High-dose Injection Research Articles

Rescue of a Mouse Model of Spinal Muscular Atrophy With Respiratory Distress Type 1 by AAV9-IGHMBP2 Is Dose Dependent

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive disease occurring during childhood. The gene responsible for disease development is a ubiquitously expressed protein, IGHMBP2. Mutations in IGHMBP2 result in the loss of α-motor neurons leading to muscle atrophy in the distal limbs accompanied by respiratory complications. Although genetically and clinically distinct, proximal SMA is also caused by the loss of a ubiquitously expressed gene (SMN). Significant preclinical success has been achieved in proximal SMA using viral-based gene replacement strategies. We leveraged the technologies employed in SMA to demonstrate gene replacement efficacy in an SMARD1 animal model. Intracerebroventricular (ICV) injection of single-stranded AAV9 expressing the full-length cDNA of IGHMBP2 in a low dose led to a significant level of rescue in treated SMARD1 animals. Consistent with drastically increased survival, weight gain, and strength, the rescued animals demonstrated a significant improvement in muscle, NMJ, motor neurons, and axonal pathology. In addition, increased levels of IGHMBP2 in lumbar motor neurons verified the efficacy of the virus to transduce the target tissues. Our results indicate that AAV9-based gene replacement is a viable strategy for SMARD1, although dosing effects and potential negative impacts of high dose and ICV injection should be thoroughly investigated.

377. AAV-Mediated Transfer of FKRP Shows Therapeutic Efficacy in a Murine Model of Limb-Girdle Muscular Dystrophy Type 2i, but Requires Tight Control of Gene Expression

Limb Girdle Muscular Dystrophies (LGMD) type 2I, a recessive autosomal muscular dystrophy, is caused by mutations in the Fukutin Related Protein (FKRP) gene. It has been proposed that FKRP, whose function remains unclear, is a participant in α-dystroglycan (αDG) glycosylation, which is important to ensure the cell/matrix anchor of muscle fibers. A knock-in mouse model of LGMD2I was generated to express the most frequent mutation (L276I) encountered in patients. The introduction of the mutation did not alter the expression of FKRP, neither at transcriptional nor at translational levels, but did alter its function since abnormal glycosylation of αDG was observed. In this model, skeletal muscles were functionally impaired from 2 months of age and a moderate dystrophic pattern was evident by histology starting from 6 months of age. Gene transfer with a rAAV2/9 vector expressing Fkrp restored the biochemical defects, corrected the histological abnormalities and improved the resistance to eccentric stress in the mouse model was obtained. However, injection of high doses of the vector induced a decrease of αDG glycosylation and laminin binding. Finally, we showed that intravenous injection of the rAAV-Fkrp vector into a dystrophic mouse model suffering of dystroglycanopathy due to skeletal muscle-specific Fukutin (Fktn) knock-out caused toxicity. The dose-dependent worsening of the dystrophic phenotype suggests requirement for a precise control of its expression.

Emulsión lipídica como antídoto al deterioro neurológico progresivo, tras la infiltración de altas dosis de anestésico local: a propósito de un caso

La infiltración de anestésico local (AL) está cada vez más extendida como indicación en el tratamiento del dolor neuropático. La intoxicación por AL es poco frecuente pero grave, potencialmente mortal y de difícil manejo. Cursa con afectación del nivel de conciencia, crisis comiciales y bloqueos de la conducción cardiaca. La emulsión lipídica (EL) al 20 % es el único tratamiento específico descrito hasta la fecha y ha mejorado el pronóstico de estos casos. Presentamos el caso de un paciente con deterioro neurológico progresivo tras la infiltración de altas dosis de AL por un dolor neuropático refractario a otros tratamientos. El paciente fue tratado con emulsión lipídica al 20 % revirtiendo el cuadro neurológico de forma precoz y completa. La aparición de síntomas neurológicos progresivos en el contexto de una infiltración de anestésico local debe alertarnos sobre una posible intoxicación para iniciar tratamiento precoz con emulsión lipídica al 20 %, evitando un pronóstico infausto. Sin embargo, la prevención sigue siendo la herramienta principal.

Tanshinone IIA increases levels of NeuN, protein disulfide isomerase, and Na+/K+-ATPase and decreases evidence of microglial activation after cerebral ischemic injury.

