Highly Efficient Ex Vivo Correction of COL7A1 through Ribonucleoprotein-Based CRISPR/Cas9 and Homology-Directed Repair to Treat Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe genetic skin disease responsible for blistering of the skin and mucosa after minor trauma. RDEB is caused by a wide variety of variants in COL7A1 encoding type VII Collagen, the major component of anchoring fibrils that form key attachment structures for dermal-epidermal adherence. In this study, we achieved highly efficient COL7A1 editing in primary RDEB keratinocytes and fibroblasts from 2 patients homozygous for the c.6508C>T (p.Gln2170∗) variant through CRISPR/Cas9-mediated homology-directed repair. Three guide RNAs targeting the c.6508C>T variant or harboring sequences were delivered together with high-fidelity Cas9 as a ribonucleoprotein complex. Among them, one achieved 73% cleavage activity in primary RDEB keratinocytes and RDEB fibroblasts. Then, we treated RDEB keratinocytes and RDEB fibroblasts with this specific ribonucleoprotein complex and the corresponding donor template delivered as single-stranded oligodeoxynucleotide and achieved up to 58% of genetic correction as well as type VII Collagen rescue. Finally, grafting of corrected 3-dimensional skin onto nude mice induced re-expression and normal localization of type VII Collagen as well as anchoring fibril formation at the dermal-epidermal junction 5 and 10 weeks after grafting. With this promising nonviral approach, we achieved therapeutically relevant specific gene editing that could be applicable to all variants in exon 80 of COL7A1 in primary RDEB cells.

Plastic surgery in bariatric patients: a nationwide study of 17,000 patients on the national administrative database.

BACKGROUND: Bariatric patients are often candidates for plastic surgery. However, the rate of postbariatric procedures is not known. OBJECTIVES: The aim of this study was to analyze the rate of plastic surgery, and factors related to surgery, in bariatric patients. SETTING: University hospital, France. METHODS: This was a cohort study based on administrative data. All adult patients who received bariatric surgery in France between 2007 and 2013 were included to estimate the rate of plastic surgery and related predictive factors. Data are reported according to the reporting of studies conducted using observational routinely collected data guidelines for observational studies on administrative data. RESULTS: Among the 183,514 patients who underwent bariatric surgery in the study period, 23,120 plastic surgeries were performed on 17,695 patients, including abdominoplasty (62%), dermolipectomy of the upper or lower limbs (25%), and reconstruction of the breast (14%). The rates of plastic surgery were 13%, 18%, and 21% at 3, 5, and 7 years post-bariatric surgery, respectively. Multivariate analysis revealed that patients who had a biliopancreatic diversion or a gastric bypass had a hazard ratio of 2.67 and 2.67 for subsequent plastic surgery, respectively, compared with patients who had adjustable gastric banding. Women had a 2-fold probability of surgery compared with men (hazard ratio 2.02). Important variability in the rate of surgery was found among different hospitals; rates ranged from 6.1% to 41.3% at 5 years. CONCLUSIONS: This study showed that 21% of bariatric patients undergo plastic surgery. Large variability exists among hospitals, suggesting that several unmeasured factors may limit access to contouring surgery.

Sarcoidosis and Wound Healing After Cellulitis of the Lower Limb: Is Methotrexate Responsible for Skin Graft Failure?

The authors report the case of a 53-year-old man with diffuse cutaneous and mediastinal pulmonary sarcoidosis and well-controlled steroid-induced diabetes. He was hospitalized for cellulitis of his left leg. His standard treatment for sarcoidosis consisted of prednisone and methotrexate. Prednisone was stopped at his admission. He received antibiotics for 4 weeks to treat the cellulitis. In parallel, the leg wound was treated with daily silver sulfadiazine applications until necrosis removal, then by skin autografting. Four successive procedures were performed, but all failed despite lack of surgical problem or local infection. Methotrexate was stopped after the fourth grafting procedure failed; the fifth, and final, autografting procedure was successfully performed.

Removal of subcutaneous lipomas: Interest of liposuction.

Subcutaneous lipomas are very common tumors. We report our experience of treatment by liposuction in carefully selected patients, initially referred for conventional surgical excision.

Indications and Results of Reconstructive Techniques with Flaps Transposition in Patients Requiring Complex Thoracic Surgery: A 12-Year Experience.

BACKGROUND: Flap transposition is an infrequent but far from exceptional thoracic surgical procedure. The aim of this retrospective study was to report our experience in a referral unit of general thoracic surgery analyzing the early results after flap transposition. METHODS: We retrospectively analyzed the clinical records, surgical notes, and postoperative results of a cohort of patients who underwent flap transposition in our unit from November 2000 to February 2013. RESULTS: Overall, a surgical approach adopting flap reconstruction techniques was performed in 81 patients (54 males, 27 females) with a median age of 62 years (range 20-87). Flap transposition was necessary to reconstruct chest wall after resection for malignancy (27 patients), to repair intrathoracic viscera perforation (15 patients), and to fill residual cavities secondary to pulmonary/pleural infection (39 patients). A pedicle muscle flap was transposed in most of cases (64 pts, 79 %), while in the remaining 17 cases (11 %), an omental flap was used. There were no immediate postoperative complications, while three in-hospital deaths occurred due to respiratory or multiorgan failure. Among patients undergone flap transposition to fill a residual cavity, we observed a recurrent bronchopleural fistula in three patients (7.7 %); such patients were treated by repeat flap transposition (2 cases) and by repeat cavernostomy (1 case). CONCLUSION: Flap transposition may be indicated as part of a multimodal treatment for severely ill patients requiring complex thoracic surgery.

