Type VII Collagen Research Articles

Genetic Insights Into Epidermolysis Bullosa: Identification of Novel Variants in Tunisian Patients.

Epidermolysis Bullosa (EB) is a group of genetic skin disorders characterized by extreme skin fragility and blistering. In North African countries, including Tunisia, complex genetic and phenotypic diversity is entangled with a scarcity of scientific research on EB. This lack of knowledge presents a distinct challenge in terms of diagnostic accuracy and patient care. Our study cohort includes 10 Tunisian patients with EB whose genetic profiles were investigated by exome sequencing. In silico analysis was conducted to determine the functional impact of three novel variants. We revealed ten genetic variants, including three novel ones within the COL7A1 and DST genes. The in silico analysis shed light on the potential structural and functional implications of these novel variants. By establishing the correlation between clinical features and genetic alterations, we have expanded the existing database of disease-causing variants associated with EB in Northern Africa. Our study fills a critical knowledge gap in the North African context, where the scarcity of clinical database and genetic testing in addition to the genetic diversity necessitates comprehensive research. Our findings have the potential to improve diagnosis and management strategies for EB patients in low and middle-income countries across the region, especially through the integration of exome sequencing and in silico analysis.

Use of multivariant enzyme-linked immunosorbent assay (ELISA) in the diagnosis of autoimmune bullous disorders in a resource-limited setting: A single-center experience.

Background Autoimmune blistering disorders (AIBD) result from the formation of auto-antibodies against adhesion proteins of the skin/mucosa(e). These auto-antibodies can be detected in the bound form in the tissue using direct immunofluorescence (DIF) or blood circulation using enzyme-linked immunosorbent assay (ELISA) or other methods. Objectives The objective of this study was to evaluate the concordance rate between the results of multivariant ELISA and the diagnosis of AIBD made using DIF and histopathology in an appropriate clinical context. Methods This was a retrospective study (December 2020 to April 2023) in which the multivariant ELISA assay (able to detect antibodies against desmoglein 1, desmoglein 3, BP180, BP230, envoplakin, and collagen VII) data were retrieved from the dermatology laboratory. Corresponding clinical and histopathology data were searched from relevant institutional databases. As per routine practice, the final diagnosis was assigned based on the clinical presentation, histopathology features and corresponding DIF report. Results After screening the records of 338 patients during the study period, 253 patients were included. Of them, 194 had AIBD and 59 had non-AIBD. In the autoimmune blistering disorder group, 122 and 72 patients had pemphigus and pemphigoid, respectively. Overall, a good level of agreement was found between multivariant ELISA results and the final diagnosis (Fleiss kappa = 0.631, p-value < 0.001). The pemphigus vulgaris group exhibited good agreement (kappa = 0.796, p < 0.001), while pemphigus foliaceous, bullous pemphigoid and non-autoimmune blistering disorders demonstrated moderate agreement (kappa = 0.641, 0.651, 0.533, respectively; p < 0.001). The mucous membrane pemphigoid group had a fair agreement (kappa = 0.289; p < 0.001). Limitations The limitations for the study were its retrospective design, fewer number of patients in certain groups like paraneoplastic pemphigus and gold-standard single antigen specific ELISA was not done. Conclusion Considering good agreement between the multivariant ELISA and the gold-standard diagnosis (clinical findings plus histopathology plus DIF), multivariant ELISA can be used for the diagnosis of AIBDs in places where facilities for DIF are unavailable. Multivariant ELISA can improve etiological diagnosis for a set of autoimmune blistering disorders whose target antigens are represented in the multivariant panel.

