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Biomedical subjects

B L Davidson

Publications and source records attributed to B L Davidson.

At least 37 records · Page 2Linked to original sources
PubMed2000

Gene therapy for amyotrophic lateral sclerosis and other motor neuron diseases.

There are several incurable diseases of motor neuron degeneration, including amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, hereditary spastic hemiplegia, spinal muscular atrophy, and bulbospinal atrophy. Advances in gene transfer techniques coupled with new insights into molecular pathology have opened promising avenues for gene therapy aimed at halting disease progression. Nonviral preparations and recombinant adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses may ultimately transduce sufficient numbers of cerebral, brainstem, and spinal cord neurons for therapeutic applications. This could be accomplished by direct injection, transduction of lower motor neurons via retrograde transport after intramuscular injection, or cell-based therapies. Studies using transgenic mice expressing mutant superoxide dismutase 1 (SOD1), a model for one form of ALS, established that several proteins were neuroprotective, including calbindin, bcl-2, and growth factors. These same molecules promoted neuronal survival in other injury models, suggesting general applicability to all forms of ALS. Potentially correctable genetic lesions have also been identified for hereditary spastic hemiplegia, bulbospinal atrophy, and spinal muscular atrophy. Finally, it may be possible to repopulate lost corticospinal and lower motor neurons by transplanting stem cells or stimulating native progenitor populations. The challenge ahead is to translate these basic science breakthroughs into workable clinical practice.

Amyotrophic Lateral Sclerosis↗
PubMed2000

Adenoviral-mediated transfer of the TNF-related apoptosis-inducing ligand/Apo-2 ligand gene induces tumor cell apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines that induces apoptosis in a variety of cancer cells. The results presented in this study demonstrate that introduction of the human TRAIL gene into TRAIL-sensitive tumor cells using an adenoviral vector leads to the rapid production and expression of TRAIL protein, and subsequent death of the tumor cells. Tumor cell death was mediated by an apoptotic mechanism, as evidenced by the activation of caspase-8, cleavage of poly(ADP-ribose) polymerase, binding of annexin V, and inhibition by caspase inhibitor zVAD-fmk. These results define a novel method of using TRAIL as an antitumor therapeutic, and suggest the potential use for an adenovirus-encoding TRAIL as a method of gene therapy for numerous cancer types in vivo.

Adenoviruses, Human↗
PubMed2000

Transduction of murine cerebellar neurons with recombinant FIV and AAV5 vectors.

Our data demonstrate that vectors derived from recombinant feline immunodeficiency virus (rFIV) and adeno-associated virus type 5 (rAAV5) transduce cerebellar cells following direct injection into the cerebellar lobules of mice. Both recombinant viruses mediated gene transfer predominantly to neurons, with up to 2500 and 1500 Purkinje cells transduced for rAAV5 or rFIV-based vectors, respectively. The vectors also transduced stellate, basket and Golgi neurons, with occasional transduction of granule cells and deep cerebellar nuclei. rAAV5 also spread outside the cerebellum to the inferior colliculus and ventricular epithelium, while rFIV demonstrated the ability to undergo retrograde transport to the physically close lateral vestibular nuclei. Thus, AAV5 and FIV-based vectors show promise for targeting neurons affected in the hereditary spinocerebellar ataxias. These vectors could be important tools for unraveling the pathophysiology of these disorders, or in testing factors which may promote neuronal survival.

Animals↗
PubMed2000

Multi-site partitioned delivery of human tyrosine hydroxylase gene with phenotypic recovery in Parkinsonian rats.

Parkinson's disease (PD) is a leading candidate for neurological gene therapy, given our increasing knowledge of the functional anatomy of the striatonigral system and the localized nature of the affected cell populations. Here we report that stereotactic introduction of a human tyrosine hydroxylase (TH-2) gene using multi-site partitioned doses resulted in behavioral recovery in 6-OHDA-lesioned rats, with transient 100% recovery observed in some animals. We also show correlation between numbers of TH-immunoreactive cells and loss of apomorphine induced rotation, with a near-linear relationship between TH expression and phenotypic recovery. Furthermore, the data suggest that only a fraction of striatal cells need to be transduced in order to exert phenotypic effects, and therefore TH partitioned gene transfer may have clinical potential in PD.