Gene-Corrected Fibroblast Therapy for Recessive Dystrophic Epidermolysis Bullosa using a Self-Inactivating COL7A1 Retroviral Vector.

Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack type VII collagen and therefore have severely impaired dermal-epidermal stability causing recurrent skin and mucosal blistering. There is currently no specific approved treatment for RDEB. We present preclinical data showing that intradermal injections of genetically corrected patient-derived RDEB fibroblasts using a Good Manufacturing Practices grade self-inactivating COL7A1 retroviral vector reverse the disease phenotype in a xenograft model in nude mice. We obtained 50% transduction efficiency in primary human RDEB fibroblasts with an average low copy number (range = 1-2) of integrated provirus. Transduced fibroblasts showed strong type VII collagen re-expression, improved adhesion properties, normal proliferative capabilities, and viability in vitro. We show that a single intradermal injection of 3 × 10(6) genetically corrected RDEB fibroblasts beneath RDEB skin equivalents grafted onto mice allows type VII collagen deposition, anchoring fibril formation at the dermal-epidermal junction, and improved dermal-epidermal adherence 2 months after treatment, supporting functional correction in vivo. Gene-corrected fibroblasts previously showed no tumorigenicity. These data show the efficacy and safety of gene-corrected fibroblast therapy using a self-inactivating vector that has now been good manufacturing grade-certified and pave the way for clinical translation to treat nonhealing wounds in RDEB patients.

Assessment of a Standardized Pre-Operative Telephone Checklist Designed to Avoid Late Cancellation of Ambulatory Surgery: The AMBUPROG Multicenter Randomized Controlled Trial.

OBJECTIVES: To assess the impact of a standardized pre-operative telephone checklist on the rate of late cancellations of ambulatory surgery (AMBUPROG trial). DESIGN: Multicenter, two-arm, parallel-group, open-label randomized controlled trial. SETTING: 11 university hospital ambulatory surgery units in Paris, France. PARTICIPANTS: Patients scheduled for ambulatory surgery and able to be reached by telephone. INTERVENTION: A 7-item checklist designed to prevent late cancellation, available in five languages and two versions (for children and adults), was administered between 7 and 3 days before the planned date of surgery, by an automated phone system or a research assistant. The control group received standard management alone. MAIN OUTCOME MEASURES: Rate of cancellation on the day of surgery or the day before. RESULTS: The study population comprised 3900 patients enrolled between November 2012 and September 2013: 1950 patients were randomized to the checklist arm and 1950 patients to the control arm. The checklist was administered to 68.8% of patients in the intervention arm, 1002 by the automated phone system and 340 by a research assistant. The rate of late cancellation did not differ significantly between the checklist and control arms (109 (5.6%) vs. 113 (5.8%), adjusted odds ratio [95% confidence interval] = 0.91 [0.65-1.29], (p = 0.57)). Checklist administration revealed that 355 patients (28.0%) had not undergone tests ordered by the surgeon or anesthetist, and that 254 patients (20.0%) still had questions concerning the fasting state. CONCLUSIONS: A standardized pre-operative telephone checklist did not avoid late cancellations of ambulatory surgery but enabled us to identify several frequent causes. TRIAL REGISTRATION: ClinicalTrials.gov NCT01732159.

Skin donors and human skin allografts: evaluation of an 11-year practice and discard in a referral tissue bank.

The Saint Louis hospital tissue bank provides skin allografts to pediatric and adult burn units in the Paris area. The aim of this study was to analyze our activity during the last 11 years focusing on the reasons for skin discard. Skin is procured solely from the back of the body, which is divided into 10 zones that are harvested and processed separately. This retrospective study included all skin donors harvested between June 2002 and June 2013, representing a total of 336 donors and 2770 zones. The donors were multiorgan heart-beating donors in 91 % of cases (n = 307). The main reason for discarding harvested skin was microbial contamination, detected in 99 donors (29 %). Most contaminants were of low pathogenicity. Other reasons for discard included positive serologic tests for 2 donors [17 zones (0.61 %)], unsuitable physical skin characteristics for 3 zones (0.11 %), the donor's medical history for 53 zones (1.91 %), and technical issues with processing or distribution for 61 zones (2.2 %). In our experience, microbial contamination continues to be the main reason for discarding potential skin allografts. However, discards are limited by separate harvesting and processing of multiple zones in each donor.

Technical note: comparison of the PrestoBlue and LDH release assays with the MTT assay for skin viability assessment.

MTT assay is the gold standard for assessing skin sample viability but it is time-consuming. Here we compared the MTT test with two other assays for the assessment of skin viability. The MTT, PrestoBlue (colorimetric method) and LDH release assays were applied to fresh and cryopreserved skin. Skin viability was considered proportional to the optical density values of the relevant analytes. PrestoBlue did not reliably distinguish between fresh and cryopreserved skin. The LDH release assay did not allow us to establish a viability index. We recommend the MTT assay for assessing skin viability.