Efficacy of rituximab in the treatment of epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is a chronic mucocutaneous disease that is mediated by antibodies that bind collagen VII. The treatment of EBA can be challenging and often multiple immunomodulatory drugs are required. Rituximab has been reported to be an effective treatment for recalcitrant EBA, although its evidence base is limited to case reports and case series. This study therefore aimed to evaluate the efficacy of rituximab in patients with EBA. EBA patients treated with rituximab were identified by searching electronic medical records. Diagnostic criteria for EBA were as follows; mechanobullous skin lesions and / or mucosal ulceration, indirect immunofluorescence localising to the base of salt split skin and positive collagen VII antibodies. Clinical disease activity, collagen VII antibody levels and serum immunoglobulins were recorded at each follow up visit over 600 day period. Treatment responses were classified as follows; complete remission (CR) as the absence of new or established lesions on minimal therapy for 2 months, partial remission (PR) as transient lesions that heal within 1 week on minimal therapy and active disease (AD) as the development of new lesions. 14 patients with EBA were treated with rituximab. CR or PR was observed in 11 patients, and the duration of response varied between 4 and 24 months. A reduction in collagen VII antibody levels was observed in all patients. No significant adverse events were reported. Rituximab is a safe and effective treatment for patients with recalcitrant EBA although there was significant heterogeneity in the disease response.

Endoplasmic reticulum exit sites are segregated for secretion based on cargo size

TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.

Twin Prime Editing Mediated Exon Skipping/Reinsertion for Restored Collagen VII Expression in Recessive Dystrophic Epidermolysis Bullosa

Gene editing nucleases, base editors, and prime editors are potential locus specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa (RDEB); however, many RDEB COL7A1 mutations are unique, making the development of personalized editing reagents challenging. 270 of the ∼320 COL7A1 EB mutations reside in exons that can be skipped, and antisense oligonucleotides (ASO) and gene editing nucleases have been used to create in-frame deletions. ASOs are transient and nucleases generate deleterious double stranded DNA breaks (DSB) and uncontrolled mixtures of allele products. We developed a twin prime editing (twinPE) strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon five. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in RDEB fibroblasts, keratinocytes, and iPSC with minimal DSBs, and collagen type VII (C7) protein was restored. TwinPE can replace the target exon with recombinase attachment sequences, and we exploited this to re-insert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twinPE can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few DSBs as a strategy that may enable a single therapeutic agent to treat multiple RDEB patient cohorts.

Methods for Decreasing Preweaning Mortality in a Fragile Mouse Model of Hypomorphic Collagen VII Deficiency.

Preweaning mortality is a widespread problem in laboratory mouse breeding, particularly in the case of fragile mouse models. While numerous studies explore alternative care methods to increase the survivability of common mouse strains, there remains a paucity of research into the care of mice with fragile health conditions that result from induced or natural genetic mutations. In this study, standard husbandry practices were enhanced by the addition of a softened diet, a nutritionally fortified dietary supplement, soft bedding, gentle handling techniques, decreased handling, lengthened weaning age, and dam productivity tracking. This alternative care plan was shown to increase the survival of a fragile recessive dystrophic epidermolysis bullosa mouse model, and some aspects could be used in developing a care plan for other fragile mouse strains.

Dual Glyoxalase-1 and β-Klotho Gene-Activated Scaffold Reduces Methylglyoxal and Reprograms Diabetic Adipose-Derived Stem Cells: Prospects in Improved Wound Healing.

Tissue engineering approaches aim to provide biocompatible scaffold supports that allow healing to progress often in healthy tissue. In diabetic foot ulcers (DFUs), hyperglycemia impedes ulcer regeneration, due to complications involving accumulations of cellular methylglyoxal (MG), a key component of oxidated stress and premature cellular aging which further limits repair. In this study, we aim to reduce MG using a collagen-chondroitin sulfate gene-activated scaffold (GAS) containing the glyoxalase-1 gene (GLO-1) to scavenge MG and anti-fibrotic β-klotho to restore stem cell activity in diabetic adipose-derived stem cells (dADSCs). dADSCs were cultured on dual GAS constructs for 21 days in high-glucose media in vitro. Our results show that dADSCs cultured on dual GAS significantly reduced MG accumulation (-84%; p < 0.05) compared to the gene-free controls. Similar reductions in profibrotic proteins α-smooth muscle actin (-65%) and fibronectin (-76%; p < 0.05) were identified in dual GAS groups. Similar findings were observed in the expression of pro-scarring structural proteins collagen I (-62%), collagen IV (-70%) and collagen VII (-86%). A non-significant decrease in the expression of basement membrane protein E-cadherin (-59%) was noted; however, the dual GAS showed a significant increase in the expression of laminin (+300%). We conclude that dual GAS-containing Glo-1 and β-klotho had a synergistic MG detoxification and anti-fibrotic role in dADSC's. This may be beneficial to provide better wound healing in DFUs by controlling the diabetic environment and rejuvenating the diabetic stem cells towards improved wound healing.

