Evaluation of Functional Erythropoietin Receptor Status in Skeletal Muscle In Vivo: Acute and Prolonged Studies in Healthy Human Subjects

BACKGROUND: Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle. METHODOLOGY/PRINCIPAL FINDINGS: The protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1-10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as activation of Epo signalling pathways (STAT5, MAPK, Akt, IKK) were analysed by western blotting. Changes in muscle protein profiles after prolonged erythropoietin treatment were evaluated by 2D gel-electrophoresis and mass spectrometry. The presence of the erythropoietin receptor in skeletal muscle was confirmed, by the M20 but not the C20 antibody. However, no significant changes in phosphorylation of the Epo-R, STAT5, MAPK, Akt, Lyn, IKK, and p70S6K after erythropoietin administration were detected. The level of 8 protein spots were significantly altered after 16 days of rHuEpo treatment; one isoform of myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased. CONCLUSIONS/SIGNIFICANCE: Acute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or downstream signalling targets in human skeletal muscle in the resting situation, whereas more prolonged exposure induces significant changes in the skeletal muscle proteome. The absence of functional Epo receptor activity in human skeletal muscle indicates that the long-term effects are indirect and probably related to an increased oxidative capacity in this tissue

Efficient and safe correction of hemophilia A by lentiviral vector-transduced BOECs in an implantable device

Hemophilia A (HA) is a rare bleeding disorder caused by deficiency/dysfunction of the FVIII protein. As current therapies based on frequent FVIII infusions are not a definitive cure, long-term expression of FVIII in endothelial cells through lentiviral vector (LV)-mediated gene transfer holds the promise of a one-time treatment. Thus, here we sought to determine whether LV-corrected blood outgrowth endothelial cells (BOECs) implanted through a prevascularized medical device (Cell Pouch™) would rescue the bleeding phenotype of HA mice. To this end, BOECs from HA patients and healthy donors were isolated, expanded and transduced with an LV carrying FVIII driven by an endothelial-specific promoter employing GMP-like procedures. FVIII-corrected HA-BOECs were either directly transplanted into the peritoneal cavity or injected into a Cell Pouch™ implanted subcutaneously in NSG-HA mice. In both cases, FVIII secretion sufficient to improve the mouse bleeding phenotype. Indeed, FVIII-corrected HA-BOECs reached a relatively short-term clinically relevant engraftment being detected up to 16 weeks after transplantation, and their genomic integration profile did not show enrichment for oncogenes, confirming the process safety. Overall, this is the first pre-clinical study showing the safety and feasibility of transplantation of GMP-like produced LV-corrected BOECs within an implantable device for the long-term treatment of HA

Sacral nerve stimulation for faecal incontinence in patients with previous partial spinal injury including disc prolapse

BACKGROUND: This study examined the use of sacral nerve stimulation (SNS) to treat faecal incontinence in patients with partial spinal injury. METHODS: Patients selected for SNS had experienced more than one episode of faecal incontinence per week to liquid or solid stool for more than 1 year and had failed maximal conservative treatment. All patients had an intact external anal sphincter. RESULTS: Temporary SNS was performed in 13 patients (median age 58.5 (range 39-73) years). The spinal insults were disc prolapse (six), trauma (four), spinal stenosis (one) or occurred during neurosurgery (two). Twelve patients (eight women and four men) had successful temporary stimulation and proceeded to permanent implantation. The median follow-up time was 12 (range 6-24) months. The mean(s.d.) number of episodes of incontinence decreased from 9.33(7.64) per week at baseline to 2.39(3.69) at last follow-up (P = 0.012). The number of days per week with incontinence and staining decreased significantly (both P < 0.001). Ability to defer defaecation improved from a median of not being able to defer (range 0-1 min) to being able to defer for 5-15 (range 0 to over 15) min (P = 0.022). CONCLUSION: SNS can benefit patients with faecal incontinence following partial spinal injury. Copyright (c) 2005 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd