Browsing by Author "Fuentes, Ignacia"
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Item APOBEC mutation drives early-onset squamous cell carcinomas in recessive dystrophic epidermolysis bullosa(American Association for the Advancement of Science, 2018) Cho, Raymond; Alexandrov, Ludmil; den Breems, Nicoline; Atanasova, Velina; Farshchian, Mehdi; Purdom, Elizabeth; Nguyen, Tran; Coarfa, Cristian; Rajapakshe, Kimal; Prisco, Marco; Sahu, Joya; Tassone, Patrick; Greenawalt, Evan; Collisson, Eric; Wu, Wei; Yao, Hui; Su, Xiaoping; Guttmann-Gruber, Christina; Piñón, Josefina; Hashmi, Raabia; Fuentes, IgnaciaRecessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light–induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide–like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus–negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.Item Betaine-urea deep eutectic solvent improves imipenem antibiotic activity(2022) Olivares, Belén; Martínez, Fabián; Ezquer, Marcelo; Morales, Bernardo; Fuentes, Ignacia; Calvo, Margarita; Campodónico, PaolaBeta-lactam antibiotics are highly unstable in aqueous media, which may lead to subclinical concentrations, antimicrobial resistance and therapeutic failure. In previous work we demonstrated that a natural deep eutectic solvent consisting of betaine and urea (BU) is capable of improving the stability of some beta-lactams, including imipenem (IMP), the most unstable antibiotic of the family. Here, IMP-BU was studied by selective protonic Nuclear Overhauser Effect Spectroscopy Magnetic Resonance (H1 NOESY NMR) to gain insight into the mechanism by which BU protects IMP. The kinetics of IMP release and its antibacterial activity were evaluated in diffusional, time-kill and antibiofilm assays. It was found that BU is a protective matrix which allows a fast release of IMP, resulting in superior antibacterial activity when compared to IMP in aqueous solution, both against bacteria growing in planktonic form and in biofilms. Furthermore, it was shown that BU is nontoxic when evaluated in fibroblast primary cell cultures and in organotypic skin cultures, and is not immunogenic when tested in vitro in macrophage cultures, suggesting that BU has potential application as a biomaterial or excipientItem Case Report: Crown Resorption in a Patient With Junctional Epidermolysis Bullosa and Amelogenesis Imperfecta With LAMB3 Gene Mutations(2021) Urzúa, Blanca; Krämer, Susanne; Morales-Bozo, Irene; Camacho, Claudia; Yubero, María Joao; Palisson, Francis; Fuentes, Ignacia; Ortega-Pinto, AnaBackground: Epidermolysis bullosa (EB) corresponds to a series of conditions characterized by extreme fragility of the skin and/or mucous membranes. Of the four main types of EB, junctional EB (JEB) is the most associated with alterations in the teeth. The purposes of this study were to determine the clinical, histopathological, and ultrastructural characteristics of teeth with amelogenesis imperfecta (AI) in a patient with JEB, and compare them with control teeth, and correlate the findings with the mutations present in the patient. Case Report: The study was conducted on a 10-year-old patient with JEB carrier of two recessive mutations in the LAMB3 gene and absence of the laminin-332 protein (LM-332), determined by immunofluorescence on a skin biopsy. The patient presents hypoplastic AI with very thin and yellow-brown colored enamel. Extraction of two permanent molars was performed due to pain and soft tissue covering the crown, resembling pulp polyp or hyperplastic gingiva. Light and scanning electron microscopy (SEM) revealed very thin enamel varying from complete absence to 60 μm, absence of normal prismatic structure, and presence of a cross-banding with a laminated appearance. The histopathological study revealed granulation tissue causing external crown resorption. Conclusion: Although coronary resorption has been reported in patients with syndromic and non-syndromic AI, this is the first clinicopathological report of coronary resorption in partially erupted teeth in patients with JEB with mutations in the LAMB3 gene and hypoplastic AI. In patients with this condition, the presence of partially erupted teeth with soft tissue covering part of the crown, without a periodontal pocket, and with a radiographic image of partial coronal radiolucency should lead to suspicion of external coronary resorption.Item Cells from discarded dressings diferentiate chronic from acute wounds in patients with Epidermolysis Bullosa(2020) Fuentes, Ignacia; Guttmann‑Gruber, Christina; Tockner, Birgit; Diem, Anja; Klausegger, Alfred; Cofré‑Araneda, Glenda; Figuera, Olga; Hidalgo, Yessia; Morandé, Pilar; Palisson, Francis; Rebolledo-Jaramillo, Boris; Yubero, María Joao; Cho, Raymond J.; Rishel, Heather I.; Marinkovich, M. Peter; Teng, Joyce M. C.; Webster, Timothy G.; Prisco, Marco; Eraso, Luis H.; Hofbauer, Josefina Piñon; South, Andrew P.