Sara Llames

The first COL7A1 mutation survey in a large Spanish dystrophic epidermolysis bullosa cohort: c.6527insC disclosed as an unusually recurrent mutation

Background  Dystrophic epidermolysis bullosa (DEB) is a genodermatosis caused by mutations in COL7A1. The clinical manifestations are highly variable from nail dystrophy to life‐threatening blistering, making early molecular diagnosis and prognosis of utmost importance for the affected families. Mutation identification is mandatory for prenatal testing.

In Vivo Assessment of Acute UVB Responses in Normal and Xeroderma Pigmentosum (XP-C) Skin-Humanized Mouse Models

In vivo studies of UVB effects on human skin are precluded by ethical and technical arguments on volunteers and inconceivable in cancer-prone patients such as those affected with Xeroderma Pigmentosum (XP). Establishing reliable models to address mechanistic and therapeutic matters thus remains a challenge. Here we have used the skin-humanized mouse system that circumvents most current model constraints. We assessed the UVB radiation effects including the sequential changes after acute exposure with respect to timing, dosage, and the relationship between dose and degree-sort of epidermal alteration. On Caucasian-derived regenerated skins, UVB irradiation (800 J/m(2)) induced DNA damage (cyclobutane pyrimidine dimers) and p53 expression in exposed keratinocytes. Epidermal disorganization was observed at higher doses. In contrast, in African descent-derived regenerated skins, physiological hyperpigmentation prevented tissue alterations and DNA photolesions. The acute UVB effects seen in Caucasian-derived engrafted skins were also blocked by a physical sunscreen, demonstrating the suitability of the system for photoprotection studies. We also report the establishment of a photosensitive model through the transplantation of XP-C patient cells as part of a bioengineered skin. The inability of XP-C engrafted skin to remove DNA damaged cells was confirmed in vivo. Both the normal and XP-C versions of the skin-humanized mice proved proficient models to assess UVB-mediated DNA repair responses and provide a strong platform to test novel therapeutic strategies.

IL‐1α (− 889) promoter polymorphism is a risk factor for osteomyelitis

As osteomyelitis (OM) induces the synthesis of inflammatory cytokines and IL‐1 mediates bone resorption by osteoclasts we determined if there is an association between certain common polymorphisms of the genes encoding proinflammatory cytokines (IL‐1α and β, IL‐6, TNF‐α) and OM in adults. The IL‐1α (− 889) TT genotype was significantly more frequent among 52 OM patients than in 109 healthy controls (13/52, [25.0%] vs. 9/109, [8.3%], P = 0.0081, χ2 = 7.01, OR = 3.7, 95% CI, 1.35–10.34). Patients who were homozygous for the T allele were younger than the rest of the OM patients (mean age 35.7 ± 11.5 vs. 58.1 ± 18.6 years, P = 0.001). IL‐1β TT (+ 3953) polymorphism was also more frequent in OM patients (P = 0.014, χ2 = 5.12, OR = 5.1, 95% CI, 1.21–52.14), but IL‐1β is in linkage disequilibrium with the IL‐1α *T (P < 0.001). Route of infection, chronicity of the infection, type of microorganism isolated, and frequency of relapses were similar in patients with and without the IL‐1α TT genotype. There were no associations between OM and polymorphisms of other cytokines genes. IL‐1α serum levels were significantly increased in all the OM patients independently of their IL‐1 genotype compared to the controls (P = 0.021). Although IL‐1α serum levels were not significantly higher in patients with the IL‐1α (− 889) polymorphism, this does not exclude a difference in production of IL‐1α by osteoclasts or other inflammatory cells at the site of infection.

The regenerative potential of fibroblasts in a new diabetes-induced delayed humanised wound healing model.

Cutaneous diabetic wounds greatly affect the quality of life of patients, causing a substantial economic impact on the healthcare system. The limited clinical success of conventional treatments is mainly attributed to the lack of knowledge of the pathogenic mechanisms related to chronic ulceration. Therefore, management of diabetic ulcers remains a challenging clinical issue. Within this context, reliable animal models that recapitulate situations of impaired wound healing have become essential. In this study, we established a new in vivo humanised model of delayed wound healing in a diabetic context that reproduces the main features of the human disease. Diabetes was induced by multiple low doses of streptozotocin in bioengineered human‐skin‐engrafted immunodeficient mice. The significant delay in wound closure exhibited in diabetic wounds was mainly attributed to alterations in the granulation tissue formation and resolution, involving defects in wound bed maturation, vascularisation, inflammatory response and collagen deposition. In the new model, a cell‐based wound therapy consisting of the application of plasma‐derived fibrin dermal scaffolds containing fibroblasts consistently improved the healing response by triggering granulation tissue maturation and further providing a suitable matrix for migrating keratinocytes during wound re‐epithelialisation. The present preclinical wound healing model was able to shed light on the biological processes responsible for the improvement achieved, and these findings can be extended for designing new therapeutic approaches with clinical relevance.

