The assessment of local tolerance, acute toxicity, and DNA biodistribution following particle-mediated delivery of a DNA vaccine to minipigs.
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Particle-mediated DNA delivery was used to administer a DNA vaccine against Hepatitis B to minipigs. The study represented one arm of the safety evaluation program for this product and was designed to assess local tolerance, acute toxicity, and biodistribution of the DNA plasmid. The vaccine was given to 4 groups of minipigs that were sacrificed at 2, 28, 56, or 141 days after treatment. The procedure was well tolerated with mild local skin reactions at 2 days postdosing and no evidence of systemic toxicity. By 28 days the skin lesions had regressed apart from a low grade perivascular mononuclear cell infiltrate in the upper dermis, together with a small number of phagocytosed gold particles. This infiltrate persisted up to 141 days. The expressed HBsAg was detected by immunohistochemistry in keratinocytes (usually in association with an intranuclear gold particle) at 2 days but not at later time points. Polymerase chain reaction (PCR) was used to assay treatment sites and selected internal organs to evaluate biodistribution and persistence of the DNA plasmid. At 2 days the plasmid was detected in the treatment sites and also in the inguinal lymph nodes. At day 57 it was present in the treatment sites only and by day 141 appeared to have cleared. The results from this study demonstrate that particle-mediated gene delivery was well tolerated in the minipig. The biodistribution and persistence of the plasmid was within acceptable limits for this type of vaccine. As the minipig is regarded as a good model for humans these data support the concept that particle-mediated DNA delivery will be safe in human clinical applications. (+info)
Gene-gun particle with pro-opiomelanocortin cDNA produces analgesia against formalin-induced pain in rats.
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Endogenous opioid peptides play an essential role in the intrinsic modulation and control of inflammatory pain, and could be therapeutically useful. These opioid peptides are synthesized as parts of larger precursor molecules. One such precursor molecule is pro-opiomelanocortin (POMC). In this study, we developed a gene-gun method for the transfer of POMC cDNA in vivo, and investigated its therapeutic effect on inflammatory pain in a rat model of formalin-induced pain. Human POMC cDNA was cloned into a modified pCMV plasmid and delivered to the skin of rats by gene gun. Three days after gene-gun injection, 1% formalin was injected. Endorphin levels were measured in the serum and skin after the formalin test, and skin histology was used to detect endorphin after green fluorescent protein (GFP; control) or POMC cDNA transfer. There was no significant difference in the results of acute nociceptive tests between the experimental and control groups. There was also no difference in response between the groups to phase 1 of the formalin test. However, rats which received POMC cDNA via gene-gun injection showed a significantly reduced response in phase 2 of the formalin test. Endorphin immunoreactivity in the skin increased approximately three- to four-fold in experimental animals compared with GFP-treated controls at day 3 after injection. The phase 2 response in animals treated with formalin and naloxone did not differ significantly from the control, implying that the analgesic effects of POMC cDNA particle injection in phase 2 of the formalin test are reversed by naloxone. There are two major findings from this study. First, in vivo DNA delivery by gene gun to the skin is feasible. Second, the production of beta-endorphin is insufficient to block phasic pain, but is effective against sensitization of the afferent neurons during phase 2 of the formalin test. (+info)
Reorganization of exuberant axonal arbors contributes to the development of laminar specificity in ferret visual cortex.
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Layer-specific cortical axons are believed to develop precisely from the outset without making exuberant branches in incorrect cortical layers. We tested this hypothesis by following the development of axonal arbors of layer 2/3 pyramidal neurons in ferret visual cortex, which in the adult have dense axonal arbors in layers 2/3 and 5 and not in layer 4. We began our studies at earlier ages and sampled at closer intervals than in previous studies. Our studies reveal that between postnatal day (P) 14 and P18 the initial growth of axonal arbors lacks specificity for layers 2/3 and 5 and involves the formation of a small number of branches incorrectly in layer 4. After P18 the number of incorrect axonal branches in layer 4 decreased, whereas there was extensive axonal growth specifically in layers 2/3 and 5. To further study the mechanisms involved in directing the layer-specific growth and elimination of axonal branches, we studied the development of layer 2/3 neurons in slices kept in culture for 5 d. In these studies the initial imprecise growth at P14-18 followed by precise growth after P18 was mimicked in vitro, but the elimination of incorrect axons from layer 4 did not occur. These studies reveal that most axonal arbors grow precisely in the correct layers, but the initial growth involves the formation of a small number of incorrect axonal branches that are later eliminated. Therefore the mechanisms that regulate axonal growth are not as precise initially as later, and mechanisms must exist to allow selective elimination of axon collaterals from incorrect layers. (+info)
Extending chestnut blight hypovirus host range within diaporthales by biolistic delivery of viral cDNA.
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Biolistic bombardment was used to successfully transform three phytopathogenic fungal species with an infectious cDNA clone of the prototypic hypovirus, CHV1-EP713, a genetic element responsible for the virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica. The fungal species included two strains each of C. parasitica and Valsa ceratosperma, as well as one strain of Phomopsis G-type (teleomorph Diaporthe Nitschke); all are members of the order Diaporthales but classified into three different genera. A subset of transformants for each of the fungal species contained CHV1-EP713 dsRNA derived from chromosomally integrated viral cDNA. As has been reported for CHV1-EP713 infection of the natural host C parasitica, biolistic introduction of CHV1-EP713 into the new fungal hosts V ceratosperma and Phomopsis G-type resulted in altered colony morphology and, more importantly, reduced virulence. These results suggest a potential for hypoviruses as biological control agents in plant-infecting fungal pathogens other than the chestnut blight fungus and closely related species. In addition, the particle delivery technique offers a convenient means of transmitting hypoviruses to potential host fungi that provides new avenues for fundamental mycovirus research and may have practical applications for conferring hypovirulence directly on infected plants in the field. (+info)
In vivo analysis of glucocorticoid-induced reporter gene expression using gene gun DNA delivery.
