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Sustained Expression (sustained + expression)
Selected AbstractsSustained expression of Epstein,Barr virus episomal vector mediated factor VIII in vivo following muscle electroporationHAEMOPHILIA, Issue 3 2006W.-H. MEI Summary., Haemophilia A treatment is an attractive candidate for gene therapy. The aim of haemophilia gene therapy is to obtain long-term therapeutic level of factor VIII (FVIII). We investigated Epstein,Barr virus (EBV)-based episomal vector combined with in vivo electroporation of naked DNA as a safe, efficient and simple method for correcting FVIII deficiency. A combinant FVIII expression EBV-based episomal vector pcDNA3-FVIII-EBVR was constructed and expressed in COS-7 cells. Then the naked plasmid DNA was injected into the quadriceps of mice following the electric pulse stimulation. Our data showed that pcDNA3-FVIII-EBVR expression in transfected COS-7 can maintain stably for at least 60 days and the hFVIII:Ag in plasma in two pcDNA3-FVIII-EBVR groups mice was higher than that in pcDNA-FVIII groups no matter with or without electric pulse stimulation. With the stimulating of electric pulse, the FVIII expression in plasma of recipient mice was increased two- to fourfolds and can be lasted for at least 90 days. No severe muscle damage was detected. So this novel strategy that FVIII expression mediated by EBV episomal vector following muscle electroporation is efficient, safe, simple and economic and may be applicable to clinical usage. [source] Different pathways leading to cutaneous leukocytoclastic vasculitis in miceEXPERIMENTAL DERMATOLOGY, Issue 6 2001C. Sunderkötter Abstract: To investigate the pathomechanisms of leukocytoclastic vasculitis (LcV) we compared mouse models of LcV with non-vasculitic irritant contact dermatitis (ICD). Criteria for LcV as met by the immune complex-mediated Arthus reaction (Art-r) were also fulfilled by the localized Shwartzman reaction (Shw-r) and by cutaneous Loxoscelism (Lox) (injection of venom from Loxosceles reclusa containing sphingomyelinase D). After depletion of PMN (by ,-irradiation) vessel damage could not be elicited in these models, distinguishing them from models of direct endothelial insult (necrotizing ICD). Depletion of complement could only delay, but not inhibit the Art-r, and did not change ICD, Lox or the Shw-r. The Shw-r exclusively revealed a sustained local expression of vascular adhesion molecules for 24 h in the preparatory phase (LPS s.c.), not observed in the Art-r, in Lox or ICD. Subsequent challenge with LPS i.p. was associated with upregulation of Mac-1 and ICAM-1 on PMN, but not of VLA-4 or LFA-1 (FACS analysis). Cytokines which were able to replace LPS in priming for LcV in the Shw-r (TNF-, and IL-1,) also induced sustained expression of adhesion molecules, whereas IL-12 and IFN-, did neither. Neutralizing IL-12 or IFN-, also inhibited neither LcV nor sustained expression of adhesion molecules, whereas anti-TNF-, inhibited both. Anti-TNF-, had no marked inhibitory effects in the Art-r, in Lox or ICD. Combined (but not separate) neutralization of both E-selectin and VCAM-1 by antibodies suppressed LcV independent from reducing influx of PMN, proving that their sustained expression is decisive for the Shw-r and interferes with normal diapedesis. Since Loxosceles venom is known to dysregulate diapedesis and degranulation of PMN in vitro, since adherent immune complexes activate PMN at the vessel wall, and since adhesion molecules are dysregulated in the Shw-r, we suggest that LcV develops when activation of PMN coincides with vascular alterations which interfere with normal diapedesis. [source] Dysfunction of the unfolded protein response increases neurodegeneration in aged rat hippocampus following proteasome inhibitionAGING CELL, Issue 6 2009María Paz Gavilán Summary Dysfunctions of the ubiquitin proteasome system (UPS) have been proposed to be involved in the aetiology and/or progression of several age-related neurodegenerative disorders. However, the mechanisms linking proteasome dysfunction to cell degeneration are poorly understood. We examined in young and aged rat hippocampus the activation of the unfolded protein response (UPR) under cellular stress induced by proteasome inhibition. Lactacystin injection blocked proteasome activity in young and aged animals in a similar extent and increased the amount of ubiquitinated proteins. Young animals activated the three UPR arms, IRE1,, ATF6, and PERK, whereas aged rats failed to induce the IRE1, and ATF6, pathways. In consequence, aged