Keratinocyte cell lines derived from severe generalized recessive Epidermolysis Bullosa patients carrying a highly recurrent COL7A1 homozygous mutation: models to assess cell and gene therapies in vitro and in vivo

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by deficiency of type VII collagen due to COL7A1 mutations such as c.6527insC, recurrently found in the Spanish RDEB population. Assessment of clonal correction–based therapeutic approaches for RDEB requires large expansions of cells, exceeding the replication capacity of human primary keratinocytes. Thus, immortalized RDEB cells with enhanced proliferative abilities would be valuable. Using either the SV40 large T antigen or papillomavirus HPV16‐derived E6‐E7 proteins, we immortalized and cloned RDEB keratinocytes carrying the c.6527insC mutation. Clones exhibited high proliferative and colony‐forming features. Cytogenetic analysis revealed important differences between T antigen‐driven and E6‐E7‐driven immortalization. Immortalized cells responded to differentiation stimuli and were competent for epidermal regeneration and recapitulation of the blistering RDEB phenotype in vivo. These features make these cell lines useful to test novel therapeutic approaches including those aimed at editing mutant COL7A1.

Two novel recessive mutations in KRT14 identified in a cohort of 21 Spanish families with epidermolysis bullosa simplex

Background  Basal epidermolysis bullosa simplex (EBS) is a group of blistering genodermatoses mostly caused by mutations in the keratin genes, KRT5 and KRT14. Recessive mutations represent about 5% of all EBS mutations, being common and specific in populations with high consanguinity, where affected patients show severe phenotypes.

Tissue-engineered oral mucosa for mucosal reconstruction in a pediatric patient with hemifacial microsomia and ankyloglossia.

Many types of soft tissue grafts have been used for the reconstruction of oral mucosal defects. The best results are achieved with mucosal grafts; however, when large areas must be grafted, sufficient donor tissue is not available. Tissue engineering represents an alternative method to obtain sufficient autologous tissue for reconstructing oral wounds. Herein we present a pediatric patient with hemifacial microsomia and congenital ankyloglossia requiring multiple surgical interventions, and in which an autologous full-thickness tissue-engineered oral mucosa was used for successful oral reconstruction. Our study demonstrates that even under challenging conditions, robust tissue-engineered products, such as the fibrin-based oral mucosa described here, can achieve successful tissue regeneration.

Identification of two rare and novel large deletions in ITGB4 gene causing epidermolysis bullosa with pyloric atresia.

Epidermolysis bullosa with pyloric atresia (EB‐PA) is a rare autosomal recessive hereditary disease with a variable prognosis from lethal to very mild. EB‐PA is classified into Simplex form (EBS‐PA: OMIM #612138) and Junctional form (JEB‐PA: OMIM #226730), and it is caused by mutations in ITGA6, ITGB4 and PLEC genes. We report the analysis of six patients with EB‐PA, including two dizygotic twins. Skin immunofluorescence epitope mapping was performed followed by PCR and direct sequencing of the ITGB4 gene. Two of the patients presented with non‐lethal EB‐PA associated with missense ITGB4 gene mutations. For the other four, early postnatal demise was associated with complete lack of β4 integrin due to a variety of ITGB4 novel mutations (2 large deletions, 1 splice‐site mutation and 3 missense mutations). One of the deletions spanned 278 bp, being one of the largest reported to date for this gene. Remarkably, we also found for the first time a founder effect for one novel mutation in the ITGB4 gene. We have identified 6 novel mutations in the ITGB4 gene to be added to the mutation database. Our results reveal genotype–phenotype correlations that contribute to the molecular understanding of this heterogeneous disease, a pivotal issue for prognosis and for the development of novel evidence‐based therapeutic options for EB management.

Clinical outcomes after the use of complete autologous oral mucosa equivalents: preliminary cases

OBJECTIVE Previously, we reported how to obtain complete autologous oral mucosa equivalents (CAOMEs) composed of an autologous plasma scaffold and fibroblasts together with immature keratinocytes able to build an oral epithelium with a structure similar to that of the oral mucosa. In this study, we present the clinical outcomes after applying our CAOMEs as grafts. STUDY DESIGN Four patients who needed a CAOME to restore a defect of oral mucosa were selected. Two of the patients suffered from ankyloglossia, and the other 2 required a restoration of the keratinized gum of the alveolar rim. To assess the outcomes, the scale designed by Ewers et al. was used. RESULTS Clinical and functional improvements were achieved in the patients with ankyloglossia. In cases of gum restoration, the mucosa was regenerated and a prosthetic restoration with implants was achieved. CONCLUSIONS The results obtained points to the potential use of CAOME in intraoral lining.