Adenoviridae↗
PubMed2000

Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system.

Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in the central nervous system (CNS), but it is not known how other rAAV serotypes perform as CNS gene transfer vectors. Serotypes 4 and 5 are distinct from rAAV2 and from each other in their capsid regions, suggesting that they may direct binding and entry into different cell types. In this study, we examined the tropisms and transduction efficiencies of beta-galactosidase-encoding vectors made from rAAV4 and rAAV5 compared with similarly designed rAAV2-based vectors. Injection of rAAV5 beta-galactosidase (betagal) or rAAV4betagal into the lateral ventricle resulted in stable transduction of ependymal cells, with approximately 10-fold more positive cells than in mice injected with rAAV2betagal. Major differences between the three vectors were revealed upon striatal injections. Intrastriatal injection of rAAV4betagal resulted again in striking ependyma-specific expression of transgene, with a notable absence of transduced cells in the parenchyma. rAAV2betagal and rAAV5betagal intrastriatal injections led to beta-gal-positive parenchymal cells, but, unlike rAAV2betagal, rAAV5betagal transduced both neurons and astrocytes. The number of transgene-positive cells in rAAV5betagal-injected brains was 130 and 5,000 times higher than in rAAV2betagal-injected brains at 3 and 15 wk, respectively. Moreover, transgene-positive cells were widely dispersed throughout the injected hemisphere in rAAV5betagal-transduced animals. Together, our data provide in vivo support for earlier in vitro work, suggesting that rAAV4 and rAAV5 gain cell entry by means of receptors distinct from rAAV2. These differences could be exploited to improve gene therapy for CNS disorders.

Animals↗
PubMed2000

Batten disease: evaluation of CLN3 mutations on protein localization and function.

Juvenile neuronal ceroid lipofuscinosis (JNCL), Batten disease, is an autosomal recessive lysosomal storage disease associated with mutations in CLN3. CLN3 has no known homology to other proteins and a function has not yet been described. The predominant mutation in CLN3 is a 1.02 kb genomic deletion that accounts for nearly 85% of the disease alleles. In this mutation, truncation of the protein by a premature stop codon results in the classical phenotype. Additional missense and nonsense mutations have been described. Some missense substitutions result in a protracted phenotype, with delays in the onset of classical clinical features, whereas others lead to classical JNCL. In this study, we examined the effect of naturally occurring point mutations on the intracellular localization of CLN3 and their ability to complement the CLN3-deficient yeast, btn1-Delta. We also examined a putative farnesylation motif thought to be involved in CLN3 trafficking. All of the point mutations, like wild-type CLN3, were highly associated with lysosome-associated membrane protein II in non-neuronal cells and with synaptophysin in neuronal cell lines. In the yeast functional assay, point mutations correlating with a mild phenotype also demonstrated CLN3 activity, whereas the mutations associated with severe disease failed to restore CLN3 function completely. CLN3 with a mutation in the farnesylation motif trafficked normally but was functionally impaired. These data suggest that these clinically relevant point mutations, causative of Batten disease, do not affect protein trafficking but rather exert their effects by impairing protein function.

Amino Acid Motifs↗
PubMed2000

Disruption of the beta-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E.

Limb-girdle muscular dystrophy type 2E (LGMD 2E) is caused by mutations in the beta-sarcoglycan gene, which is expressed in skeletal, cardiac, and smooth muscle. beta-sarcoglycan-deficient (Sgcb-null) mice developed severe muscular dystrophy and cardiomyopathy with focal areas of necrosis. The sarcoglycan-sarcospan and dystroglycan complexes were disrupted in skeletal, cardiac, and smooth muscle membranes. epsilon-sarcoglycan was also reduced in membrane preparations of striated and smooth muscle. Loss of the sarcoglycan-sarcospan complex in vascular smooth muscle resulted in vascular irregularities in heart, diaphragm, and kidneys. Further biochemical characterization suggested the presence of a distinct epsilon-sarcoglycan complex in skeletal muscle that was disrupted in Sgcb-null mice. Thus, perturbation of vascular function together with disruption of the epsilon-sarcoglycan-containing complex represents a novel mechanism in the pathogenesis of LGMD 2E.