Efficacy of human living skin equivalent in the treatment of inherited epidermolysis bullosa

Background: inherited epidermolysis bullosa is a group of genetic skin disorders caused by mutations in genes encoding structural proteins of epidermis and dermo-epidermal junction. Clinical manifestations are characterized by spontaneous or trauma-induced skin and/or mucosal blistering, and extensive wounds. Cell therapy is considered to be a perspective therapeutic approach in improving wound healing process.&#x0D; Aims: to assess safety and efficacy of human skin equivalent in the treatment of inherited epidermolysis bullosa patients&#x0D; Materials and methods: 7 patients (5 female and 2 male subjects from the age of 20 to 55) with inherited epidermolysis bullosa with different clinical subtypes were enrolled in the study: 3 patients with intermediate recessive dystrophic epidermolysis bullosa, 2 patients with severe recessive dystrophic epidermolysis bullosa, 1 patient with dominant dystrophic epidermolysis bullosa and 1 patient with junctional epidermolysis bullosa. Transplantation of composite allogeneic living skin equivalent comprising allogeneic keratinocytes and fibroblasts in low concentration (5 mg/ml) embedded within a type I collagen gel matrix was performed. The living skin equivalent was developed at N.K. Koltsov Institute of Developmental Biology. 19 erosions and ulcers with a surface area between 0,4 cm2 and 120 cm2 were evaluated. At day 14 clinical assessment was performed. To assess level of expression immunofluorescence antigen mapping was used.&#x0D; Results: at day 14 complete erosion closure was achieved in 10 (53%) erosions. 4 (21%) erosions reduced in size 75%. Size reduction between 25% and 75% was shown in a single (5%) case. No clinical efficacy was demonstrated in 4 (21%) cases. Collagen VII expression increased comparing to baseline level and accompanied clinical improvement.&#x0D; Conclusions: the obtained data showed clinical efficacy of topical treatment with living skin equivalent, although no statistically significant difference was seen between living skin equivalent and atraumatic non-adhesive dressings.&#x0D; Keywords: inherited epidermolysis bullosa, junctional epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, erosions, healing, topical treatment.

Epidermal or Dermal Collagen VII Is Sufficient for Skin Integrity: Insights to Anchoring Fibril Homeostasis

Collagen VII forms anchoring fibrils that are essential for the stability of the skin and other epithelial organs. In addition to such structural functions, it is emerging that collagen VII fills instructive functions. Collagen VII is synthesized by both epithelial cells and fibroblasts. Genetic loss of collagen VII causes dystrophic epidermolysis bullosa, which manifests with chronic skin fragility and fibrosis. Significant progress has been made in developing therapies for dystrophic epidermolysis bullosa; however, such work has also raised questions on the importance of the cellular source of collagen VII for maintenance of tissue integrity and homeostasis. Toward this end, we engineered mice that kept the physiological expression of collagen VII only in epithelial cells or in fibroblasts. Our study revealed that production of collagen VII either by keratinocytes or fibroblasts alone is sufficient for creation of mechanically robust skin. Importantly, we also show tissue-diverse dependence on epithelial and mesenchymal production of collagen VII and provide support for limited amounts of collagen VII being sufficient for tissue protection. Furthermore, a disconnect between collagen VII abundance and anchoring fibril numbers supports the concept that restoration of fully physiological collagen VII levels may not be needed to achieve complete mechanical protection of dystrophic epidermolysis bullosa skin.