This study was designed to clarify the neuroprotective effects of tanshinone IIA (TSA) following cerebral ischemic insult. Adult Sprague-Dawley rats were operated upon to achieve a middle cerebral artery occlusion to cause transient focal cerebral ischemia, which were then randomly divided into the sham-operated control group and cerebral ischemia/reperfusion (I/R) groups receiving a 2 h occlusion. The treatment groups received daily intraperitoneal injections of high or low doses of TSA, for 7 or 15 days. NeuN immunostaining revealed neuronal loss following I/R, which was partially prevented with subsequent TSA dosing. Protein disulfide isomerase and adenosine triphosphatase (Na(+)/K(+)-ATPase) levels were all depressed by means of I/R. TSA treatment markedly reversed the depression of all indices examined. The intensity of microglial activation, as evidenced with CD11b staining, was increased by means of cerebral artery occlusion, but this was partially reversed with subsequent TSA treatment. TSA may affect neuroprotection by way of minimizing deficits in energy metabolism and reduction of the extent of cell death within affected regions.

Expansion and Hepatic Differentiation of Adult Blood-Derived CD34+ Progenitor Cells and Promotion of Liver Regeneration After Acute Injury

The low availability of functional hepatocytes has been an unmet demand for basic scientific research, new drug development, and cell-based clinical applications for decades. Because of the inability to expand hepatocytes in vitro, alternative sources of hepatocytes are a focus of liver regenerative medicine. We report a new group of blood-derived CD34(+) progenitor cells (BDPCs) that have the ability to expand and differentiate into functional hepatocyte-like cells and promote liver regeneration. BDPCs were obtained from the peripheral blood of an adult mouse with expression of surface markers CD34, CD45, Sca-1, c-kit, and Thy1.1. BDPCs can proliferate in vitro and differentiate into hepatocyte-like cells expressing hepatocyte markers, including CK8, CK18, CK19, α-fetoprotein, integrin-β1, and A6. The differentiated BDPCs (dBDPCs) also display liver-specific functional activities, such as glycogen storage, urea production, and albumin secretion. dBDPCs have cytochrome P450 activity and express specific hepatic transcription factors, such as hepatic nuclear factor 1α. To demonstrate liver regenerative activity, dBDPCs were injected into mice with severe acute liver damage caused by a high-dose injection of carbon tetrachloride (CCl4). dBDPC treatment rescued the mice from severe acute liver injury, increased survival, and induced liver regeneration. Because of their ease of access and application through peripheral blood and their capability of rapid expansion and hepatic differentiation, BDPCs have great potential as a cell-based therapy for liver disease. Hematopoietic stem/progenitor cell expansion and tissue-specific differentiation in vitro are challenges in regenerative medicine, although stem cell therapy has raised hope for the treatment of liver diseases by overcoming the scarcity of hepatocytes. This study identified and characterized a group of blood-derived progenitor cells (BDPCs) from the peripheral blood of an adult mouse. The CD34(+) progenitor-dominant BDPCs were rapidly expanded and hepatically differentiated into functional hepatocyte-like cells with our established coculture system. BDPC treatment increased animal survival and produced full regeneration in a severe liver injury mouse model caused by CCl4. BDPCs could have potential for liver cell therapies.

Characterization of a novel high-dose ovalbumin-induced murine model of allergic sinonasal inflammation.

Few efficacious topical therapies exist for chronic rhinosinusitis (CRS). The lack of a reproducible mouse model of CRS limits the pilot testing of potential novel anti-inflammatory therapies. Although the ovalbumin-induced mouse model of sinonasal inflammation is commonly used, it is difficult to reproduce and can generate variable histologic results. In this study, we explore a variation of this model in different strains of mice and explore various inflammatory cytokines as reproducible molecular markers of inflammation. Allergic sinonasal inflammation was generated in BALB/c and C57BL/6 mice using intraperitoneal high-dose injections of ovalbumin (Ova; Sigma Chemical Co.) followed by 10 days of high-dose intranasal sensitization. Real-time polymerase chain reaction (RT-PCR) for eotaxin, interleukin 4 (IL-4), and IL-13 were measured from sinonasal mucosa. We also pilot tested a known topical budesonide to characterize the anti-inflammatory response. Histological sections were analyzed for epithelial thickness and eosinophilia. Both BALB/c and C57BL/6 mice consistently showed increases in T helper 2 (Th2) cytokines after sensitization with high-dose Ova (p < 0.0001) when compared to controls. There were also significant increases in epithelial thickening in Ova-sensitized mice and eosinophilia in both BALB/c and C57BL/6 strains. In addition, topical budesonide significantly reduced anti-inflammatory cytokines, eosinophilia, and epithelial thickness. Our variation of the ovalbumin-induced mouse model of sinonasal inflammation in both BALB/c and C57BL/6 mice provides an efficacious model for testing potential topical anti-inflammatory therapies for CRS. The utilization of sinonasal mucosal Th2 cytokines along with histologic markers provides a consistent and quantifiable marker of inflammation in assessing the efficacy of candidate drugs.