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.Publication Characterisation of the pathophysiology of neuropathy and sensory dysfunction in a mouse model of recessive dystrophic epidermolysis bullosa(2022) Schmidt, Daniela; Díaz, Paula; Muñoz, Daniela; Espinoza, Fernanda; Nystrom, Alexander; Fuentes, Ignacia; Ezquer, Marcelo; Bennett, David L.; Calvo, MargaritaRecessive 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.Publication Characterization of the epidermal-dermal junction in hiPSC-derived skin organoids(2022) Ramovs, Veronika; Janssen, Hans; Fuentes, Ignacia; Pitaval, Amandine; Rachidi, Walid; Chuva de Sousa Lopes, Susana M.; Freund, Christian; Gidrol, Xavier; Mummery, Christine L.; Raymond, KarineHuman 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 β1-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.Item Clinical Practice Guidelines for Epidermolysis Bullosa Laboratory Diagnosis(2019) Has, Cristina; Liu, Lu; Bolling, Marieke; Charlesworth, Alexandra V.; Hachem, May El; Escámez, María José; Fuentes, Ignacia; Büche, Sarah; Hiremagalore, Ravi; Pohla-Gubo, Gabriele; Akker, Peter van den; Wertheim-Tysarowska, Katarzyna; Zambruno, GiovannaThe overall objective of this guideline is to provide the user with information on the laboratory diagnosis of inherited epidermolysis bullosa (EB) to improve outcomes (Table 1). An accurate diagnosis and subclassification of EB enables (i) early prognostication of disease severity, (ii) decision making for patient management, (iii) informed genetic counselling for the patient and family and DNA‐based prenatal or preimplantation genetic diagnosis, (iv) long‐term surveillance and management of possible complications, (v) inclusion in clinical trials and (vi) precision medicine.Item Collagen VII maintains proteostasis in dermal fibroblasts by scaffolding TANGO1 cargo(2022) Cao, Qingqing; Tartaglia, Grace; Alexander, Michael; Park, Pyung Hung; Poojan, Shiv; Farshchian, Mehdi; Fuentes, Ignacia; Chen, Mei; McGrath, John A.; Palisson, Francis; Salas -Alanis, Julio; South, Andrew P.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 TGFβ 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 TGFβ 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.Item Early teeth extraction in patients with generalized recessive dystrophic epidermolysis bullosa: A case series(2020) Véliz, Sebastián; Huber, Hinrich; Yubero, María Joao; Fuentes, Ignacia; Alsayer, Fatimah; Krämer, Susanne M.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.Item Epidermolysis bullosa simplex with KLHL24 mutations is associated with dilated cardiomyopathy(2019) Schwieger-Briel, A.; Fuentes, Ignacia; Castiglia, D.; Barbato, A.; Greutmann, M.; Leppert, J.; Duchatelet, S.; Hovnanian, A.; Burattini, S.; Yubero, María; Ibañez-Arenas, Rodrigo; Rebolledo-Jaramillo, Boris; Gräni, C.; Ott, H.; Theiler, M.; Weibel, L.; Paller, A.S.; Zambruno, G.; Fischer, J.; Palisson, Francis; Has, C.Inherited epidermolysis bullosa (EB) comprises rare heterogeneous disorders characterized by cutaneous and mucosal fragility. Most of the 20 proteins affected have structural functions. Recently, a previously undescribed type of EB simplex (EBS), caused by gain-of-function mutations in KLHL24, encoding KLHL24 has been identified (He et al., 2016; Lin et al., 2016). This protein seems to be involved in protein ubiquitination. Patients carrying monoallelic mutations in the translation initiation codon of KLHL24 have a characteristic clinical phenotype, showing skin defects and blistering at birth and unusual stellate scarring, skin fragility, and whorled or macular hyperpigmentation or hypopigmentation in childhood (Figure 1a–e).Item Epidermolysis bullosa simplex-generalized severe due to p.Glu477Lys mutation in keratin 5: a genotype-phenotype correlation with in silico modeling analysis(2019) Lalor, Leah; Titeux, Matthias; Palisson, Francis; Fuentes, Ignacia; Yubero, María; Tasanen, Kaisa; Huilaja, Laura; Has, Cristina; Tadini, Gianluca; Haggstrom, Anita N.; Hovnanian, Alain; Lucky, Anne W.Background/Objectives: Epidermolysis bullosa is a group of diseases caused by mutations in skin structural proteins. Availability of genetic sequencing makes identification of causative mutations