Expression of Collagen VI, Anticollagenase, Laminin, MM9, Claudins 1 and 5, N and E Cadherins in Choroid Plexus Tumors

Background: CPTs are rare intraventricular papillary neoplasms derived from the choroid plexus epithelium. Anti-collagenase and extracellular matrix which have not been expressed in brain tumors. Objective: The purpose of this study was to investigate the expression levels of collagen type VI, anti-collagenase, laminin, MM9, claudins 1 and 5, N and E cadherins, and collagen VII, tejido, and collagen degradation enzyme complexes in choroid plexus tumors. Materials and methods: We studied the expression of adhesion molecules, extracellular matrix, and anticollagenase with an immunohistochemistry approach and electron microscopy analysis in 42 choroid plexus tumors. Results: 28(67%) were choroid plexus papillomas, 8 (19%) were atypical choroid plexus papillomas and 6 (14%) were choroid plexus carcinomas. The Ki67-li and MVD increased from CPC to ACPP, being the highest in malignant tumors as well as a strong immunoexpression of anti-collagenase and were inverse correlation with claudin 5, E, and N cadherin and collagen IV immunoexpressions which added further significant information to the prognosis and varied according to the histologic classification. By ultrastructure, the loss of basal membrane and cilia, disorganization, and proliferation of ECM were observed in CPC. Cerebral homeostasis largely results from the ability of both the Blood–Brain Barrier (BBB) at the brain microvascular endothelium and the Blood–Cerebrospinal Fluid Barrier (BCSFB) at the epithelium of the Choroid Plexuses (CPs), to control the composition of the CSF and cerebral extracellular fluid. Under expression of the tight junction proteins occludin, claudin-1 and claudin-5 are key molecular abnormalities responsible for the increased permeability of tumor endothelial tight junctions. Conclusion: The loss of basement membrane and ECM overexpression could be considered as a poor prognosis predictor in CPT. Anti-collagenase and MMP9 overexpression could be related to basal membrane and BBB plasticity in CPTs.

1122 ZKN-0013 promotes premature termination codon readthrough to restore collagen VII protein expression and function in recessive dystrophic epidermolysis bullosa (RDEB)

1122 ZKN-0013 promotes premature termination codon readthrough to restore collagen VII protein expression and function in recessive dystrophic epidermolysis bullosa (RDEB)

Fibrosis as a Risk Factor for Cutaneous Squamous Cell Carcinoma in Recessive Dystrophic Epidermolysis Bullosa: A Systematic Review.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe subtype of epidermolysis bullosa caused by changes in collagen VII with a high risk of early development of cutaneous squamous cell carcinoma (cSCC). This review aimed to discuss the relationship between the recurrent healing process, the appearance of fibrosis, and malignant epithelial transformation in RDEB. We searched PubMed, the Regional Portal of the Virtual Health Library, and Embase for articles on the relationship between blistering, recurrent scarring, and fibrosis in the context of cSCC and RDEB. That alterations of collagen VII result in blister formation, scar deficiency associated with inflammation, and increased expression of transforming growth factor β. These events promote the differentiation of myofibroblasts and the expression of profibrotic proteins, leading to structural changes and the establishment of a microenvironment favorable to carcinogenesis. Patients with RDEB and areas of recurrent scarring and fibrosis may be more prone to the development of cSCC.

Epidermolysis Bullosa Acquisita-Current and Emerging Treatments.