Effects of transgene expression level per cell in mice livers on induction of transgene-specific immune responses after hydrodynamic gene transfer.

We previously showed that high and sustained transgene expression of antigenic proteins induced transgene-specific immune responses. In the present study, a detailed relationship between the level of transgene expression per cell and immune response after hydrodynamic gene transfer was investigated. Cypridina luciferase (cLuc), a secretory antigenic reporter protein, was selected as a model antigen, and pROSA-cLuc, a plasmid expressing cLuc, was constructed. A fixed dose (30 μg) of pROSA-cLuc was delivered to mice by a single hydrodynamic injection or three injections at 24-h intervals because the number of cells transfected with plasmids is dependent on the number of hydrodynamic injections. Serum cLuc activity, an indicator of the total amount of cLuc transgene expression, was almost equal between these two groups. In contrast, the high-dose single injection induced higher levels of cLuc-specific humoral and cellular immune responses than the three low-dose injections. Moreover, the serum cLuc activity of the high-dose single injection group began to decline ~10 days after injection, whereas the activity remained constant in the three low-dose injection group. These results indicate that it is preferable to reduce the level of transgene expression per cell to avoid induction of the transgene-specific immune response after hydrodynamic gene transfer.

Venom conjugated polylactide applied as biocompatible material for passive and active immunotherapy against scorpion envenomation

Scorpion envenoming represents a public health issue in subtropical regions of the world. Treatment and prevention need to promote antitoxin immunity. Preserving antigenic presentation while removing toxin effect remains a major challenge in toxin vaccine development. Among particulate adjuvant, particles prepared with poly (d,l-lactide) polymer are the most extensively investigated due to their excellent biocompatibility and biodegradability. The aim of this study is to develop surfactant-free PLA nanoparticles that safely deliver venom toxic fraction to enhance specific immune response. PLA nanoparticles are coated with AahG50 (AahG50/PLA) and BotG50 (BotG50/PLA): a toxic fraction purified from Androctonus australis hector and Buthus occitanus tunetanus venoms, respectively. Residual toxicities are evaluated following injections of PLA-containing high doses of AahG50 (or BotG50). Immunization trials are performed with the detoxified fraction administered alone without adjuvant. A comparative study of the effect of Freund is also included. The neutralizing capacity of sera is determined in naive mice. Six months later, immunized mice are challenged subcutaneously with increased doses of AahG50.Subcutaneous lethal dose 50 (LD50) of AahG50 and BotG50 is of 575μg/kg and 1300μg/kg respectively. By comparison, BotG50/PLA is totally innocuous while 50% of tested mice survive 2875μg AahG50/kg. Alhydrogel and Freund are not able to detoxify such a high dose. Cross-antigenicity between particulate and soluble fraction is also, ensured. AahG50/PLA and BotG50/PLA induce high antibody levels in mice serum. The neutralizing capacity per mL of anti-venom was 258μg/mL and 186μg/mL calculated for anti-AahG50/PLA and anti-BotG50/PLA sera, respectively. Animals immunized with AahG50/PLA are protected against AahG50 injected dose of 3162μg/kg as opposed all non-immunized mice died at this dose. We find that the detoxification approach based PLA nanoparticles, benefit the immunogenicity and protective efficacy of venom immunogen.

An acute, epitope-specific modification in the dopamine transporter associated with methamphetamine-induced neurotoxicity.