Animals↗
PubMed2000

A simple method for the rapid generation of recombinant adenovirus vectors.

Recombinant adenoviruses are useful vectors for basic research. When the vectors are used for delineating protein function, several viruses, each containing a mutated version of the transgene are compared at the same time. However, methods to generate multiple vectors simultaneously within a short time period are cumbersome. In this report, we show that a novel backbone plasmid, when cotransfected with routinely used shuttle vectors into HEK293 cells allowed for production of recombinant viruses in an average of 14 days. The recombinant viruses had no detectable wild-type virus contamination by A549 plaque assay and only three to 300 E1a copies per 109 adenovirus genomes by a sensitive PCR-based assay. Further culturing or serial amplification did not result in wild-type revertants nor did cultures show increased levels of E1a copy number by quantitative PCR. Thus, recombinant adenovirus vectors can be produced very simply, rapidly and with little to no contaminating wild-type particles. This system should facilitate the generation of multiple genetic variants by eliminating the need for time-consuming plaque purification and the need to manipulate and screen very large plasmids. We call this the RAPAd.I system.

Adenoviridae↗
PubMed2000

Early adenovirus-mediated gene transfer effectively prevents muscular dystrophy in alpha-sarcoglycan-deficient mice.

Limb-girdle muscular dystrophy type 2D (LGMD 2D) is the most common cause of LGMD with a sarcoglycan defect. We recently engineered a murine model for this progressive disease and we investigated the possibility of preventing the development of muscular dystrophy in these animals by adenovirus-mediated gene transfer of human alpha-sarcoglycan. Here we report that a single intramuscular injection of a first generation adenovirus into the skeletal muscle of neonate mice led to sustained expression of alpha-sarcoglycan at the sarcolemma of transduced myofibers for at least 7 months. The morphology of transduced muscles was consequently preserved. In addition, we have used contrast agent-enhanced magnetic resonance imaging (MRI) to investigate sarcolemmal integrity in adenovirus-injected animals and have thereby demonstrated maintenance of sarcolemmal function. In conclusion, we provide evidence that early virus-mediated gene transfer of a sarcoglycan protein constitutes a promising therapeutic strategy for LGMDs and that the benefits of this approach can easily and effectively be monitored by noninvasive methodologies such as MRI.

Adenoviridae↗
PubMed2000

Human coronavirus 229E infects polarized airway epithelia from the apical surface.

Gene transfer to differentiated airway epithelia with existing viral vectors is very inefficient when they are applied to the apical surface. This largely reflects the polarized distribution of receptors on the basolateral surface. To identify new receptor-ligand interactions that might be used to redirect vectors to the apical surface, we investigated the process of infection of airway epithelial cells by human coronavirus 229E (HCoV-229E), a common cause of respiratory tract infections. Using immunohistochemistry, we found the receptor for HCoV-229E (CD13 or aminopeptidase N) localized mainly to the apical surface of airway epithelia. When HCoV-229E was applied to the apical or basolateral surface of well-differentiated primary cultures of human airway epithelia, infection primarily occurred from the apical side. Similar results were noted when the virus was applied to cultured human tracheal explants. Newly synthesized virions were released mainly to the apical side. Thus, HCoV-229E preferentially infects human airway epithelia from the apical surface. The spike glycoprotein that mediates HCoV-229E binding and fusion to CD13 is a candidate for pseudotyping retroviral envelopes or modifying other viral vectors.

Cell Line↗
PubMed2000

Recombinant human adenovirus: targeting to the human transferrin receptor improves gene transfer to brain microcapillary endothelium.

