Closure of a Large Chronic Wound through Transplantation of Gene-Corrected Epidermal Stem Cells

Generalized junctional epidermolysis bullosa (JEB) is caused by mutations in LAMA3,LAMB3,or LAMC2,which together encode laminin-332, a hetero-trimeric protein consisting ofa3,b3, andg2chain. In nonlethal generalized intermediate JEB, laminin-332 is highly reduced, and hemidesmosomes are rudimentary or completely absent, leading to blister formation within the lamina lucida of the basement membrane upon minor trauma. The resulting chronic skin wounds invariably develop recurrent infections and scarring, which greatly impair patients’ quality of life. We report on a patient in whom gene-corrected epidermal sheets were transplanted onto a large nonhealing epidermal ulceration following a good manufacturing practice protoco

Antidiabetic Drug Prescription Pattern in Hospitalized Older Patients with Diabetes

Objective: To describe the prescription pattern of antidiabetic and cardiovascular drugs in a cohort of hospitalized older patients with diabetes. Methods: Patients with diabetes aged 65 years or older hospitalized in internal medicine and/or geriatric wards throughout Italy and enrolled in the REPOSI (REgistro POliterapuie SIMI—Società Italiana di Medicina Interna) registry from 2010 to 2019 and discharged alive were included. Results: Among 1703 patients with diabetes, 1433 (84.2%) were on treatment with at least one antidiabetic drug at hospital admission, mainly prescribed as monotherapy with insulin (28.3%) or metformin (19.2%). The proportion of treated patients decreased at discharge (N = 1309, 76.9%), with a significant reduction over time. Among those prescribed, the proportion of those with insulin alone increased over time (p = 0.0066), while the proportion of those prescribed sulfonylureas decreased (p < 0.0001). Among patients receiving antidiabetic therapy at discharge, 1063 (81.2%) were also prescribed cardiovascular drugs, mainly with an antihypertensive drug alone or in combination (N = 777, 73.1%). Conclusion: The management of older patients with diabetes in a hospital setting is often sub-optimal, as shown by the increasing trend in insulin at discharge, even if an overall improvement has been highlighted by the prevalent decrease in sulfonylureas prescription

The “Diabetes Comorbidome”: A Different Way for Health Professionals to Approach the Comorbidity Burden of Diabetes

(1) Background: The disease burden related to diabetes is increasing greatly, particularly in older subjects. A more comprehensive approach towards the assessment and management of diabetes’ comorbidities is necessary. The aim of this study was to implement our previous data identifying and representing the prevalence of the comorbidities, their association with mortality, and the strength of their relationship in hospitalized elderly patients with diabetes, developing, at the same time, a new graphic representation model of the comorbidome called “Diabetes Comorbidome”. (2) Methods: Data were collected from the RePoSi register. Comorbidities, socio-demographic data, severity and comorbidity indexes (Cumulative Illness rating Scale CIRS-SI and CIRS-CI), and functional status (Barthel Index), were recorded. Mortality rates were assessed in hospital and 3 and 12 months after discharge. (3) Results: Of the 4714 hospitalized elderly patients, 1378 had diabetes. The comorbidities distribution showed that arterial hypertension (57.1%), ischemic heart disease (31.4%), chronic renal failure (28.8%), atrial fibrillation (25.6%), and COPD (22.7%), were the more frequent in subjects with diabetes. The graphic comorbidome showed that the strongest predictors of death at in hospital and at the 3-month follow-up were dementia and cancer. At the 1-year follow-up, cancer was the first comorbidity independently associated with mortality. (4) Conclusions: The “Diabetes Comorbidome” represents the perfect instrument for determining the prevalence of comorbidities and the strength of their relationship with risk of death, as well as the need for an effective treatment for improving clinical outcomes

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

Bases moléculaires de l’adhésion cellulaire à la laminine 332 induite par le syndecan-1

