Nonviral glial cell-derived neurotrophic factor gene transfer enhances survival of cultured dopaminergic neurons and improves their function after transplantation in a rat model of Parkinson's disease

Transplantation of dopaminergic fetal mesencephalic tissue into the striatum is currently being developed for treatment of patients with advanced Parkinson's disease. Ethical concerns regarding the use of human fetal tissue, and the limited availability as well as poor survival and differentiation of dopaminergic neurons after transplantation have reduced the extent and outcome of this approach so far. With the purpose of finding means to increase the yield of dopaminergic neurons in transplants, and to reduce the amount of fetal tissue needed for each transplanted patient, we transfected rat fetal ventral mesencephalic (VM) tissue grown as organotypic free-floating roller tube (FFRT) cultures with a vector encoding human glial cell-derived neurotrophic factor (hGDNF). For transfer of an episomal expression vector (pRep7-GDNF8) a nonviral, nonliposomal cationic transfection technique was applied and optimized. Recombinant hGDNF expression resulted in a higher number of TH-positive neurons in the cultures as measured 6 days after transfection. Ventral mesencephalic cultures expressing hGDNF were then grafted into the striatum of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. Grafting of genetically modified VM cultures resulted in earlier functional recovery compared with grafting nontransfected cultures. We conclude that organotypic free-floating roller tube cultures can be successfully transfected to produce hGDNF with effects on TH-expressing neurons in vitro and functional effects after grafting in a rat Parkinson's disease model.     

Borocaptate sodium: a potential boron delivery compound for boron neutron capture therapy evaluated in dogs with spontaneous intracranial tumors.

Borocaptate sodium (Na2B12H11SH) is a boron-carrying compound under consideration for use in boron neutron capture therapy. The biodistribution of boron from borocaptate sodium administration will partly determine boron neutron capture therapy efficacy and normal tissue radiation tolerance. The biodistribution of boron was determined in 30 dogs with spontaneous intracranial tumors at 2, 6, or 12 hr after intravenous borocaptate sodium infusion. Blood and tissue boron concentrations were measured using inductively coupled plasma atomic emission spectroscopy. Mean tumor boron concentration (mean +/- standard error) was 35.9 +/- 4.6 (n = 15), 22.5 +/- 6.0 (n = 9), and 7.0 +/- 1.1 micrograms of boron per g (n = 6) at 2, 6, and 12 hr, respectively, after borocaptate sodium infusion. Peritumor boron concentrations were elevated above that of normal brain in half of the dogs. Normal brain boron concentration (mean +/- standard error) was 4.0 +/- 0.5, 2.0 +/- 0.4, and 2.0 +/- 0.3 micrograms of boron per g at 2, 6, and 12 hr after infusion, respectively. Some cranial and systemic tissues, and blood, had high boron concentration relative to tumor tissue. Geometric dose sparing should partly offset these relatively high normal tissue and blood concentrations. Borocaptate sodium biodistribution is favorable because tumor boron concentrations of recommended magnitude for boron neutron capture therapy were obtained and there was a high tumor-to-normal brain boron concentration ratio.     

Prevalence of THAP1 sequence variants in German patients with primary dystonia

Primary dystonias are a clinically and genetically heterogeneous group of movement disorders, but only for two of them, i.e., dystonia 1 and dystonia 6, the disease causing gene has been identified. Dystonia 1 is characterized by an early onset and is caused by a mutation in the TOR1A gene. Only recently, mutations in THAP1 have been shown to be the cause of DYT6 dystonia. We analyzed 610 patients with various forms of dystonia for sequence variants in the THAP1 gene by means of high resolution melting to delineate the prevalence of sequence variants and phenotypic variability. We identified seven sequence variants in patients and one sequence variant in a control. The sequence variants were not detected in 537 healthy controls. Four patients present with generalized dystonia with speech involvement of early onset, another three patients suffered exclusively from cervical dystonia of adult onset. These findings suggest that THAP1 sequence variations seem to be associated with different ages of onset and distribution of symptoms. Consequently, the phenotypic spectrum might be broader than previously assumed. © 2010 Movement Disorder Society     

Regional cerebral glucose metabolism and anxiety symptoms in bipolar depression: Effects of levothyroxine

