Regeneration of the entire human epidermis using transgenic stem cells.

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Effects of catecholamines on microcirculation during general inhalation anesthesia.

OBJECTIVE: The aim of this study was to investigate the effects of clinical dosages of norepinephrine and dobutamine on sublingual microcirculation during general anesthesia with sevoflurane in minor surgical procedures. DESIGN: This prospective study was performed on patients scheduled for breast cancer surgery. SETTING: Tertiary care university hospital. PARTICIPANTS: Twenty patients undergoing elective surgery. INTERVENTIONS: Patients received a continuous infusion of norepinephrine (0.1 µg/kg/min) and afterwards, following a 15-minute interval, a continuous infusion of dobutamine (5 µg/kg/min). Prior to and at the end of each drug infusion period, hemodynamic parameters were measured using an esophageal Doppler probe (ED), and 5 sidestream darkfield (SDF) sublingual microcirculation video recordings were taken. MEASUREMENTS AND MAIN RESULTS: No significant changes to total vessel density (TVD)(mm/mm(2)), perfused vessel density (PVD) (mm/mm(2)), proportion of perfused vessels (PPV) (percentage), or microvascular flow index (MFI) (arbitrary units) were measured at the end of each drug infusion period versus pre-infusion data and no differences were observed between the effects of norepinephrine versus dobutamine. Mean arterial pressure (APm) (mmHg) was significantly greater following both norepinephrine and dobutamine infusions compared to pre-infusion values, while peak velocity (PV) (cm/sec) and the stroke volume index (SVI) (mL/m(2)) only showed a significant increase following the dobutamine infusion. No change in corrected flow time (FTc) (msec) was observed. CONCLUSIONS: During general anesthesia with sevoflurane, the infusion of clinical dosages of norepinephrine and dobutamine did not alter sublingual perfusion, although the expected systemic hemodynamic alterations were induced.

Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells.

Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.

An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model.

The purpose of this study was to investigate the response of porcine corneal organ cultures to riboflavin/UV-A phototherapy in the injury healing of induced lesions. A porcine corneal organ culture model was established. Corneal alterations in the stroma were evaluated using an assay system, based on an automated image analysis method able to (i) localize the holes and gaps within the stroma and (ii) measure the brightness values in these patches. The analysis has been performed by dividing the corneal section in 24 regions of interest (ROIs) and integrating the data analysis with a "multi-aspect approach." Three group of corneas were analyzed: healthy, injured, and injured-and-treated. Our study revealed a significant effect of the riboflavin/UV-A phototherapy in the injury healing of porcine corneas after induced lesions. The injured corneas had significant differences of brightness values in comparison to treated (p < 0.00) and healthy (p < 0.001) corneas, whereas the treated and healthy corneas showed no significant difference (p = 0.995). Riboflavin/UV-A phototherapy shows a significant effect in restoring the brightness values of damaged corneas to the values of healthy corneas, suggesting treatment restores the injury healing of corneas after lesions. Our assay system may be compared to clinical diagnostic methods, such as optical coherence tomography (OCT) imaging, for in vivo damaged ocular structure investigations.

Engineering partial resistance to cucumber mosaic virus in tobacco using intrabodies specific for the viral polymerase.

A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) '2a' consisting of the full-length 2a gene (839 amino acids, aa), (ii) 'Motifs' covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) 'GDD' located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the '2a' and 'Motifs' constructs interacted with 96 and 25 library constructs, respectively, while the 'GDD' construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants.

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.