Preclinical studies demonstrate that repeated, high-dose methamphetamine administrations rapidly decrease plasmalemmal dopamine uptake, which may contribute to aberrant dopamine accumulation, reactive species generation, and long-term dopaminergic deficits. The present study extends these findings by demonstrating a heretofore unreported, epitope-specific modification in the dopamine transporter caused by a methamphetamine regimen that induces these deficits. Specifically, repeated, high-dose methamphetamine injections (4 × 10 mg/kg/injection, 2-h intervals) rapidly decreased immunohistochemical detection of striatal dopamine transporter as assessed 1 h after the final methamphetamine exposure. In contrast, neither a single high dose (1 × 10 mg/kg) nor repeated injections of a lower dose (4 × 2 mg/kg/injection) induced this change. The high-dose regimen-induced alteration was only detected using antibodies directed against the N-terminus. Immunohistochemical staining using antibodies directed against the C-terminus did not reveal any changes. The high-dose regimen also did not alter dopamine transporter expression as assessed using [(125) I]RTI-55 autoradiography. These data suggest that the repeated, high-dose methamphetamine regimen alters the N-terminus of the dopamine transporter. Further, these data may be predictive of persistent dopamine deficits caused by the stimulant. Future studies of the signaling cascades involved should provide novel insight into potential mechanisms underlying the physiological and pathophysiological regulation of the dopamine transporter.

High-density lipoprotein modulates thrombosis by preventing von Willebrand factor self-association and subsequent platelet adhesion

AbstractThe ability of von Willebrand factor (VWF) to initiate platelet adhesion depends on the number of monomers in individual VWF multimers and on the self-association of individual VWF multimers into larger structures. VWF self-association is accelerated by shear stress. We observed that VWF self-association occurs during adsorption of VWF onto surfaces, assembly of secreted VWF into hyperadhesive VWF strings on the endothelial surface, and incorporation of fluid-phase VWF into VWF fibers. VWF adsorption under static conditions increased with increased VWF purity and was prevented by a component of plasma. We identified that component as high-density lipoprotein (HDL) and its major apolipoprotein ApoA-I. HDL and ApoA-I also prevented VWF on the endothelium from self-associating into longer strands and inhibited the attachment of fluid-phase VWF onto vessel wall strands. Platelet adhesion to VWF fibers was reduced in proportion to the reduction in self-associated VWF. In a mouse model of thrombotic microangiopathy, HDL also largely prevented the thrombocytopenia induced by injection of high doses of human VWF. Finally, a potential role for ApoA-I in microvascular occlusion associated with thrombotic thrombocytopenic purpura and sepsis was revealed by the inverse relationship between the concentration of ApoA-I and that of hyperadhesive VWF. These results suggest that interference with VWF self-association would be a new approach to treating thrombotic disorders.

Resistant and Recurrent Late Reaction to Hyaluronic Acid-Based Gel.

Late reactions to hyaluronic acid (HA)-based fillers have a recorded rate of 0.02%. The recent experience with a newly introduced filler in the tear trough area and the lips showed higher rate of reactions with a tendency to persistency and recurrences. To delineate the features of reactions to this newly introduced filler. Medical records of 400 patients (360 women and 40 men; average age = 49.6 years) were examined in this retrospective study. Juvéderm Volbella (HA-Vb) filler was injected only into the tear trough area or lips. Other HA-based products were used in other areas of the face. Seventeen patients (4.25%) developed prolonged (up to 11 months) and recurrent (average: 3.17 episodes) late (average onset: 8.41 weeks after the injection) inflammatory cutaneous reactions. The incidence of late reactions to HA-based fillers varies between products. The authors are reporting an exceptionally high rate of cutaneous reactions for this newly introduced filler. In the authors' experience, broad-spectrum antibiotics in conjunction with repeated high-dose hyaluronidase injections into the inflammatory nodules were effective treatments.

The Effect of nef-Deleted SIV Administration on Disease Progression in SIVInfected Rhesus Macaques