Some inborn errors of metabolism due to deficiencies of soluble lysosomal enzymes cause global neurodegenerative disease. Representative examples include the infantile and late infantile forms of the ceroid lipofuscinoses (CLN1 or CLN2 deficiency, respectively) and mucopolysaccharidoses type VII (MPS VII), a deficiency of beta-glucuronidase. Treatment of the central nervous system component of these disorders will require widespread protein or enzyme replacement, either through dissemination of the protein or through dissemination of a gene encoding it. We hypothesize that transduction of brain microcapillary endothelium (BME) with recombinant viral vectors, with secretion of enzyme product basolaterally, could allow for widespread enzyme dissemination. To achieve this, viruses should be modified to target the BME. This requires (i) identification of a BME-resident target receptor, (ii) identification of motifs targeted to that molecule, (iii) the construction of modified viruses to allow for binding to the target receptor, and (iv) demonstrated transduction of receptor-expressing cells. In proof of principal experiments, we chose the human transferrin receptor (hTfR), a molecule found at high density on human BME. A nonamer phage display library was panned for motifs which could bind hTfR. Forty-three clones were sequenced, most of which contained an AKxxK/R, KxKxPK/R, or KxK motif. Ten peptides representative of the three motifs were cloned into the HI loop of adenovirus type 5 fiber. All motifs tested retained their ability to trimerize and bind transferrin receptor, and seven allowed for recombinant adenovirus production. Importantly, the fiber-modified viruses facilitated increased gene transfer (2- to 34-fold) to hTfR expressing cell lines and human brain microcapillary endothelia expressing high levels of endogenous receptor. Our data indicate that adenoviruses can be modified in the HI loop for expanded tropism to the hTfR.

Adenoviruses, Human↗
PubMed2000

Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer.

In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring beta-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5-derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2.

Animals↗
PubMed2000

Gene transfer of calcitonin gene-related peptide to cerebral arteries.

Overexpression of calcitonin gene-related peptide (CGRP), an extremely potent vasodilator, to blood vessels is a possible strategy for prevention of vasospasm. We constructed an adenoviral vector that encodes prepro-CGRP (Adprepro-CGRP) and examined the effects of gene transfer on cultured cells and cerebral arteries. Transfection of Adprepro-CGRP to Cos-7 and NIH-3T3 cells increased CGRP-like immunoreactivity in media and produced an increase in cAMP in recipient cells. Five days after injection of Adprepro-CGRP into the cisterna magna of rabbits, the concentration of CGRP-like immunoreactivity increased by 93-fold in cerebrospinal fluid. In basilar artery, cAMP increased by 2.3-fold after Adprepro-CGRP compared with a control adenovirus. After transfection of Adprepro-CGRP, contraction of basilar artery in vitro to histamine and serotonin was attenuated, and relaxation to an inhibitor of cyclic nucleotide phosphodiesterase 3-isobutyl-1-methylxanthine was augmented compared with nontransduced arteries or arteries transfected with a control gene. Altered vascular responses were restored to normal by pretreatment with a CGRP(1) receptor antagonist CGRP-(8-37). Thus gene transfer of prepro-CGRP in vivo overexpresses CGRP in cerebrospinal fluid and perivascular tissues and modulates vascular tone. We speculate that this approach may be useful in prevention of vasospasm after subarachnoid hemorrhage.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Risk assessment and prophylaxis of venous thromboembolism in acutely and/or critically ill patients.

Both undetected and clinically evident venous thrombosis and venous thromboembolism (VTE) can seriously impact the prognosis of acutely and/or critically ill patients. Pulmonary embolism (PE) is harder to diagnose in the acutely and/or critically ill, many of whom also have developed respiratory failure for other reasons. Deep vein thrombosis (DVT) of the upper and lower extremities can subsequently complicate insertion of central venous catheters, leading to PE, sepsis and septic shock. Recovery from the original critical illness (e.g. weaning from mechanical ventilation) can be adversely affected by these complications. There are recent data suggesting that, for prophylaxis, low-molecular-weight heparin (LMWH) is more effective than unfractionated heparin (UFH) in critically ill trauma patients, and that high-dose LMWH is more effective than placebo or low-dose LMWH in seriously ill medical patients. In both populations, LMWH appeared safe. While LMWH appears superior to UFH in acute stroke patients to prevent venographically-proven lower-extremity DVT, whether it provides a superior long-term outcome after acute stroke is uncertain. One study found that a high dosage of the LMWH dalteparin was more effective than placebo in preventing left ventricular thrombi after acute myocardial infarction, but there was a significant safety cost. Current questions surrounding prophylaxis of VTE and the use of LMWH in acutely and/or critically ill patients include whether monitoring levels and dosage adjustment in some of these patients would improve outcome, and whether the diagnosis of VTE can be improved so that treatment can be instituted when prophylaxis has failed.

