Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation

Keratins 8 and 18 belong to the keratin family of intermediate filament (IF) proteins and constitute a hallmark for all simple epithelia, including the liver. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/K18). In these cells, the loss of one partner via a targeted null mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins. Here, we report that K8-null mouse hepatocytes in primary culture and in vivo are three- to fourfold more sensitive than wild-type (WT) mouse hepatocytes to Fas-mediated apoptosis after stimulation with Jo2, an agonistic antibody of Fas ligand. This increased sensitivity is associated with a higher and more rapid caspase-3 activation and DNA fragmentation. In contrast, no difference in apoptosis is observed between cultured K8-null and WT hepatocytes after addition of the Fas-related death-factors tumor necrosis factor (TNF) α or TNF-related apoptosis-inducing ligand. Analyses of the Fas distribution in K8-null and WT hepatocytes in culture and in situ demonstrate a more prominent targeting of the receptor to the surface membrane of K8-null hepatocytes. Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8-null versus WT hepatocytes. Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface

Accuracy of unguided and ultrasound guided Coracohumeral ligament infiltrations – a feasibility cadaveric case series

Coracohumeral ligament (CHL) thickening, contracture, and fibroplasia have been identified in glenohumeral idiopathic adhesive capsulitis (GHIAC). The CHL is the main structure responsible for the range of motion limitations. Favorable outcomes have been reported with CHL surgical release. Intra-articular glenohumeral joint corticosteroid infiltrations are utilized to disrupt the inflammatory process and reduce pain in GHIAC. The aim of this study was to investigate whether the CHL could be accurately targeted with a periligamentous infiltration

Water analysis with the help of tensor canonical decompositions

Coopération universitaire et scientifique Franco-VietnamienneInternational audienceRaw data are collected in five measurement locations along the Var river. It is assumed that some locations interact with each other, whereas others do not. In such a context, we are interested in determining the contribution of each location and in better understanding the water exchanges that are involved. Organic components can also be identified thanks to methods such as Canonical Polyadic decompositions (CP) (sometimes known as Parafac), applied to 3D fluorescence spectra calculated from the collected samples. The expected impact is a more efficient detection of polluting matters in water

Scalable lentiviral vector production using stable producer cell lines in perfusion mode

Lentiviral vectors (LVs) are becoming an important tool in gene and cell therapy and are being utilized in several clinical studies for rare and more frequent genetic and acquired diseases, as well as in cancer therapies. However, two major challenges need to be overcome in order to generate enough material to treat patients: First, current production platforms result in low titers (stable producer cell lines from adherent cell lines) or are not amenable to large scale production (LV produced by transfection). Next, LVs are known to have a low temperature stability. To address these two challenges, the National Research Council Canada has developed packaging cell lines and stable producer cell lines for the production of LVs which can grow in suspension in serum-free media and produce LV in the 106 TU/ml range without optimization. Furthermore, productions are performed in perfusion mode in order to operate at high cell densities and address the low LV stability. Please click Additional Files below to see the full abstrac

Surgical Stabilization for Multiple Rib Fractures: Whom the Benefit? -A Prospective Observational Study

Background: Surgical repair has demonstrated a beneficial effect on outcome for patients presenting with flail chest or with multiple rib fractures. We hypothesized that benefit on outcome parameters concerns predominantly patients being extubated within 24 hours post-operatively. Methods: We prospectively recorded all patients presenting with chest traumatism eligible for surgical repair with anticipated early extubation according to our institutional consensus (flail chest, major deformity, poor pain control, associated lesions requiring thoracotomy). We compared outcomes of patients extubated within 24 hours post-operatively to those who required prolonged ventilator support. We tested predictive factors for prolonged intubation with univariate and multivariate analysis. Results: From 2010 to 2014, 132 patients required surgical repair. Two thirds were extubated within 24 hours following surgical repair. Pneumonia was the main complication and occurred in 30.3% of all patients. Patients extubated within 24 hours following surgical repair had significantly shorter ICU stay and shorter in-hospital stay (P<0.0001 both). Pneumonia occurred significantly more often in patients with longer mechanical ventilation (over 24 hours) (P<0.0001) and the overall post-operative complications rate was higher (P=0.0001). Main independent risk factors for delayed extubation were bilateral chest rib fractures and initially associated pneumothorax. Conclusions: We conclude that patients extubated within 24 hours after repair have an improved outcome with reduced complication rate and shorter hospital stay. The initial extent of the trauma is an important risk factor for delayed extubation and high complication rate despite surgical stabilization