animals did not induce the expression of pro-survival factors (chaperones, Bcl-XL and Bcl-2), displayed a more sustained expression of pro-apoptotic markers (CHOP, Bax, Bak and JKN), an increased caspase-3 processing. At the cellular level, proteasome inhibition induced neuronal damage in young and aged animals as assayed using Fluorojade-B staining. However, degenerating neurons were evident as soon as 24 h postinjection in aged rats, but it was delayed up to 3 days in young animals. Our findings show evidence supporting age-related dysfunctions in the UPR activation as a potential mechanism linking protein accumulation to cell degeneration. An imbalance between pro-survival and pro-apoptotic proteins, because of noncanonical activation of the UPR in aged rats, would increase the susceptibility to cell degeneration. These findings add a new molecular vision that might be relevant in the aetiology of several age-related neurodegenerative disorders. [source] Molecular architecture of myelinated peripheral nerves is supported by calorie restriction with agingAGING CELL, Issue 2 2009Sunitha Rangaraju Summary Peripheral nerves from aged animals exhibit features of degeneration, including marked fiber loss, morphological irregularities in myelinated axons and notable reduction in the expression of myelin proteins. To investigate how protein homeostatic mechanisms change with age within the peripheral nervous system, we isolated Schwann cells from the sciatic nerves of young and old rats. The responsiveness of cells from aged nerves to stress stimuli is weakened, which in part may account for the observed age-associated alterations in glial and axonal proteins in vivo. Although calorie restriction is known to slow the aging process in the central nervous system, its influence on peripheral nerves has not been investigated in detail. To determine if dietary restriction is beneficial for peripheral nerve health and glial function, we studied sciatic nerves from rats of four distinct ages (8, 18, 29 and 38 months) kept on an ad libitum (AL) or a 40% calorie restricted diet. Age-associated reduction in the expression of the major myelin proteins and widening of the nodes of Ranvier are attenuated by the dietary intervention, which is paralleled with the maintenance of a differentiated Schwann cell phenotype. The improvements in nerve architecture with diet restriction, in part, are underlined by sustained expression of protein chaperones and markers of the autophagy,lysosomal pathway. Together, the in vitro and in vivo results suggest that there might be an age-limit by which dietary intervention needs to be initiated to elicit a beneficial response on peripheral nerve health. [source] Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosisJOURNAL OF NEUROCHEMISTRY, Issue 5 2002Makoto Urushitani Abstract Accumulating evidence indicates that abnormal conformation of mutant superoxide dismutase 1 (SOD1) is an essential feature underlying the pathogenesis of mutant SOD1-linked familial amyotrophic lateral sclerosis (ALS). Here we investigated the role of ubiquitin-proteasome pathway in the mutant SOD1-related cell death and the effect of oxidative stress on the misfolding of mutant SOD1. Transient overexpression of ubiquitin with human SOD1 (wild-type, ala4val, gly85arg, gly93ala) in Neuro2A cells decreased the amount of mutant SOD1, but not of wild-type, while only mutants were co-immunoprecipitated with poly-ubiquitin. Proteasome inhibition by lactacystin augmented accumulation of mutant SOD1 in the non-ionic detergent-insoluble fraction. The spinal cord lysates from mutant SOD1 transgenic mice showed multiple carbonylated proteins, including mutant SOD1 with SDS-resistant dimer formation. Furthermore, the treatment of hSOD1-expressing cells with hydrogen peroxide promoted the oligomerization, and detergent-insolubility of mutant SOD1 alone, and the oxidized mutant SOD1 proteins were more heavily poly-ubiquitinated. In Neuro2A cells stably expressing human SOD1 protein, the proteasome function measured by chymotrypsin-like activity, was decreased over time without a quantitative alteration of the 20S proteasomal component. Finally, primary motor neurons from the mouse embryonic spinal cord were more vulnerable to lactacystin than non-motor neurons. These results indicate that the sustained expression of mutant SOD1 leads to proteasomal inhibition and motor neuronal death, which in part explains the pathogenesis of mutant SOD1-linked ALS. [source] Improved neuronal transgene expression from an AAV-2 vector with a hybrid CMV