The effect of a live nef-deleted SIVmac 239 construct (SIVΔ nef) was evaluated in rhesus macaques infected with wild-type SIVmac 239 . Rhesus macaques were inoculated by intravenous administration of 100 tissue culture infectious doses 50% (TCID 50 ) of SIVmac 239 . All animals had detectable viremia at 1 week post-inoculation (pi), with peak viremia (6 to 100 million copies viral RNA/ml plasma) two to three weeks pi. From week 4 to week 8 pi, virus levels ranged from ~ 10 4 to ~ 10 6 copies/ml, which is the expected range for SIVmac 239 . MHC type 1 Phenotyping and week 4 viral load data were used to stratify the animals into 3 groups, which all received 2 consecutive intravenous injections of Placebo or SIV Δnef at weeks 8 and 10 pi. The control group received Placebo vehicle (RPMI1640 medium). The Low Dose group received 4e5 TCID 50 units/dose of SIV Δnef, while the log- fold High Dose group received 4e6 TCID 50 units/dose. Animals were monitored daily for clinical signs. At defined regular time points, weight and body temperatures were recorded and blood and urine samples were collected for viral load analysis, clinical haematology, clinical chemistry, urinalysis and immunological assay. No obvious differences were observed in any parameters between the three experimental groups.We conclude that SIV Δnef injection in rhesus macaques infected with wild-type SIV did not increase the progression to disease compared to Placebo control. We also investigated whether intradermal injection of rhesus macaques with nef-deleted SIV is a suitable route to induce infection with this live-attenuated SIV. Two animals that received a high-dose intradermal injection of SIVΔnef became infected and had a pattern of viremia that is indistinguishable from historical data for animals inoculated intravenously with this virus. These findings confirm the attenuated phenotype of SIVΔnef.

Elusive sources of variability of dystrophin rescue by exon skipping.

BackgroundSystemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials.MethodsIn the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays.ResultsThe triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression.ConclusionsWhile we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0070-6) contains supplementary material, which is available to authorized users.

Recombinant Fusion Allergens, Cry j 1 and Cry j 2 from Japanese Cedar Pollen, Conjugated with Polyethylene Glycol Potentiate the Attenuation of Cry j 1-Specific IgE Production in Cry j 1-Sensitized Mice and Japanese Cedar Pollen Allergen-Sensitized Monkeys

Background: Japanese cedar (Cryptomeria japonica) pollinosis is the most prevalent seasonal rhinitis in Japan. A standardized Japanese cedar pollen extract (CPE) containing 1.5-4.2 μg of Cry j 1 is currently the highest-concentration extract available for allergen-specific immunotherapy (SIT) against this pollinosis. Therefore, we developed a PEGylated fusion protein as a more effective SIT vaccine against Japanese cedar pollinosis. Methods: The fusion protein of major allergens for Japanese cedar pollen, Cry j 1 and Cry j 2, was expressed in Escherichia coli and conjugated with polyethylene glycol (PEG). The purified PEGylated Cry j 1/2 fusion protein (PEG-fusion) was subcutaneously injected four times into Cry j 1- sensitized mice and CPE-sensitized monkeys. The mice were then subcutaneously challenged with Cry j 1 and serum levels of Cry j 1-specific immunoglobulin, and the proliferation and cytokine production of splenocytes were analyzed. The monkeys were intranasally challenged with CPE and analyzed for Cry j 1-specific immunoglobulin levels in plasma. Results: Cry j 1-specific IgE was significantly attenuated in the PEG-fusion-treated group after Cry j 1-challenge and Cry j 1-specific IgG was significantly increased following PEG-fusion treatment in mice and monkeys. Proliferation and Th2-type cytokine production in splenocytes stimulated with Cry j 1 were also reduced in PEG-fusion-treated mice. IL10 and IL2 production were reduced, but not significantly, while IFN-γ was significantly increased in the PEG-fusion-treated group. Conclusions: A high-dose injection of PEG-fusion appears to be a valid candidate for a safer and more effective vaccine than the conventional SIT extract for Japanese cedar pollinosis.

Levetiracetam Prevents Perforin Mediated Neuronal Injury Induced by Acute Cerebral Ischemia Reperfusion.

The purpose of this study is to explore the neuroprotection mechanism of levetiracetam (LEV) with acute focal cerebral ischemia-reperfusion (I/P) mouse. The cerebral artery I/P animal model was prepared with a middle artery cerebral occlusion method. For drug intervention, mice were intraperitoneally injected with LEV with a dose of either 15 or 150mg/kg. Neuronal injury was evaluated by measuring the infarct area, apoptosis ratio, and observation of blood-brain barrier ultrastructure with transmission electron microscope. CD8(+) antibody and perforin antibody were used to make cross-reference screen through flow cytometry to determine a perforin-positive rate in CD8(+) T lymphocytes (PFN + %). Injection of LEV can reduce infarct area, apoptosis ratio, and blood-brain barrier damage 24h later after acute I/P in WT mice. In vitro, perforin can lower hippocampal neuron viability. In vivo, removing perforin can relieve neuronal injury. High dose injection of LEV (150mg/kg) can inhibit perforin secreting from CD8(+)T lymphocytes. In addition, LEV can still protect neurons with perforin knockout mice. Therefore, our results suggested that LEV may contribute to neuron protection after cerebral ischemia reperfusion. The possible mechanism may be related with perforin release. However, we cannot roll out other mechanisms.