Sexual violence in older adults : a Belgian prevalence study

Background: Sexual violence (SV) is an important public health problem which may cause long-lasting health problems. SV in older adults remains neglected in research, policies and practices. Valid SV prevalence estimates and associated risk factors in older adults are currently unavailable. Objective: To measure lifetime and past 12-months sexual victimisation in older adults living in Belgium, its correlates, assailant characteristics and the way that victims framed their SV experiences. Design: Cross-sectional general population study. Setting: Community-dwelling, assisted living and nursing homes. Participants: 513 people of 70 years and older living in Belgium. Methods: SV was measured using behaviourally specific questions based on a broad definition of SV. Participants were selected via a cluster random probability sampling with a random route finding approach. Information on sexual victimisation, correlates, assailant characteristics and framing was collected via structured face-to-face interviews. Results: Lifetime SV prevalence was 44% (55% F, 29% M). Past 12-months prevalence was 8% (9% F, 8% M). Female sex and a higher number of sexual partners were associated with lifetime SV (p <.05), non-heterosexual sexual orientation with past 12-months SV (p <.05). Correlates generally linked to elder abuse and neglect were not linked with SV. ‘Someone unknown’ was identified as most common assailant. Conclusions: SV appears to be common in older adults in Belgium. Both correlates and assailant characteristics seem to differ from previous studies on elder abuse and neglect. Recognising older adults as a risk group for sexual victimisation in research, policies and practices is of the utmost importance

292. Towards Large-Scale Manufacturing of Adeno-Associated Virus by Transient Transfection of HEK293 Suspension Cells in a Stirred Tank Bioreactor Using Serum-Free Medium

Adeno-Associated Virus (AAV) vectors showing safety profile in phase I clinical trials and its ability to transduce gene expression in various tissues have made it a vector of choice for gene delivery. There are different modes of AAV vector production and each has advantages and disadvantages. Here we demonstrated that the production of AAV by transient transfection in a serum-free medium using NRC's patented cGMP compliant human embryonic kidney HEK293 cell line (clone HEK293SF-3F6) adapted for growth in suspension can be readily scaled-up in stirred tank bioreactors. We employed triple-plasmid / polyethylenimine (PEI) based transient transfection technique. As a proof of concept, we demonstrated that nine serotypes of AAV (AAV-1 to AAV-9) encoding GFP can be produced by our cell line HEK293SF with yields of about 1E+13 genome-containing particles per liter (Vg/L). Depending on the serotypes 4-30% of AAV is present in the supernatant of the cell culture at 48hpt. The presence of plasmids and plasmid polyplexes that were not taken up by the cells or were not brought into the cell nucleus were removed by Iodixanol-ultracentrifugation method and Benzonase treatment before analyzing by real-time PCR. About 25% loss in genome containing viral particle counts were observed by Iodixanol purification method based on infectivity assay. Productions of AAV2 and AAV6 encoding GFP were demonstrated in 3L stirred tank bioreactors. Purification scheme was based on column chromatography - a scalable process. Different chromatography media, such as cation exchanger, anion exchanger and hydrophobic interaction chromatography, were tested with each AAV serotypes for their ability to adsorb and elute efficiently. The purification scheme was then adopted by integrating best chromatography medium and sequence dependent upon the AAV serotype in use. We demonstrated the purification scheme for AAV2 based on ion-exchange and hydrophobic interaction chromatography steps. The SDS-PAGE showed the purity of the final product and the presence of three capsid proteins VP1, VP2 and VP3 on Western blot corresponding to the only three bands present in the final product on SDS-PAGE. To extend the storage life of AAV we explored lyophilization technique to study the stability of AAV2 and AAV6 under lyophilized conditions. The AAV2 and AAV6 were stable for over 40 weeks based on infectivity assay. We demonstrated the scalability of the process up to 45L. Productions tested in 20 and 500 mL cultures in shake flasks were scaled up in 2 and 45L cultures (in 3- and 60-L stirred tank bioreactors, respectively). The volumetric yields and purification recoveries were comparable at all of these production scale levels demonstrating scalability of transient transfection at even larger scale is possible to generate material necessary for dosages required for gene therapy application

