RESUMEN
We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy for neurodegenerative diseases, neurotrauma, and stroke. In this study, using a mini pig model of spinal cord injury, we proposed for the first time the use of gene-modified leucoconcentrate prepared from peripheral blood in the plastic blood bag for personalized ex vivo gene therapy. Leucoconcentrate obtained from mini pig peripheral blood was transduced with a chimeric adenoviral vector (Ad5/35F) that carried an enhanced green fluorescent protein (EGFP) reporter gene in the plastic blood bag. The day after blood donation, the mini pigs were subjected to moderate SCI and four hours post-surgery they were intravenously autoinfused with gene-modified leucoconcentrate. A week after gene-modified leucoconcentrate therapy, fluorescent microscopy revealed EGFP-expressing leucocytes in spinal cord at the site of contusion injury. In the spleen the groups of EGFP-positive cells located in the lymphoid follicles were observed. In vitro flow cytometry and fluorescent microscopy studies of the gene-modified leucoconcentrate samples also confirmed the production of EGFP by leucocytes. Thus, the efficacy of leucocytes transduction in the plastic blood bag and their migratory potential suggest their use for temporary production of recombinant biologically active molecules to correct certain pathological conditions. This paper presents a proof-of-concept of simple, safe and effective approach for personalized ex vivo gene therapy based on gene-modified leucoconcentrate autoinfusion. The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee (approval No. 5) on May 27, 2014.
RESUMEN
Natural brain repair after stroke is extremely limited, and current therapeutic options are even more scarce with no clinical break-through in sight. Despite restricted regeneration in the central nervous system, we have previously proved that human umbilical cord blood mono-nuclear cells (UCB-MC) transduced with adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) successfully rescued neurons in amyotrophic lateral sclerosis and spinal cord injury. This proof-of-principle project was aimed at evaluating the beneficial effects of the same triple-gene approach in stroke. Rats subjected to distal occlusion of the middle cerebral artery were treated intrathecally with a combination of these genes either directly or using our cell-based (UCB-MC) approach. Various techniques and markers were employed to evaluate brain injury and subsequent recovery after treatment. Brain repair was most prominent when therapeutic genes were delivered via adenoviral vector- or UCB-MC-mediated approach. Remodeling of brain cortex in the stroke area was confirmed by reduction of infarct volume and attenuated neural cell death, depletion of astrocytes and microglial cells, and increase in the number of oligodendroglial cells and synaptic proteins expression. These results imply that intrathecal injection of genetically engineered UCB-MC over-expressing therapeutic molecules (VEGF, GDNF, and NCAM) following cerebral blood vessel occlusion might represent a novel avenue for future research into treating stroke.
RESUMEN
BACKGROUND AND AIMS: Small gastric or colorectal tumours can be visually undetectable during laparoscopic surgeries, and available methods still do not provide a 100% localisation rate. Thus, new methods for further improvements in tumour localisation are highly desirable. In this study, we evaluated the usage of the Medical Tactile Endosurgical Complex (MTEC) in gastrointestinal surgery for localisation of tumours. The MTEC provides the possibility of instrumental mechanoreceptoric palpation, which serves as an analogue of conventional manual palpation. METHODS: Ninety-six elective surgeries were performed, including 48 open surgeries, 43 laparoscopies, and 5 robot-assisted surgeries. The 20 mm version of the MTEC tactile mechanoreceptor was used in open surgeries, and the 10 mm version in laparoscopic and robot-assisted surgeries. RESULTS: The mean time of instrumental mechanoreceptoric palpation was 3 minutes 12 seconds for open surgeries, which constituted the early stage of the learning curve, and 3 minutes 34 seconds for laparoscopic surgeries. No side effects or postoperative complications related to instrumental mechanoreceptoric palpation were observed, and this procedure provided data sufficient for tumour localisation in more than 95% of cases. CONCLUSION: Instrumental mechanoreceptoric palpation performed using MTEC is a simple, safe, and reliable method for tumour localisation in gastrointestinal laparoscopic surgery.
RESUMEN
The gene therapy has been successful in treatment of spinal cord injury (SCI) in several animal models, although it still remains unavailable for clinical practice. Surprisingly, regardless the fact that multiple reports showed motor recovery with gene therapy, little is known about molecular and cellular changes in the post-traumatic spinal cord following viral vector- or cell-mediated gene therapy. In this study we evaluated the therapeutic efficacy and changes in spinal cord after treatment with the genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), angiogenin (ANG), and neuronal cell adhesion molecule (NCAM) applied using both approaches. Therapeutic genes were used for viral vector- and cell-mediated gene therapy in two combinations: (1) VEGF+GDNF+NCAM and (2) VEGF+ANG+NCAM. For direct gene therapy adenoviral vectors based on serotype 5 (Ad5) were injected intrathecally and for cell-mediated gene delivery human umbilical cord blood mononuclear cells (UCB-MC) were simultaneously transduced with three Ad5 vectors and injected intrathecally 4 h after the SCI. The efficacy of both treatments was confirmed by improvement in behavioral (BBB) test. Molecular and cellular changes following post-traumatic recovery were evaluated with immunofluorescent staining using antibodies against the functional markers of motorneurons (Hsp27, synaptophysin, PSD95), astrocytes (GFAP, vimentin), oligodendrocytes (Olig2, NG2, Cx47) and microglial cells (Iba1). Our results suggest that both approaches with intrathecal delivery of therapeutic genes may support functional recovery of post-traumatic spinal cord via lowering the stress (down regulation of Hsp25) and enhancing the synaptic plasticity (up regulation of PSD95 and synaptophysin), supporting oligodendrocyte proliferation (up regulation of NG2) and myelination (up regulation of Olig2 and Cx47), modulating astrogliosis by reducing number of astrocytes (down regulation of GFAP and vimetin) and microglial cells (down regulation of Iba1).
RESUMEN
BACKGROUND: Robotic surgery has gained wide acceptance due to minimizing trauma in patients. However, the lack of tactile feedback is an essential limiting factor for the further expansion. In robotic surgery, feedback related to touch is currently kinesthetic, and it is mainly aimed at the minimization of force applied to tissues and organs. Design and implementation of diagnostic tactile feedback is still an open problem. We hypothesized that a sufficient tactile feedback in robot-assisted surgery can be provided by utilization of Medical Tactile Endosurgical Complex (MTEC), which is a novel specialized tool that is already commercially available in the Russian Federation. MTEC allows registration of tactile images by a mechanoreceptor, real-time visualization of these images, and reproduction of images via a tactile display. MATERIALS AND METHODS: Nine elective surgeries were performed with da Vinci™ robotic system. An assistant performed tactile examination through an additional port under the guidance of a surgeon during revision of tissues. The operating surgeon sensed registered tactile data using a tactile display, and the assistant inspected the visualization of tactile data. First, surgeries where lesion boundaries were visually detectable were performed. The goal was to promote cooperation between the surgeon and the assistant and to train them in perception of the tactile feedback. Then, instrumental tactile diagnostics was utilized in case of visually undetectable boundaries. RESULTS: In robot-assisted surgeries where lesion boundaries were not visually detectable, instrumental tactile diagnostics performed using MTEC provided valid identification and localization of lesions. The results of instrumental tactile diagnostics were concordant with the results of intraoperative ultrasound examination. However, in certain cases, for example, thoracoscopy, ultrasound examination is inapplicable, while MTEC-based tactile diagnostics can be efficiently utilized. CONCLUSION: The study proved that MTEC can be efficiently used in robot-assisted surgery allowing correct localization of visually undetectable lesions and visually undetectable boundaries of pathological changes of tissues.