigated to determine the origins of these mutations and the extent to which shared ancestry contributes to disease prevalence. The results demonstrate both shared European and American origins of RDEB mutations in distinct populations in the Americas and suggest the influence of Sephardic ancestry in at least some RDEB mutations of European origins. Knowledge of ancestry and relatedness among RDEB patient populations will be crucial for the development of future clinical trials and the advancement of novel therapeutics.

Melasma en el 2021: actualización en la patogénesis y en el enfrentamiento terapéutico / Melasma in 2021: update on the pathogenesis and therapeutic coping

El Melasma es una patología con alta prevalencia a nivel mundial presente en alrededor de un 10% de la población Latinoamericana. Se caracteriza por ser una hipermelanosis cutánea adquirida que ocurre con más frecuencia en cara y cuello de mujeres con fototipos de piel III-VI de Fitzpatrick. Su patogenia aún no ha sido completamente dilucidada; sin embargo, existe evidencia que respalda la asociación del melasma con la radiación ultravioleta, la luz visible, la estimulación hormonal, factores genéticos y procesos inflamatorios. Su diagnóstico es fundamentalmente clínico, y es apoyado por instrumentos de medición que nos permiten objetivar la severidad e impacto en la calidad de vida de los pacientes afectados. El tratamiento continúa siendo un desafío ya que, si bien existen múltiples terapias que han demostrado efectividad, aún no han logrado una remisión completa, presentando una alta tasa de recurrencia. Dentro de las opciones terapéuticas destacan los tratamientos tópicos combinados, los peelings químicos y las terapias basadas en láser, sin embargo, lo más importante es hacer énfasis en la fotoprotección como medida preventiva. En esta revisión pretendemos actualizar sobre los últimos avances tanto de la fisiopatología como del tratamiento del melasma

Melasma is a pathology with a high prevalence worldwide, present in approximately 10% of the Latin American population. It is a cutaneous hypermelanosis that presents itself more frequently on the face and neck of women with Fitzpatrick skin phototypes III-VI. Its pathogenesis has not yet been fully elucidated, however, there is evidence that supports its association with ultraviolet radiation, hormonal stimulation, genetic factors, and inflammatory processes. Its diagnosis is fundamentally clinical, and is supported by clinical scores that allow us to objectify the severity and impact on the quality of life of patients who suffer from it. Treatment continues to be a challenge since, although there are multiple therapies that have demonstrated effectiveness, they have not yet achieved a complete and / or definitive remission of the disease, presenting a high recurrence rate. Treatment options include combined topical therapy, chemical peels and laser-based treatments. Much emphasis has been placed lately on photoprotection of the skin as a preventive measure. In this review we intend to update the latest advances in both the pathophysiology and treatment of melasma

Early teeth extraction in patients with generalized recessive dystrophic epidermolysis bullosa: A case series.

OBJECTIVES: To present early teeth extractions as a treatment option in severe dental crowding in patients with generalized recessive dystrophic epidermolysis bullosa (RDEB). MATERIALS AND METHODS: Three patients with generalized RDEB were treated with early teeth extractions to prevent severe dental crowding. RESULTS: Two patients had bilateral upper first premolars extraction, and the third patient had permanent maxillary canine extraction. Crowding was avoided, and no further orthodontic treatment was necessary. CONCLUSION: Considering the challenges of severe mucosal fragility and microstomia in patients with generalized RDEB, early teeth extractions are a reasonable option as an orthodontic management. This approach reduces the severity of dental crowding as the child gets older and reduces the need for orthodontic appliances. Individual factors such as access to dental care, general health, and oral health have an important impact on the decision-making process. Orthodontic treatment planning should include a multidisciplinary team.

Cells from discarded dressings differentiate chronic from acute wounds in patients with Epidermolysis Bullosa.

Impaired wound healing complicates a wide range of diseases and represents a major cost to healthcare systems. Here we describe the use of discarded wound dressings as a novel, cost effective, accessible, and non-invasive method of isolating viable human cells present at the site of skin wounds. By analyzing 133 discarded wound dressings from 51 patients with the inherited skin-blistering disease epidermolysis bullosa (EB), we show that large numbers of cells, often in excess of 100 million per day, continually infiltrate wound dressings. We show, that the method is able to differentiate chronic from acute wounds, identifying significant increases in granulocytes in chronic wounds, and we show that patients with the junctional form of EB have significantly more cells infiltrating their wounds compared with patients with recessive dystrophic EB. Finally, we identify subsets of granulocytes and T lymphocytes present in all wounds paving the way for single cell profiling of innate and adaptive immune cells with relevance to wound pathologies. In summary, our study delineates findings in EB that have potential relevance for all chronic wounds, and presents a method of cellular isolation that has wide reaching clinical application.

Outcomes and Predictors for Re-stenosis of Esophageal Stricture in Epidermolysis Bullosa: A Multicenter Cohort Study.

BACKGROUND: Esophageal strictures are the common gastrointestinal complications in patients with epidermolysis bullosa (EB) requiring dilation. There is limited information on the best type of intervention, outcomes, and predictors for re-stenosis. OBJECTIVES: We aimed to investigate the frequency, clinical presentation of esophageal strictures in EB patients, and to ascertain the predictors of re-stenosis. METHODS: We conducted a retrospective, multicenter cohort study involving 7 specialized, international EB centers on patients who were 0 to 50 years of age. Descriptive statistics and hazard risks for re-stenosis were calculated. RESULTS: We identified 125 patients with 497 esophageal stricture episodes over a mean period of observation of 17 (standard deviation [SD] = 11.91) years. Dilations were attempted in 90.74% of episodes, using guided fluoroscopy 45.23%, retrograde endoscopy 33.04%, and antegrade endoscopy 19.07%. Successful dilation was accomplished in 99.33% of attempts. Patients experienced a median of 2 (interquartile range [IQR]: 1-7) stricture episodes with a median interval between dilations of 7 (IQR: 4-12) months. Predictors for re-stenosis included: number of strictures (2 vs 1 stricture: χ = 4.293, P = 0.038, hazard ratio [HR] = 1.294 (95% confidence interval [CI]: 1.014--1.652 and 3 vs 1 stricture:χ = 7.986, P = 0.005, HR = 1.785 [95% CI: 1.194, 2.667]) and a long (≥1 cm) segment stricture (χ = 4.599, P = 0.032, HR = 1.347 (95% CI: 1.026--1.769). Complications were more common with the endoscopic approach (8/86, antegrade endoscopy; 2 /149, retrograde endoscopy vs 2/204, fluoroscopy; χ = 17.39, P-value <0.000). CONCLUSIONS: We found excellent dilation outcomes irrespective of the dilation procedure; however, with higher complications in the endoscopic approach. Long (>1 cm) segment involvement and multiple locations were predictive of stricture reoccurrence.

Epithelial HMGB1 Delays Skin Wound Healing and Drives Tumor Initiation by Priming Neutrophils for NET Formation.

Regenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a danger-associated molecular pattern contributing to inflammatory pathologies. We show that HMGB1 derived from keratinocytes, but not myeloid cells, delays cutaneous wound healing and drives tumor formation. In wounds of mice lacking HMGB1 selectively in keratinocytes, a marked reduction in neutrophil extracellular trap (NET) formation is observed. Pharmacological targeting of HMGB1 or NETs prevents skin tumorigenesis and accelerates wound regeneration. HMGB1-dependent NET formation and skin tumorigenesis is orchestrated by tumor necrosis factor (TNF) and requires RIPK1 kinase activity. NETs are present in the microenvironment of keratinocyte-derived tumors in mice and lesional and tumor skin of patients suffering from recessive dystrophic epidermolysis bullosa, a disease in which skin blistering predisposes to tumorigenesis. We conclude that tumorigenicity of the wound microenvironment depends on epithelial-derived HMGB1 regulating NET formation, thereby establishing a mechanism linking reparative inflammation to tumor initiation.