enhancer/PDGF-, promoterTHE JOURNAL OF GENE MEDICINE, Issue 7 2005C. Y. Wang Abstract Background Adeno-associated virus type 2 (AAV-2) vectors are highly promising tools for gene therapy of neurological disorders. After accommodating a cellular promoter, AAV-2 vectors are able to drive sustained expression of transgene in the brain. This study aimed to develop AAV-2 vectors that also facilitate a high level of neuronal expression by enhancing the strength of a neuron-specific promoter, the human platelet-derived growth factor ,-chain (PDGF) promoter. Methods and results A hybrid promoter approach was adopted to fuse the enhancer of human cytomegalovirus immediately early (CMV) promoter to the PDGF promoter. In cultured cortex neurons, AAV-2 vectors containing the hybrid promoter augmented transgene expression up to 20-fold over that mediated by titer-matched AAV-2 vectors with the PDGF promoter alone and 4-fold over the CMV enhancer/promoter. Injection of AAV-2 vectors with the hybrid promoter into the rat striatum resulted in neuron-specific transgene expression, the level of which was about 10-fold higher than those provided by the two control AAV-2 expression cassettes at 4 weeks post-injection and maintained for at least 12 weeks. Gene expression in the substantia nigra through possible retrograde transport of the AAV-2 vectors injected into the striatum was not obvious. After direct injection of AAV-2 vectors into the substantia nigra, transgene expression driven by the hybrid promoter was observed specifically in dopaminergic neurons and its level was about 3 and 17 times higher than that provided by the PDGF promoter alone and the CMV enhancer/promoter, respectively. Conclusions Enhanced transgene capacity plus neuron-specificity of the AAV-2 vectors developed in this study might prove valuable for gene therapy of Parkinson's disease. Copyright © 2005 John Wiley & Sons, Ltd. [source] Comparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epitheliumTHE JOURNAL OF GENE MEDICINE, Issue 12 2003Nils Loewen Abstract Background In neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2,5 days post-natal). In non-retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol -encoded functions intact. For HIV-1 vectors injected into brain, these have been used to simultaneously control for pseudotransduction and verify that long-term expression requires integration. Such experiments compare particles that differ only in a single amino acid within a single enzyme that forms a very small molar fraction of the virion. Class I integrase mutants have not been described for feline immunodeficiency virus (FIV) integrase, or tested in the eye for any lentiviral vector. Methods We compared subretinally and intravitreally injected FIV vectors and followed animals for up to 7 months, a duration that exceeds prior studies. We also compared the wild-type (WT) vector with one incorporating a single class I amino acid mutation in FIV integrase (D66V). A mock vector (packaging construct absent) was an alternative control. All vectors were vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped and were injected on day 7 of life. One group of animals received either subretinal or intravitreal injections of WT vector in the right eyes. Control left eyes were injected with mock vector. These animals were sacrificed at 2 or 7 days post-injection. A second group received subretinal injections of either WT vector or equivalent D66V vector (reverse transcriptase-normalized to WT), and were analyzed after 2, 3 and 7 months. All eyes were scored for marker gene (,-galactosidase) expression by an observer blinded to vector assignments. Results Subretinal FIV vector injections were much more effective than intravitreal injections. The RPE was the principal retinal layer transduced by the WT vector, and at least 50% of the area of the retina expressed the marker gene at 3 and 7 months. Occasional cells in inner retinal layers also expressed ,-galactosidase at these time points. The sustained retinal expression produced by subretinally injected vector was blocked by the D66V mutation. Conclusions These results show that class I integrase mutant FIV vectors are useful control vectors, and that VSV-G-pseudotyped FIV vectors produce extensive retinal expression for at least 215 days, the longest duration yet reported for lentiviral vectors in retina. Transgene expression is mostly restricted to RPE after post-natal day 7 in rats, suggesting that FIV vectors could be used to target RPE for gene therapy. Copyright © 2003 John Wiley & Sons, Ltd. [source] | |||||||||||||