easier, and genotype‐phenotype description and correlation are important. We describe six patients with a keratin 5 mutation resulting in a glutamic acid to lysine substitution at position 477 (p.Glu477Lys) who have a distinctive, severe and sometimes fatal phenotype. We also perform in silico modeling to show protein structural changes resulting in instability. Methods: In this case series, we collected clinical data from six patients with this mutation identified from their national or local epidermolysis bullosa databases. We performed in silico modeling of the keratin 5‐keratin 14 coil 2B complex using CCBuilder and rendered with Pymol (Schrodinger, LLC, New York, NY). Results: Features include aplasia cutis congenita, generalized blistering, palmoplantar keratoderma, onychodystrophy, airway and developmental abnormalities, and a distinctive reticulated skin pattern. Our in silico model of the keratin 5 p.Glu477Lys mutation predicts conformational change and modification of the surface charge of the keratin heterodimer, severely impairing filament stability. Conclusions: Early recognition of the features of this genotype will improve care. In silico analysis of mutated keratin structures provides useful insights into structural instability.Item Epithelial HMGB1 Delays Skin Wound Healing and Drives Tumor Initiation by Priming Neutrophils for NET Formation(2019) Hoste, Esther; Maueröder, Christian; Hove, Lisette van; Catrysse, Leen; Vikkula, Hanna-Kaiza; Sze, Mozes; Maes, Bastiaan; Karjosukarso, Dyah; Martens, Liesbet; Goncalves, Amanda; Parthoens, Eef; Roelandt, Ria; Declercq, Wim; Fuentes, Ignacia; Palisson, Francis; Gonzalez, Sergio; Salas-Alanis, Julio C.; Boon, Louis; Huebener, Peter; Mulder, Klaas Willem; Ravichandran, Kodi; Saeys, Yvan; Schwabe, Robert Felix; Loo, Geert vanRegenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a dangerassociated 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.Item 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(2021) Ramovs, Veronika; Fuentes, Ignacia; Freund, Christian; Mikkers, Harald; Mummery, Christine L.; Raymond, KarineFibroblasts 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.Item Identification of Rigosertib for the Treatment of Recessive Dystrophic Epidermolysis Bullosa-Associated Squamous Cell Carcinoma.(2019) Atanasova, Velina S.; Pourreyron, Celine; Farshchian, Mehdi; Christian A., Brown IV; Watt, Stephen A.; Wright, Sheila; Warkala, Michael; Guttmann-Gruber, Christina; Piñón Hofbauer, Josefina; Fuentes, Ignacia; Prisco, Marco; Rashidghamat, Elham; Has, Cristina; Palisson, Francis; Hovnanian, Alain; McGrath, John A.; Mellerio, Jemima E.; Bauer, Johann; South, Andrew P.Purpose: Squamous cell carcinoma (SCC) of the skin is the leading cause of death in patients with the severe generalized form of the genetic disease recessive dystrophic epidermolysis bullosa (RDEB). Although emerging data are identifying why patients suffer this fatal complication, therapies for treatment of RDEB SCC are in urgent need. Experimental Design: We previously identified polo-like kinase 1 (PLK1) as a therapeutic target in skin SCC, including RDEB SCC. Here, we undertake a screen of 6 compounds originally designated as PLK1 inhibitors, and detail the efficacy of the lead compound, the multipathway allosteric inhibitor ON-01910, for targeting RDEB SCC in vitro and in vivo. Results: ON-01910 (or rigosertib) exhibited significant specificity for RDEB SCC: in culture rigosertib induced apoptosis in 10 of 10 RDEB SCC keratinocyte populations while only slowing the growth of normal primary skin cells at doses 2 orders of magnitude higher. Furthermore, rigosertib significantly inhibited the growth of two RDEB SCC in murine xenograft studies with no apparent toxicity. Mechanistically, rigosertib has been shown to inhibit multiple signaling pathways. Comparison of PLK1 siRNA with MEK inhibition, AKT inhibition, and the microtubule-disrupting agent vinblastine in RDEB SCC shows that only PLK1 reduction exhibits a similar sensitivity profile to rigosertib. Conclusions: These data support a "first in RDEB" phase II clinical trial of rigosertib to assess tumor targeting in patients with late stage, metastatic, and/or unresectable SCC. Translational Relevance Collectively, our data support a clinical trial of rigosertib for treatment of recessive dystrophic epidermolysis bullosa–associated squamous cell carcinoma, an inherently aggressive subtype of squamous cell carcinoma with extremely low 5-year survival. Currently, there are no effective treatments for this