We examined the relationships between regional brain activity and anxiety in bipolar depressed patients receiving adjunctive treatment with levothyroxine. Regional brain activity was assessed with positron emission tomography and [¹⁸F]fluorodeoxyglucose in 10 euthyroid, depressed bipolar women before and after 7 weeks of adjunctive therapy with levothyroxine. The primary biological measures were relative (to global) regional radioactivity as a surrogate index of glucose metabolism in pre-selected brain regions. Relationships were assessed between regional brain activity and anxiety symptoms while controlling for depression severity. At baseline, Trait Anxiety Inventory measures covaried positively with relative brain activity bilaterally in the dorsal anterior cingulate, superior temporal gyri, parahippocampal gyri, amygdala, hippocampus, ventral striatum, and right insula; state anxiety showed a similar pattern. After treatment anxiety was improved significantly. Change in trait anxiety covaried positively with changes in relative activity in right amygdala and hippocampus. Change in state anxiety covaried positively with changes in relative activity in the hippocampus bilaterally and left thalamus, and negatively with changes in left middle frontal gyrus and right dorsal anterior cingulate. Results indicate that comorbid anxiety symptoms have specific regional cerebral metabolic correlates in bipolar depression and cannot only be explained exclusively by the depressive state of the patients.     

AAV based gene delivery to myocardium in rodents and pigs

Objective: To optimize transgene expression levels after AAV‐mediated gene transfer different delivery methods were compared in a rat (A) and porcine (B) heterotopic heart transplantation model. Methods: (A) Heterotopic abdominal heart transplantations were performed in male Lewis rats. After harvesting the donor hearts, the viral vectors were delivered to the graft by the following methods: (1) 0.35 ml saline solution containing AAV2/9‐LacZ (2 × 10¹¹ vector genome, vg) was injected directly into the myocardium (apex) immediately after reperfusion. (2) cardioplegic solution (0.3 ml) containing AAV‐2(HBSD), 2/9‐LacZ vectors was rapidly injected into the aortic root with the pulmonary trunk clamped. Before transplantation the transfected heart was incubated for 20 min in iced cardioplegia. (3) A reperfusion system was applied: For 20 min a cold solution of cardioplegia (5 ml) and AAV‐2(HBSD) or AAV2/9‐LacZ vectors were recirculated through the donor heart. Transplanted grafts were explanted after 3 weeks. To detect and measure marker gene expression X‐gal staining or a luciferase assay was performed. In a second series we compared the effects of the transduction of PD‐L1 to LacZ using the optimum method (intraaortic root injection) in the same heart transplantation model. (B) Heterotopic abdominal HTX was performed in pigs (Landrace, 10–18 kg) following vector application to the donor heart in an in situ‐Langendorff perfusion system (AAV2/GFP and AAV2/Luciferase). Recipients were given tacrolimus (0.3 mg/kg BW), after 21 days the transplanted hearts were explanted for transgene expression analysis. In a second series we compared AAV2/9‐mediated transduction of PD‐L1 and LacZ in the in situ‐Langendorff model and consequent allogeneic heterotopic abdominal heart transplantation. Results: (A) Highest transfection efficiency was observed in the grafts treated with intracoronary infusion of AAV2/9 at the higher dosage, and the expression pattern was global and homogenous in the grafts. hPD‐L1 transduction resulted in no significant difference of survival time and signs of rejection after allogeneic rat heart transplantation. (B) AAV2‐mediated gene delivery was unable to yield sufficient transgene expression after in situ‐Langendorff perfusion of porcine hearts. AAV2/9 based gene transfer of LacZ and hPD‐L1 led to excellent myocardial gene expression lasting up to 2 months. Due to species incompatibility no protective effects were observed in our allogeneic porcine transplantation model. Conclusions: (A) We demonstrated that infusion of AAV vectors into the aortic root with the pulmonary trunk clamped is a simple and efficient method for gene delivery to the donor heart in an allogeneic rat heart transplantation setting. Gene transfer of hPD‐L1 was ineffective to protect against allorejection, on the contrary there was a trend to aggravated rejection. Therefore the exact mechanism of hPD‐L1 in allograft rejection needs further investigation. (B) The in situ‐Langendorff model was developed to allow isolated target organ perfusion with high vector concentrations under physiological conditions. Using this method AAV2/9 mediated gene delivery into porcine hearts proved effective to induce excellent marker gene expression. Expression of hPD‐L1 could also be achieved but was ineffective in pig allotransplantation due to species incompatibility. Double transgenic pig hearts (e.g. Gal‐KO+CD46) can now efficiently be transduced with hPD‐L1 or hCTLA4‐Ig to further optimize long‐term survival after pig‐to‐primate cardiac xenotransplantation.     

Comparison between bovine bone and titanium interference screws for implant fixation in ACL reconstruction: a biomechanical study

Introduction

The application of interference screws for the fixation of bone-patellar tendon-bone (BPTB) grafts is a well-established technique in anterior-cruciate ligament reconstruction. Interference screws derived from bovine compact bone are a biological alternative to metallic or biodegradable polymer interference screws.

