Translating the combination of gene therapy and tissue engineering for treating recessive dystrophic epidermolysis bullosa.

The combination of gene therapy and tissue engineering is one of the most promising strategies for the treatment of recessive dystrophic epidermolysis bullosa (RDEB). RDEB is a rare genetic disease characterised by mutations in the COL7A1 gene, encoding type VII collagen (COLVII), which forms anchoring fibrils at the dermal-epidermal junction of the skin. This disease causes severe blistering and only palliative treatments are offered. In this study, the base of a strategy combining gene therapy and a tissue-engineered skin substitute (TES), which would be suitable for the permanent closure of skin wounds, was set-up. As a high transduction efficiency into fibroblasts and/or keratinocytes seems to be a prerequisite for a robust and sustained correction of RDEB, different envelope pseudotyped retroviral vectors and the transduction enhancer EF-C were tested. When green fluorescent protein (GFP) was used as a reporter gene to evaluate the retroviral-mediated gene transfer, the fibroblast infection efficiency was 30 % higher with the Ampho pseudotyped vector as compared with the other pseudotypes. At least a 3.1-fold and a 1.3-fold increased transduction were obtained in fibroblasts and keratinocytes, respectively, with EF-C as compared with polybrene. A continuous and intense deposit of haemagglutinin (HA)-COLVII was observed at the dermal-epidermal junction of self-assembled TESs made of cells transduced with a HA-tagged COL7A1 vector. Furthermore, HA-tagged basal epidermal cells expressing keratin 19 were observed in TESs, suggesting stem cell transduction. This approach could be a valuable therapeutic option to further develop, in order to improve the long-term life quality of RDEB patients.

Scrotal centesis: a broader role beyond the confines of the scrotum.

An elderly man was treated for severe acute scrotum pain with centesis. We report the diagnosis, underlying causes and management, and discuss the procedure. Centesis is performed rarely, but could be undertaken more often given the added benefits.

TENS for surgical stabilisation of acetabular fracture in the skeletally immature: A novel technique.

BACKGROUND: Acetabular fractures in childhood are rare and the literature is scarce to describe a standard protocol in surgical management of these injuries. As the patient is still growing, it warrants a detailed assessment with a sound surgical plan if operative intervention is deemed necessary to prevent late complications. Throughout literature, most fixation rely on using pins, screws, plates or combination of the three which require large surgical exposure and risk of secondary physeal injury, hence we come up with a method of using the Titanium Elastic Nail System (TENS) to overcome this issue. We describe a novel technique in managing acetabular fractures in this group of patients using the TENS. METHOD: An 8 year old girl with a diagnosis of right anterior column posterior hemitransverse acetabular fracture was fixed with 3 TENS for supra-acetabular, anterior column and posterior column fragments. Surgery was performed in a minimally invasive manner. No drilling was performed during the surgery and implant insertion is done manually. RESULTS: Advantages of this procedure include minimally invasive surgery with smaller wounds, minimal intraoperative bleeding and theoretically reduces the risk of premature fusion of the triradiate cartilage. Patient is allowed early rehabilitation with this method. CONCLUSION: This novel method provides an alternative to traditional usage of wires, pins, plates and screws as is described in most literature. However, it requires the surgeon to appreciate that the safe corridors for the implant are much narrower than adults. We recommend this technique for fractures that are deemed suitable for intramedullary fixation and further research in the future will be needed.

Hyperamylasaemia in ureteric colic.

Hyperamylasaemia is classically associated with acute pancreatitis. Hyperamylasaemia may be associated with many other clinical conditions. However, ureteric colic has never been reported to cause hyperamylasaemia. We describe a 47-year-old woman who presented with an atypical history of left ureteric colic. Radiological investigations confirmed an upper ureteric stone with urinary extravasation. At presentation, the serum amylase was elevated but normalised after 24 h. In conclusion, ureteric colic may cause hyperamylasaemia and this is likely a result of pancreatic irritation due to urinary extravasation. Patients presenting with ureteric colic and elevated concentrations of serum amylase should raise the clinical suspicion of urinary extravasation.

Variation in the use of postoperative radiotherapy among high-risk patients following radical prostatectomy.

BACKGROUND: We used data from the Michigan Urological Surgery Improvement Collaborative (MUSIC) to investigate the use of adjuvant and salvage radiotherapy (ART, SRT) among patients with high-risk pathology following radical prostatectomy (RP). METHODS: For patients with pT3a disease or higher and/or positive surgical margins, we examined post-RP radiotherapy administration across MUSIC practices. We excluded patients with <6 months follow-up, and those that failed to achieve a postoperative PSA nadir ⩽0.1. ART was defined as radiation administered within 1 year post RP, with all post-nadir PSA levels <0.1 ng ml(-1). Radiation administered >1 year post RP and/or after a post-nadir PSA ⩾0.1 ng ml(-1) was defined as SRT. We used claims data to externally validate radiation administration. RESULTS: Among 2337 patients undergoing RP, 668 (28.6%) were at high risk of recurrence. Of these, 52 (7.8%) received ART and 56 (8.4%) underwent SRT. Patients receiving ART were younger (P=0.027), more likely to have a greater surgical Gleason sum (P=0.009), higher pathologic stage (P<0.001) and received treatment at the smallest and largest size practices (P=0.011). Utilization of both ART and SRT varied widely across MUSIC practices (P<0.001 and P=0.046, respectively), but practice-level rates of ART and SRT administration were positively correlated (P=0.003) with lower ART practices also utilizing SRT less frequently. Of the 88 patients not receiving ART and experiencing a PSA recurrence ⩾0.2 ng ml(-1), 38 (43.2%) progressed to a PSA ⩾0.5 ng ml(-1) and 20 (22.7%) to a PSA ⩾1.0 ng ml(-1) without receiving prior SRT. There was excellent concordance between registry and claims data κ=0.98 (95% CI: 0.94-1.0). CONCLUSIONS: Utilization of ART and SRT is infrequent and variable across urology practices in Michigan. Although early SRT is an alternative to ART, it is not consistently utilized in the setting of post-RP biochemical recurrence. Quality improvement initiatives focused on current postoperative radiotherapy administration guidelines may yield significant gains for this high-risk population.