devastating cancer, and often times, initial squamous cell carcinoma will recur and readily metastasize; any effective systemic therapy that reduces tumor burden will improve quality of life in this patient population.Publication Longitudinal study of wound healing status and bacterial colonisation of Staphylococcus aureus and Corynebacterium diphtheriae in epidermolysis bullosa patients(2022) Fuentes, Ignacia; Joao, María; Morandé, Pilar; Varela, Carmen; Orostica, Karen; Acevedo, Francisco; Rebolledo, Boris; Arancibia, Esteban; Porte, Lorena; Palisson, FrancisEpidermolysis 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.Publication Maintenance of chronicity signatures in fibroblasts isolated from recessive dystrophic epidermolysis bullosa chronic wound dressings under culture conditions(2023) De Gregorio, Cristian; Catalán, Evelyng; Garrido, Gabriel; Morandé, Pilar; Castillo, Jimena; Muñoz, Catalina; Cofré, Glenda; Huang, Ya-Lin; Cuadra, Bárbara; Murgas, Paola; Calvo, Margarita; Altermatt, Fernando; Joao, María; Palisson, Francis; South, Andrew; Ezquer, Marcelo; Ezquer, Marcelo; Fuentes, IgnaciaBackground: 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-β1 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-1β 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.Item Outcomes and Predictors for Re-stenosis of Esophageal Stricture in Epidermolysis Bullosa: A Multicenter Cohort Study(2020-09) Pope, Elena; Mansour, Mark; Berseneva, María; Liy-Wong, Carmen; Salas, Julio; Fuentes, Ignacia; Yubero, Maria Joao; Palisson, Francis; Martinez, Anna; Mellerio, Jemima; Lara-Corrales, Irene; Yang, Anes; Murrell, Dedee; Torres-Pradilla, Mauricio; Lucky, AnneBackground: 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]¼ 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:x2¼ 4.293,P¼ 0.038, hazard ratio [HR]¼ 1.294 (95% confidence interval [CI]: 1.014–1.652 and 3 vs 1 stricture:x2¼ 7.986, P¼ 0.005, HR¼ 1.785 [95% CI: 1.194, 2.667]) and a long (1 cm) segment stricture (x2¼ 4.599, P¼ 0.032, HR¼ 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; x2¼ 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.Item Recessive dystrophic epidermolysis bullosa results in painful small fibre neuropathy(Springer, 2017) von Bischhoffshausen, Sofia; Ivulic, Dinka; Alvarez, Paola; Schuffeneger, Victor C.; Idiaquez, Juan; Fuentes, Constanza; Morande, Pilar; Fuentes, Ignacia; Palisson, Francis; Bennett, David L. H.; Calvo, MargaritaSmall fibres in the skin are vulnerable to damage in metabolic or toxic conditions such as diabetes mellitus or chemotherapy resulting in small fibre neuropathy and associated neuropathic pain. Whether injury to the most distal portion of sensory small fibres due to a primary dermatological disorder can cause neuropathic pain is still unclear. Recessive dystrophic epidermolysis bullosa (RDEB) is a rare condition in which mutations of proteins of the dermo-epidermal junction lead to cycles of blistering followed by regeneration of the skin. Damage is exclusive to the skin and mucous membranes, with no known direct compromise of the nervous system. It is increasingly recognized that most RDEB patients experience daily pain, the aetiology of which is unclear but may include inflammation (in the wounds), musculoskeletal (due to atrophy and retraction scars limiting movement) or neuropathic pain. In this study we investigated the incidence of neuropathic pain and examined the presence of nerve dysfunction in RDEB patients. Around three quarters of patients presented with pain of neuropathic characteristics, which had a length-dependent distribution. Quantitative sensory testing of the foot revealed striking impairments in thermal detection thresholds combined with an increased mechanical pain sensitivity and wind up ratio (temporal summation of noxious mechanical stimuli). Nerve conduction studies showed normal large fibre sensory and motor nerve conduction; however, skin biopsy showed a significant decrease in intraepidermal nerve fibre density. Autonomic nervous system testing revealed no abnormalities in heart rate and blood pressure variability however the sympathetic skin response of the foot was impaired and sweat gland innervation was reduced. We conclude that chronic cutaneous injury can lead to injury and dysfunction of the most distal part of small sensory fibres in a length-dependent distribution resulting in disabling neuropathic pain. These findings also support the use of neuropathic pain screening tools in these patients and treatment algorithms designed to target neuropathic pain.