Materials and methods

In 60 porcine specimens, the tibial part of an anterior-cruciate ligament reconstruction was performed using a BPTB graft. To secure the graft, either an 8-mm titanium interference screw or a self-made bovine interference screw (BC), or a commercial bovine compact bone screw (Tutofix^®) was used. The maximum failure load was determined by means of a universal testing machine with computer interface at a testing speed of 50 mm/min. In a second test series, cyclic sub-maximal load was applied to the test specimen from 40 to 400 N with a number of 1,000 load cycles and a frequency of 1 Hz. Subsequently, the maximum failure load was determined. The stiffness of the test specimen was investigated in both test series. Each type of interference screw was tested 10 times.

Results

A secure fixation of the grafts was achieved with all interference screws. In the experiments on the maximum load to failures, the titanium screws showed significantly higher failure loads than the Tutofix^® screws (P = 0.005). The stiffness of the grafts fixed with BC screws was significantly higher as compared to the fixation with Tutofix^® screws (P = 0.005). After cyclic sub-maximal loading, the maximum failure load of the titanium screws was significantly higher than that of the Tutofix^® screws (P = 0.033). The fixation of the BC screws showed a significantly higher failure load (P = 0.021) and stiffness (P = 0.032) than the Tutofix^® screw fixation. Except for two screw head fractures and two intra-tendinous graft ruptures, the failure mode was slippage in the interface between interference screw and bone plug.

Conclusion

Interference screws derived from bovine compact bone show similar good results as the titanium interference screws. Therefore, the safety and in vivo performance of products derived from xenogenic bone should be the focus of further investigations.     

Healing process after surgical treatment with scalpel, electrocautery and laser radiation: histomorphologic and histomorphometric analysis

The aim of this study was to evaluate and compare the healing process after surgical treatment of chemically induced lesions in the lateral edge of tongue of hamsters performed with scalpel, electrocautery, carbon dioxide (CO₂) laser radiation or neodymium:yttrium–aluminum–garnet (Nd:YAG) laser radiation. Eighty hamsters of both sexes were used and examined at postoperative days 7, 14, 21 and 28 by histological and histomorphometric analysis of the skeletal muscle fibers. In the histological analysis it was observed that the dynamics of the healing process was faster in the group treated by scalpel than in the other groups. The histomorphometric observation of the skeletal muscle fibers was submitted to one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test, with a level of significance set at P?<?0.05, which showed that the amount of skeletal muscle fiber formed had significantly increased in the group treated by scalpel in comparison with that in the groups treated by electrocautery (P?<?0.01), CO₂ laser irradiation (P?<?0.001) and Nd:YAG laser irradiation (P?<?0.01) on the 14th postoperative day. A gradual increase in skeletal muscle fibers formed during the healing process was observed in all groups. When the laser irradiated groups were compared, it was possible to conclude that tissue organization and vascularization were faster and more intense in the Nd:YAG laser irradiated group than in the CO₂ laser irradiated group.     

Repeat Retrourethral Transobturator Sling in the Management of Recurrent Postprostatectomy Stress Urinary Incontinence After Failed First Male Sling

Background

A failure rate between 20% and 45.5% after retrourethral transobturator sling (RTS) is reported. Recommendations for the management of persistent or recurrent postprostatectomy stress urinary incontinence (SUI) after failed male sling do not exist.

Objective

The aim of this study was the prospective evaluation of the efficacy of repeat RTS (RRTS) in patients after failed first RTS.

Design, setting, and participants

Between March 2007 and August 2009, 35 patients with mild to severe SUI after failed first RTS were treated with a second AdVance sling (American Medical Systems, Minnetonka, MN, USA).

Measurements

Preoperative and postoperative evaluation included daily pad use, 1-hr pad test, postvoiding residual (PVR) urine, uroflowmetry, and quality-of-life (QoL) scores.

Results and limitations

After 6 mo, 45.5% (15 of 33 patients) showed no pad use; 30.3% (10 of 33 patients), one dry “security” pad; 3% (1 of 33 patients), one wet pad; 6.1% (2 of 33 patients), two pads; 3% (1 of 33 patients), pad reduction ≥50%; and 12.1% (4 of 33 patients), treatment failure. After 16.6 mo, 34.5% (10 of 29 patients) showed no pad use; 37.9% (11 of 29 patients), one dry “security” pad; 3.4% (1 of 29 patients), one wet pad; 3.4% (1 of 29 patients), two pads; 10.3% (3 of 29 patients), pad reduction ≥50%; and 10.4% (3 of 29 patients), treatment failure. Daily pad use and pad weight decreased significantly. PVR and uroflowmetry results showed no significant change. QoL improved significantly. Postoperative acute urinary retention was observed in 23.6% of patients.

Conclusions

RRTS is an effective and safe treatment option for the management of SUI after failed first RTS.