Bystander effect in glioblastoma cells with a predominant cytoplasmic localization of connexin43.

Herpes simplex virus thymidine kinase (TK) gene transfer followed by ganciclovir (GCV) administration is an approach investigated for glioblastoma treatment. The bystander effect (BE) enhances the cytotoxic effect of this strategy by allowing the diffusion of phosphorylated GCV from TK-expressing cells toward neighboring TK negative cells. This transfer of toxic metabolites is mainly mediated via gap junctions that are composed of connexins. Downregulation and/or cytoplasmic localization of connexins are common in tumors, and should be detrimental to the success of the TK/GCV strategy. In this study, we investigated the level of expression, the localization and the functionality of connexin43 (Cx43) in three glioblastoma cell lines. We showed that Cx43 was predominantly located in lysosomes and late endosomes, with only few gap junctions present at the cell surface. Surprisingly, the gap-junctional intercellular communication (GJIC) and the BE capacity were preserved, and in two of the cell lines analyzed, it was at least twice as high as compared to a control HeLa transfectant that expresses high levels of Cx43 at the cell membrane. Experiments performed in the presence of alpha-glycyrrhetinic acid or small interfering RNA confirmed that Cx43 was responsible for the GJIC and the BE. Our results indicate for the first time that the very limited numbers of gap junctions present in glioblastoma cells are highly functional. We thus conclude that the TK/GCV strategy is still a valuable therapeutic option to be developed for the treatment of glioblastoma patients.

Generation of a high-titer packaging cell line for the production of retroviral vectors in suspension and serum-free media.

Several patients with severe combined immunodeficiency-X1 disease and adenosine deaminase deficiency have been cured by retroviral-mediated gene therapy. Despite the earlier success, the production of retroviral vectors for clinical gene therapy is cumbersome, costly and lacks safety features because of the adherent nature of packaging cells and the necessity to supplement the culture media with bovine serum. The aim of this study was to generate a retrovirus packaging cell line that could be used for the production of large clinical batch vectors. Bicistronic vectors containing an internal ribosomal entry site followed by a selection gene were used to express Moloney murine leukemia gag-pol and amphotropic envelope viral proteins in HEK293 cells. The candidate clone (293GP-A2) that was selected as the packaging cell line could release recombinant green fluorescent protein retroviruses at 4x10(7) infectious viral particles per ml. Similar titers were achieved after these cells were adapted to grow in suspension and serum-free media. Furthermore, using the same culture conditions viral titers proved to be stable for a 3-month culture period. The 293GP-A2 packaging cell line has the potential to be cultured in bioreactors, opening the possibility for large-scale use of retroviral vectors in late stage clinical trials.

Transient femoral nerve palsy following ilio-inguinal nerve blockade for day case inguinal hernia repair.

BACKGROUND: Transient femoral nerve palsy (TFNP) has been reported in patients undergoing inguinal hernia repair involving the use of ilio-inguinal nerve block. Ilio-inguinal nerve blocks can be administered under vision by the surgeon or by the anaesthetist using a standard blind technique. There has been no study that has specifically examined the incidence of this complication and whether its development is related to the type of method used to administer the block. PATIENTS AND METHODS: Data on patients undergoing surgery in the Royal Infirmary Edinburgh Day Case Unit are collected prospectively. All patients who undergo inguinal hernia repair are given ilio-inguinal field blocks, either pre-operatively by anaesthetists (blind technique) or peri-operatively under direct vision by surgeons. Several cases of TFNP were initially identified during the process of surgical audit and this led to a retrospective analysis over a period of one year. RESULTS: During a 12-month period, 194 patients underwent 200 open inguinal hernia repairs (188 unilateral and 6 bilateral), under general anaesthesia. Ten patients (5%) developed TFNP resulting in overnight admission. Surgeons administered 101 blocks under direct vision of which 4 (4%) resulted in TFNP, whereas 6 out of 99 (6%) blind blocks resulted in TFNP (p=0.49, df=1, Chi2 test). DISCUSSION AND CONCLUSION: TFNP is a recognised complication following ilioinguinal nerve blockade for inguinal hernia surgery. Our series shows that ilio-inguinal block given under direct vision does not appear to reduce the chance of this complication occurring. This may result from the fact that this complication could be due to local infiltration into the operative field rather than direct infiltration around the femoral nerve. As inguinal hernia repair undertaken as a day case procedure increases, the awareness of this complication is important to avoid morbidity

Histiocytosis X: evidence for a genetic etiology.

Histiocytosis X is a rare disorder with no particular predilection for race, age or sex. Since its discovery by Hand in 1893, the etiology has remained unknown, although viruses, bacteria and genetic factors have been implicated. Familial occurrence of this disease is very rare, and only a handful of such cases have been reported. The present study adds further evidence to support the influence of genetic factors in the etiology of histiocytosis X.