Prokineticin 2 facilitates mechanical allodynia induced by α,β-methylene ATP in rats

Prokineticin 2 (PK2), a new chemokine, causes mechanical hypersensitivity in the rat hind paw, but little is known about the molecular mechanism. Here, we have found that ionotropic P2X receptor is essential to mechanical allodynia induced by PK2. First, intraplantar injection of high dose (3 or 10pmol) of PK2 significantly increased paw withdrawal response frequency (%) to innocuous mechanical stimuli (mechanical allodynia). And the mechanical allodynia induced by PK2 was prevented by co-administration of TNP–ATP, a selective P2X receptor antagonist. Second, although low dose (0.3 or 1pmol) of PK2 itself did not produce an allodynic response, it significantly facilitated the mechanical allodynia evoked by intraplantar injection of α,β-methylene ATP (α,β-meATP). Third, PK2 concentration-dependently potentiated α,β-meATP-activated currents in rat dorsal root ganglion (DRG) neurons. Finally, PK2 receptors and intracellular signal transduction were involved in PK2 potentiation of α,β-meATP-induced mechanical allodynia and α,β-meATP-activated currents, since the potentiation were blocked by PK2 receptor antagonist PKRA and selective PKC inhibitor GF 109203X. These results suggested that PK2 facilitated mechanical allodynia induced by α,β-meATP through a mechanism involved in sensitization of cutaneous P2X receptors expressed by nociceptive nerve endings.

Long-term in vivo expression of trastuzumab following intramuscular electrotransfer of the encoding DNA in mice

In vivo antibody expression is at crossroads between monoclonal antibody (mAb) and gene therapy. Following a single intramuscular injection of the DNA that encodes a therapeutic mAb, the muscle is turned into a ‘bioreactor’, resulting in prolonged mAb secretion in circulation [1-3]. This innovative approach addresses several challenges associated with conventional mAb proteins. In R&D, in vivo mAb expression allows production and evaluation of leads directly into animal models. In the clinic, it can improve treatment efficacy and patient comfort by avoiding repeated high-dose injections, and provide a cost-effective answer to the increasing need for mAb combination therapies - in the field of cancer immunotherapy and beyond. This study outlines the development and delivery of a trastuzumab-encoding plasmid for in vivo mAb expression. To improve intramuscular DNA delivery in mice, we first established an optimal electrotransfer protocol using novel reporter plasmids. Following the optimized intramuscular electrotransfer of the trastuzumab-encoding plasmid, trastuzumab was detected with a commercial ELISA at therapeutically relevant concentrations (1-15 µg/ml) in the sera of athymic nude mice (n=16) for the full duration of the ongoing follow-up (>3 months). A cell viability assay demonstrated similar activity of the expressed versus commercial trastuzumab in the BT-4747 breast cancer cell line. In conclusion, we achieved proof of concept for the long-term in vivo expression of biologically active trastuzumab in mice. Ongoing work focuses on optimizing in vivo mAb expression for clinical application and evaluation for combination therapy.

Repeated low doses of morpholino antisense oligomer: an intermediate mouse model of spinal muscular atrophy to explore the window of therapeutic response.