Epidermolysis bullosa acquisita (EBA) is a rare chronic autoimmune subepidermal blistering disease of the skin and mucous membranes, usually beginning in adulthood. EBA is induced by autoantibodies to type VII collagen, a major component of anchoring fibrils in the dermal-epidermal junction (DEJ). The binding of autoantibodies to type-VII collagen subsequently leads to the detachment of the epidermis and the formation of mucocutaneous blisters. EBA has two major clinical subtypes: the mechanobullous and inflammatory variants. The classic mechanobullous variant presentation consists of skin fragility, bullae with minimal clinical or histological inflammation, erosions in acral distribution that heal with scarring, and milia formation. The inflammatory variant is challenging to differentiate from other autoimmune bullous diseases, most commonly bullous pemphigoid (BP) but also mucous membrane pemphigoid (MMP), Brunsting-Perry pemphigoid, and linear IgA dermatosis. Due to its recalcitrance conventional treatment of epidermolysis bullosa acquisita is shown to be demanding. Here we discuss novel therapeutic strategies that have emerged and which could potentially improve the quality of life in patients with EBA.

Epidermolysis Bullosa Acquisita Presenting a Mix of Classical and Atopiform Features

Epidermolysis bullosa acquisita (EBA) is a rare acquired autoimmune bullous disorder. This disease is characterised by the presence of autoantibodies targeted against Collagen VII of the anchoring fibrils in the basement membrane zone of skin and mucous membranes. The two most common presentations of EBA are the classical or mechano-bullous and the bullous pemphigoid (BP) like forms. Patients with BP-like form of EBA have generally profuse skin lesions suggestive of BP in some areas and EBA in others. We present a case of inflammatory BP like EBA, in a young woman, who presented with classical and also atypical atopiform features.

218 The Impact of Tissue Biomechanics on Epidermal Integrity in Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited skin disorder, characterized by mutations in the gene encoding for collagen VII. DEB manifests in chronic skin fragility with blistering at the dermal-epidermal junction. Unremitting injury and inflammation evoke progressive fibrosis of skin and other epithelia in DEB. Here, we aimed to understand consequences of this with respect to biomechanics, epidermal integrity and barrier function. To this end, we followed changes in skin biomechanics with age in DEB mice using atomic force microscopy and its consequences using lipidomics, followed by validating molecular analyses in human DEB donor skin.

062 Phage display-based isolation of collagen VII-specific monoclonal antibodies from mice with actively induced Epidermolysis Bullosa Acquisita

To establish an entirely murine antibody transfer model for Epidermolysis Bullosa Acquisita (EBA) research, we generated a defined array of monoclonal antibodies (mAbs) against the second von Willebrand factor type A-like domain of murine collagen VII (mCOL7vWFA2), the immunodominant epitope in EBA. To identify mCOL7vWFA2 binding antibodies, we generated two single-chain fragment variable (scFv) immune libraries from the spleen and inguinal lymph nodes of a mCOL7vWFA2 immunized mouse with active EBA disease, confirmed by disease affected body area (10%) and anti-mCOL7vWFA IgG serum titer.

281 Normalization of MCP1 and sCD40L after systemic treatment with bone marrow derived mesenchymal stromal cells correlated with clinical benefits in patients with recessive dystrophic epidermolysis bullosa

RDEB is an incurable rare blistering disorder due to a lack of collagen VII, causing mucocutaneous damage complicated with systemic inflammation. Mesenchymal stromal cells (MSC) have immunomodulatory, antinflammatory and tissue-regenerative properties. Four clinical trials of intravenous MSC in RDEB, one of them by us (Mesensistem-EB, NCT04153630), found temporary clinical benefits but the therapeutic mechanism remain elusive. We have reported the regulation of MCP1 and TGFβ correlating with the health improvement of an RDEB patient after systemic MSC.