Scalable lentiviral vector production using stable producer cell lines in perfusion mode

Lentiviral vectors (LVs) are becoming an important tool in gene and cell therapy and are being utilized in several clinical studies against genetic and acquired diseases, as well as in cancer therapies. To address the challenges linked to the generation of preclinical and clinical supply, the National Research Council Canada has developed packaging cell lines and stable producer cell lines for the production of LVs which can grow in suspension in serum-free media and produce LV in the 106 TU/ml range without optimization. We focus on the development of perfusion processes to both intensify the process and harvest produced LV rapidly

Glenohumeral joint capsular tissue tension loading correlates moderately with shear wave elastography: a cadaveric investigation

Purpose The purpose of this study was to investigate changes in the mechanical properties of capsular tissue using shear wave elastography (SWE) and a durometer under various tensile loads, and to explore the reliability and correlation of SWE and durometer measurements to evaluate whether SWE technology could be used to assess tissue changes during capsule tensile loading. Methods The inferior glenohumeral joint capsule was harvested from 10 fresh human cadaveric specimens. Tensile loading was applied to the capsular tissue using 1-, 3-, 5-, and 8-kg weights. Blinded investigators measured tissue stiffness and hardness during loading using SWE and a durometer, respectively. Intraobserver reliability was established for SWE and durometer measurements using intraclass correlation coefficients (ICCs). The Pearson product-moment correlation was used to assess the associations between SWE and durometer measurements. Results The ICC3,5 for durometer measurements was 0.90 (95% confidence interval [CI], 0.79 to 0.96; P<0.001) and 0.95 (95% CI, 0.88 to 0.98; P<0.001) for SWE measurements. The Pearson correlation coefficient values for 1-, 3-, and 5-kg weights were 0.56 (P=0.095), 0.36 (P=0.313), and -0.56 (P=0.089), respectively. When the 1- and 3-kg weights were combined, the ICC3,5 was 0.72 (P<0.001), and it was 0.62 (P<0.001) when the 1-, 3-, and 5-kg weights were combined. The 8-kg measurements were severely limited due to SWE measurement saturation of the tissue samples. Conclusion This study suggests that SWE is reliable for measuring capsular tissue stiffness changes in vitro at lower loads (1 and 3 kg) and provides a baseline for the non-invasive evaluation of effects of joint loading and mobilization on capsular tissues in vivo

Production of rVSV-ZEBOV in serum-free suspension culture of HEK 293SF cells.

Abstract Ebola virus disease is an urgent international priority. Promising results for several vaccine candidates have been reported in non-human primate studies and clinical trials with the most promising being the rVSV-ZEBOV vaccine. In this study, we sought to produce rVSV-ZEBOV in HEK 293SF cells in suspension and serum-free media. The purpose of this study was to establish a process using the HEK 293SF production platform, optimise the production titre, demonstrate scalability and the efficiency of the generated material to elicit an immune reaction in an animal model. Critical process parameters were evaluated to maximize production yield and process robustness and the following operating conditions: 1–2 × 106 cells/mL grown in HyClone HyCell TransFx-H media infected at an MOI of 0.001 with a temperature shift to 34 °C during the production phase and a harvest of the product after 48 h. Using these conditions, scalability in a 3.5 L controlled bioreactor was shown reaching a titre of 1.19 × 108 TCID50/mL at the peak of production, the equivalent of 4165 doses of vaccine per litre. The produced virus was shown to be thermostable in the culture media and, when concentrated, purified and administered to mice, demonstrated the ability to induce a ZEBOV-specific immune response