The human SMN2 transgenic mice are well-established models of spinal muscular atrophy (SMA). While the severe type I mouse model has a rapidly progressive condition mimicking type I SMA in humans, the mild type III mice do not faithfully recapitulate chronic SMA variants affecting children. A SMA mouse model that clinically mimics the features of type II and III SMA in human is therefore needed. In this study, we generated intermediately affected SMA mice by delivering low-dose morpholino oligomer (PMO25) into the existing severe SMA mice. We show that a single low-dose administration of PMO25 moderately extended the survival of severe type I SMA mice. The neuromuscular pathology is also modestly but significantly improved in these mice. A second administration of PMO25 at postnatal day 5 (PND5) demonstrated an additive effect on survival. Additional systemic administration of low-dose PMO25 at 2-week intervals suppressed the occurrence of distal necrosis beyond postnatal day 100, and induced more complete phenotypic rescue than a single bolus high-dose injection at PND0. Our study demonstrates that survival of motor neuron (SMN) is required early at a critical threshold to prevent symptoms and suggests that subsequent systemic administration of low-dose PMO25 in SMA mice can provide therapeutic benefit and phenotypic rescue, presumably via peripheral SMN restoration. Our work also provides additional insight into the time window of response to administration of antisense oligonucleotides to SMA mice with an intermediate phenotype. This information is crucial at a time when a number of therapeutic interventions are in clinical trials in SMA patients.

Electrophysiological, haemodynamic, and mitochondrial alterations induced by levobupivacaine during myocardial ischemia in a pig model: protection by lipid emulsions?

Accidental intravascular or high-dose injection of local anesthetics (LA) can result in serious, potentially life-threatening complications. Indeed, adequate supportive measures and the administration of lipid emulsions are required in such complications. The study's objectives were threefold: (i) evaluate the myocardial toxicity of levobupivacaine when administered intravenously; (ii) investigate levobupivacaine toxicity on cardiomyocytes mitochondrial functions and cellular structure; (iii) assess the protective effects of a lipid emulsion in the presence or absence of myocardial ischemia. Domestic pigs randomized into two groups of 24 animals each, with either preserved coronary circulation or experimental myocardial ischemia. Six animals from each group received either: (i) single IV injection of saline, (ii) lipid emulsion (Intralipid(®) ), (iii) levobupivacaine, (iv) combination levobupivacaine-Intralipid(®) . Serially measured endpoints included: heart rate, duration of the monophasic action potentials (dMAP), mean arterial pressure, and peak of the time derivative of left ventricular pressure (LV dP/dtmax ). In addition, the following cardiomyocytes mitochondrial functions were measured: reactive oxygen species (ROS) production, oxidative phosphorylation, and calcium retention capacity (CRC) as well as the consequences of ROS production on lipids, proteins, and DNA. IV injection of levobupivacaine induced sinus bradycardia and reduced dMAP and LV dP/dtmax . At the mitochondrial level, oxygen consumption and CRC were decreased. In contrast, ROS production was increased leading to enhanced lipid peroxidation and structural alterations of proteins and DNA. Myocardial ischemia was associated with global worsening of all changes. Intralipid(®) quickly improved haemodynamics. However, beneficial effects of Intralipid(®) were less clear after myocardial ischemia.

The endocannabinoid, endovanilloid and nitrergic systems could interact in the rat dorsolateral periaqueductal gray matter to control anxiety-like behaviors

Cannabinoid compounds usually produce biphasic effects in the modulation of emotional responses. Low doses of the endocannabinoid anandamide (AEA) injected into the dorsolateral periaqueductal gray matter (dlPAG) induce anxiolytic-like effects via CB1 receptors activation. However, at higher doses the drug loses this effect, in part by activating Transient Receptor Potential Vanilloid Type 1 (TRPV1). Activation of these latter receptors could induce the formation of nitric oxide (NO). Thus, the present study tested the hypothesis that at high doses AEA loses it anxiolytic-like effect by facilitating, probably via TRPV1 receptor activation, the formation of NO. Male Wistar rats received combined injections into the dlPAG of vehicle, the TRPV1 receptor antagonist 6-iodo-nordihydrocapsaicin or the NO scavenger carboxy-PTIO (c-PTIO), followed by vehicle or AEA, and were submitted to the elevated plus maze (EPM) or the Vogel conflict test (VCT). A low dose (5pmol) of AEA produced an anxiolytic-like effect that disappeared at higher doses (50 and 200pmol). The anxiolytic-like effects of these latter doses, however, were restored after pre-treatment with a low and ineffective dose of c-PTIO in both animal models. In addition, the combined administration of ineffective doses of 6-iodo-nordihydrocapsaicin (1nmol) and c-PTIO (0.3nmol) produced an anxiolytic-like response. Therefore, these results support the hypothesis that intra-dlPAG injections of high doses of AEA lose their anxiolytic effects by favoring TRPV1 receptors activity and consequent NO formation, which in turn could facilitate defensive responses.