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Glucocorticoid regulates various physiological processes via the activation and repression of gene expression. The anti-inflammatory effects and the adverse effects are believed to be dependent on the repression and the activation of genes, respectively. Reporter gene assay is a useful technique to separately evaluate these two functions and has been used for in vitro screening of novel ligands for the glucocorticoid receptor (GR). We report here the application of a reporter gene assay for the in vivo determination of the GR-mediated gene activation. A reporter plasmid containing glucocorticoid response elements was introduced to abdominal mouse skin using a gene gun. Administration of prednisolone induced the expression of the reporter gene, only when the GR expression plasmid was co-transfected with the reporter plasmid. Endogenous levels of corticosterone appeared to be negligible in this protocol. The dose response for this induction was comparable to those for the decreases in thymus weight and serum corticosterone. These results suggest that gene gun-mediated skin transfection enables the in vivo reporter gene assay and that this technique can be used to predict the potency of ligands for the GR-mediated gene activation. (+info)
Distally directed dendrotoxicity induced by kainic Acid in hippocampal interneurons of green fluorescent protein-expressing transgenic mice.
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Excitotoxicity, resulting from the excessive release of glutamate, is thought to contribute to a variety of neurological disorders, including epilepsy. Excitotoxic damage to dendrites, i.e., dendrotoxicity, is often characterized by the formation of large dendritic swellings, or "beads." Here, we show that hippocampal interneurons that express the neuropeptide somatostatin are highly vulnerable to the excitotoxic effects of the ionotropic glutamate receptor agonist kainate. Brief, focal iontophoretic application of kainate rapidly induced bead formation in dendrites of somatostatinergic interneurons that express green fluorescent protein (GFP) from mice of the transgenic line GIN (GFP-expressing inhibitory neurons). Surprisingly, beads often did not form at the site of kainate application or even in the dendritic segment to which kainate was applied; instead, dendritic beading occurred more distally, often encompassing all branches distal to the application site. We have termed this phenomena, "distally directed dendrotoxicity." Distally directed beading was induced regardless of the branch order of the site of application and was found to be dependent on activation of voltage-gated sodium channels. Subsequent to induction, distally directed beading would reverse in most cells; in other cells, however, beading irreversibly invaded proximal dendritic segments and gradually encompassed the entire dendritic tree. These results demonstrate that distal dendritic segments are highly vulnerable to excitotoxic injury and imply that excessive excitatory activity originating in one synaptic pathway can impact synapses at more distal dendritic segments of the same neuron. The discovery of this phenomenon will likely be important in understanding interneuronal dysfunction following excitotoxic injury. (+info)
Cell-to-cell movement of the PVX 12K, 8K, or coat proteins may depend on the host, leaf developmental stage, and the PVX 25K protein.
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The requirements for intercellular movement of Potato virus X (PVX) 12K, 8K, and coat proteins (CP) differed in two Nicotiana spp. Plasmids containing the green fluorescent protein (GFP) gene fused to PVX 12K, 8K, or CP genes were bombarded to tobacco leaves. Protein movement was observed in N. benthamiana but not N. tabacum leaves. GFP:12K and GFP:8K moved cell-to-cell in 25K-expressing transgenic N. tabacum source but not sink leaves. In N. tabacum, GFP:12K and GFP:8K intercellular movement depends on the 25K and leaf developmental stage. Leaves were bombarded using two biolistic delivery methods and the results were similar indicating that movement of GFP:12K or GFP:8K is independent of the delivery system. Mutations in 12K, 8K, and CP genes within the PVX genome inhibited viral intercellular movement in both Nicotiana spp. Thus plasmodesmata gating is not an essential function of these proteins for virus cell-to-cell movement. These proteins likely provide additional activities for virus cell-to-cell movement. (+info)
Increased expression of brain-derived neurotrophic factor induces formation of basal dendrites and axonal branching in dentate granule cells in hippocampal explant cultures.
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During limbic epileptogenesis in vivo the dentate granule cells (DGCs) exhibit increased expression of brain-derived neurotrophic factor (BDNF), followed by striking morphologic plasticities, namely the formation of basal dendrites and the sprouting of mossy fibers. We hypothesized that increased expression of BDNF intrinsic to DGCs is sufficient to induce these plasticities. To test this hypothesis, we transfected DGCs in rat hippocampal slice cultures with BDNF or nerve growth factor (NGF) via particle-mediated gene transfer, and we visualized the neuronal processes with cotransfected green fluorescent protein. Transfection with BDNF produced significant increases in axonal branch and basal dendrite number relative to NGF or empty vector controls. Structural changes were prevented by the tyrosine kinase inhibitor K252a. Thus increased expression of BDNF within DGCs is sufficient to induce these morphological plasticities, which may represent one mechanism by which BDNF promotes limbic epileptogenesis. (+info)