Acute Disease↗
PubMed2000

Increasing epithelial junction permeability enhances gene transfer to airway epithelia In vivo.

Gene transfer to airway epithelia is the most direct approach for treating the progressive lung disease associated with cystic fibrosis. However, the transduction efficiency is poor when viral vectors are applied to the mucosal surface. We reported previously that gene transfer via the apical surface of human airway epithelia in vitro was improved by formulating vectors with ethyleneglycol-bis-(2-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA) in a hypotonic buffer. First, we investigated the mechanism for this enhancement. When 100-nm fluorescent beads were applied to the apical surface in the presence of EGTA, paracellular deposition of the particles was noted. Transmission electron microscopy verified that the epithelial junction complex was disrupted under these conditions. The Ca(2+) chelators EGTA, 1,2-bis (2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ethylenediaminetetraacetic acid all caused a rapid, reversible drop in transepithelial resistance and facilitated gene transfer with retrovirus or adenovirus in vitro. When Ca(2+) chelators were applied to rabbit tracheal epithelia or human nasal epithelia in vivo, the transepithelial voltage decreased, and amiloride sensitivity was lost, suggesting that epithelial junctions opened. Importantly, this novel formulation enhanced both retroviral- and adenoviral-mediated gene transfer to rabbit tracheal epithelia in vivo. This technique may have applications for vector or drug delivery to airway epithelia and other polarized cells.

Amiloride↗
PubMed2000

Immunolocalization and adenoviral vector-mediated manganese superoxide dismutase gene transfer to experimental oral tumors.

The anti-oxidant enzyme system protects cellular macromolecules against damage from reactive oxygen species. One component of this system, manganese superoxide dismutase (MnSOD), has also been shown to display tumor suppressor gene-like activity. The purpose of this study was to examine changes in MnSOD expression during hamster cheek pouch carcinogenesis, and the effects of MnSOD overexpression using an adenoviral vector. Tumor induction was carried out using 7,12-dimethylbenz[alpha]anthracene. Animals were killed at periodic intervals, and cheek pouch tissues were excised and examined for MnSOD expression by immunohistochemistry and digital image analysis. We observed a reduction in MnSOD expression as early as 2 weeks after the start of carcinogen application. Low MnSOD expression persisted until the end of the 23-week experimental period. Solid hamster cheek pouch carcinoma xenografts were then established in nude mice. An adenoviral vector encoding the human MnSOD gene was delivered to the xenografts by direct injection. We observed high, immediate expression of MnSOD in the xenografts that persisted for 10 days following cessation of viral construct delivery. Delivery of the MnSOD construct resulted in a maximal 50% reduction in tumor growth compared with untreated controls. Our results suggest that MnSOD may be a tumor suppressor gene in the hamster cheek pouch model system.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2000

DVT treatment in 2000: state of the art.

In the past, only symptomatic, objectively confirmed, deep vein thrombosis (DVT) was treated with intravenous (or less commonly, twice-daily subcutaneous) unfractionated heparin in the hospital. Now, asymptomatic DVT (especially calf DVT) is also diagnosed by venography in clinical trials of antithrombotic therapies. Magnetic resonance imaging, ultrasound tests, and occasionally venography are identifying symptomatic calf DVT. Upper extremity and superior vena cava DVT are also being diagnosed more frequently, especially in hospitalized patients. For all of these patients, hospitalized or not, subcutaneous low-molecular-weight heparin likely represents the best therapeutic alternative. Twice-daily dosing is probably safest, but evidence supports once-daily dosing with some agents in certain circumstances. Outpatient treatment is proven safe in reliable patients with minimal comorbidities and significant cost-saving implications. The duration of anticoagulant treatment (usually, but not necessarily, oral) for different patient categories is the next area requiring disciplined scientific investigation.

Anticoagulants↗