L’interaction du récepteur syndecan-1 de la famille des héparanes sulfates protéoglycanes avec le fragment carboxy-terminal alpha3LG4/5 de la protéine d’adhérence matricielle, la laminine 332, induit une réorganisation du cytosquelette de la cellule conduisant à la formation de filopodes et de microspicules, caractéristiques de la migration cellulaire. Notre laboratoire a mis en évidence que l’adhésion cellulaire syndecan-1 dépendante implique les chaînes d’héparanes sulfates et chondroïtine sulfate. Afin d’identifier le (les) zone(s) impliquée(s) dans l’interaction domaine LG4/5-syndécan-1, une approche de mutagenèse dirigée a été mise en place sur le fragment LG4/5 recombinant. Les résidus conservés parmi les laminines, identifiés dans la littérature comme liant l’héparine, aussi bien que des résidus basiques spécifiques à la chaine α3 identifiés par des approches prédictives, ont été remplacés par le résidu neutre glutamine. Toutes les protéines couplées avec l’étiquette 6-Histidine ont été produites dans des cellules de mammifère, purifiées par chromatographie d'affinité et caractérisées biochimiquement et par dichroïsme circulaire. L’évaluation de l’affinité des protéines produites pour l’héparine nous a permis d’identifier un site d’interaction majeur avec les glycosaminoglycanes dans le domaine LG4/5, entouré par des résidus à mineur affinité. La technique de résonance plasmonique de surface et des tests d’adhérence cellulaire nous ont permis de confirmer ce résultat puisque l’absence du site d’interaction majeur avec l’héparine a produit une inhibition totale de l’adhérence. Des expériences de pull-down nous ont montré que ce site est aussi impliqué dans l’interaction avec le syndecan-4, indiquant que cette séquence pourrait ainsi jouer un rôle dans différents processus cellulaires. Une collaboration avec des bio-informaticiens nous a permis de proposer un modèle structural du domaine LG4/5 et de montrer que la zone identifiée est localisée dans une boucle exposée à l’extérieure du module LG4, entourée par des résidus à plus faible affinitéThe HSPG receptor syndecan-1 interacts with the carboxy-terminal LG4/5 domain in laminin 332 to participate in keratinocyte migration by inducing formation of cytoskeleton related protrusive structures. We have shown that syndecan-1 mediated cell adhesion occurs in heparan sulphate and chondroitin sulphate dependent manner and that these two glycosaminoglycan (GAG) chains bind independently to LG4/5 with different affinities. To identify residues involved in the interaction of the LG4/5 domain with syndecan-1 and to apprehend the molecular basis of the GAGs interaction specificity, we have used a site-directed mutagenesis approach of the recombinant LG4/5 fragment. The residues identified as conserved heparin binding residues throughout laminins, as well as “candidate” basic residues identified through predictive approaches, have been replaced by the neutral residue glutamine. All LG4/5 proteins carrying a hexa-histidine tag at their C-terminal end were expressed in mammalian cells. The produced proteins were purified and characterized biochemically. Circular dichroism studies performed on all mutagenised proteins showed that the overall structure of each mutant is comparable to that of the wild type protein. Heparin affinity chromatography analysis allowed us to identify a major heparin binding site in the LG4/5 domain surrounded by several minor GAG binding sites. Surface plasmon resonance analysis of mutated LG4/5 proteins-heparan sulphate interaction confirmed these results. These findings were well correlated with our in cellulo syndecan-1 mediated cell adhesion as the lack of this major heparin binding site totally abrogated cell adhesion. Pull down experiments allowed us to show that this heparin binding site sequence is responsible not only for the interaction of the receptors syndecan-1 but also for syndecan-4 suggesting that additional cellular functions may be carried by this sequence. Our structural predictions suggest that the LG4/5 in laminin 332 encompasses a major GAG binding site surrounded by a track of converging positively charged residue

The long and winding road that leads to a cure for epidermolysis bullosa

Inherited epidermolysis bullosa (EB) is a family of rare genetic skin disorders characterized by structural and mechanical fragility of skin and mucosal membranes. The main feature of EB is the presence of recurrent skin blistering or erosions, which have a profound impact in the quality of life of EB patients and, in the most severe forms, cause early lethality. During the past two decades, it became possible to identify mutations in genes responsible for different types of EB and characterize the abnormalities of the related proteins. Nowadays, there is no cure for EB; all the treatments are palliative and focused on the relief of the devastating EB clinical picture. Recent advancements in molecular biology, stem cell biology and regenerative medicine have fostered new therapeutic approaches for EB. This review is focused on recent developments in gene therapy, protein replacement and cell-based therapy for EB, all aimed at finding a cure for this devastating disease

Cell surface proteoglycans syndecan-1 and -4 bind overlapping but distinct sites in laminin α3 LG45 protein domain.

International audienceKeratinocyte migration during epidermal repair depends on interactions between cellular heparan sulfate proteoglycan receptors, syndecan-1 and -4, and the C-terminal globular domains (LG45) of the extracellular matrix protein laminin 332. This study investigates the molecular basis of the binding specificity of the syndecan-1 and -4 receptors expressed by human keratinocytes. We used site-directed mutagenesis to alter a recombinant LG45 protein by substituting the most critical basic residues with glutamine. All proteins were expressed in mammalian cells, purified, and characterized biochemically. We used in vitro binding assays, including surface plasmon resonance, to examine interactions between mutated LG45 and heparan sulfates, syndecan-1 and -4. We identify a major heparin binding domain on the outer edge of a β-strand of LG45 surrounded by a track of converging low affinity residues. This domain harbors distinctive syndecan-1 and -4 binding-specific sequences. This is the first study to demonstrate a binding specificity of two proteoglycans produced by a single cell type. In addition, we found that although syndecan-1 interacts exclusively through its glycosaminoglycan chains, syndecan-4 binding relies on both its core protein and its heparan sulfate chains. These results suggest that LG45 may trigger different signals toward keratinocytes depending on its interaction with syndecan-1 or -4

Isolation of human keratinocyte stem cells and high-throughput screening approach for their characterization

In the last three decades, regenerative medicine has opened new horizons for the in vitro reconstruction of epithelial tissues and gene therapy treatment of skin disorders involving the use of adult keratinocyte stem cells (KSCs). Although the ability to identify and isolate these cells represents an important prerequisite for the development of these approaches, molecular markers and their precise in vivo localization are still lacking. In order to define genes involved in the control of stemness and commitment of KSCs, we developed a non-invasive, stem cell-preserving magnetic micro beads based method in order to obtain a KSCs enriched population for high throughput screening experiments. After 3T3 murine fibroblast feeder layer depletion from our keratinocyte cultures, we isolated a subpopulation of basal epithelial cells on the basis of the different expression levels of the a6β4 integrin. By using different approaches, including clonal analysis and p63 bright cells quantification, we clearly showed that a6β4 integrin bright cells have greater growth potential and clonogenic capacity compared to the remaining cell fraction and they include the KSCs population. Comparing gene expression profile of a KSCs-enriched and a terminally differentiated cell population coming from the same original primary cell culture we defined a set of genes most probably involved in stemness maintenance. Ongoing gene profiling on single clone type will allow us to validate this gene signature and to start functional studies on selected genes. Extending this approach to different ectodermal derived tissues will provide a genome wide signature of the molecular pathways underlying self-renewal, commitment and differentiation of KSCs