Fibroblast morphology, growth rate and gene expression in facial melasma

BackgroundIn addition to melanocytic hyperfunction, changes are observed in the upper dermis of melasma, and fibroblasts play a central role in collagen synthesis and pigmentation induction. ObjectiveTo explore the morphology, growth rate, and gene expression profile of fibroblasts from the skin with melasma in comparison to fibroblasts from the adjacent healthy skin. MethodsTen women with facial melasma were biopsied (lesion and adjacent healthy skin), and the fragments were processed for fibroblast culture. Samples from five participants were seeded to evaluate growth (days 2, 5 and 8) and senescence (SA-β-gal) curves. The samples from the other participants were submitted to real-time PCR to comparatively evaluation of the expression of 39 genes. ResultsCultured fibroblasts from melasma skin were morphologically less fusiform in appearance and on average a 34% (95% CI 4%‒63%) greater proportion of cells labeled with SA-β-gal than the fibroblasts from the adjacent skin. The cell growth rate was lower for the melasma samples after eight days (p < 0.01). TheWNT3A, EDN3, ESR2, PTG2, MMP1, and SOD2 genes were up-regulated, whereas the COL4A1, CSF2, DKK3, COL7A1, TIMP4, CCL2, and CDH11 genes were down-regulated in melasma skin fibroblasts when compared to the ones from adjacent healthy skin. Study limitationsSmall sample size; absence of functional tests. ConclusionsFibroblasts from the skin with melasma showed a lower growth rate, less fusiform morphology and greater accumulation of SA-β-gal than those from adjacent photo exposed skin. Moreover, their gene expression profile comprised factors that may contribute to upper dermis damage and sustained melanogenesis.

Facial skin ageing: Key concepts and overview of processes.

IntroductionThe face is a cosmetically sensitive region where the process of ageing is most clearly manifested. With increased focus on anti‐ageing and longevity, more anti‐senescent treatments are being proposed despite limited evidence. This study outlines the pathways and mechanisms underpinning the biological process of ageing in the face.MethodsComprehensive searches of MEDLINE, EMBASE, Cochrane Library and CINAHL from inception to 2020. Inclusion criteria included all empirical human research studies specific to facial ageing features, written in the English language.ResultsA total of 65 papers met inclusion criteria for analysis. Pathways were subdivided into intrinsic and extrinsic senescence mechanisms. Intrinsic pathways included genetics, generation of reactive oxygen species and hormonal changes. Extrinsic pathways included photoageing and damage to skin layers. The combined intrinsic and extrinsic pathway alterations result in wrinkles, higher laxity, slackness and thinning of the skin. Skin functions such as barrier immune function, wound healing, thermoregulation and sensory function are also impaired.ConclusionThe ageing process is unique to the individual and depends on the interplay between an individual's genetics and external environmental factors. Through understanding the molecular and cellular mechanisms, an appreciation of the consequent structural and functional changes can be achieved. Based on this knowledge, further research can focus on how to slow or impede the ageing process and identify specific targets to develop and evolve new treatment strategies.

Bullous pemphigoid and milia: prevalence and clinical laboratory findings in a Brazilian sample

BackgroundBullous pemphigoid (BP) associated with milia lesions has been increasingly reported, but its prevalence has not been reported in a Brazilian BP population yet. ObjectivesTo describe the occurrence and clinical-laboratorial findings of BP-milia association in a southeastern Brazilian sample. MethodsA descriptive study based on the medical charts of 102 BP patients was accomplished. Clinical and laboratory data of BP-milia patients were compiled. Total serum IgE measurements, immunoblot assays based on basement membrane zone antigens, and HLA-DQ alleles typing were performed. ResultsMilia was evident in 8 (7.8%) BP patients, five males, aged between 46 and 88 years. Increased total IgE levels were determined in 7 (87.5%) of the eight patients. In five of eight patients, immunoblotting showed IgG reactivity against the BP180-NC16a domain but not against collagen VII or laminin-332; it also revealed reactivity against the BP180 C-terminal domain or LAD-1, or both in four of them. The HLA-DQB1*03:01 and HLA-DQA1*05:05 alleles were identified in three of five BP-milia patients. Moreover, three of five cases presented the HLA-DQB1*06 allelic group. Study limitationsHLA determination was performed in five patients. ConclusionsMilia formation in BP patients seems to be less uncommon than previously admitted. Laboratory data revealed increased IgE; autoantibodies against the BP180 C-terminal domain or LAD-1, or both; and the HLA-DQB1*06 allelic group, described for the BP-milia association. Careful determination of antibodies against basement membrane zone molecules and HLA characterization in different populations may provide further insights into this association.