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Biomedical subjects

D S Latchman

Publications and source records attributed to D S Latchman.

At least 73 records · Page 4Linked to original sources
PubMed2001

Multiple immediate-early gene-deficient herpes simplex virus vectors allowing efficient gene delivery to neurons in culture and widespread gene delivery to the central nervous system in vivo.

Herpes simplex virus (HSV) has several potential advantages as a vector for delivering genes to the nervous system. The virus naturally infects and remains latent in neurons and has evolved the ability of highly efficient retrograde transport from the site of infection at the periphery to the site of latency in the spinal ganglia. HSV is a large virus, potentially allowing the insertion of multiple or very large transgenes. Furthermore, HSV does not integrate into the host chromosome, removing any potential for insertional activation or inactivation of cellular genes. However, the development of HSV vectors for the central nervous system that exploit these properties has been problematical. This has mainly been due to either vector toxicity or an inability to maintain transgene expression. Here we report the development of highly disabled versions of HSV-1 deleted for ICP27, ICP4, and ICP34.5/open reading frame P and with an inactivating mutation in VP16. These viruses express only minimal levels of any of the immediate-early genes in noncomplementing cells. Transgene expression is maintained for extended periods with promoter systems containing elements from the HSV latency-associated transcript promoter (J. A. Palmer et al., J. Virol. 74:5604-5618, 2000). Unlike less-disabled viruses, these vectors allow highly effective gene delivery both to neurons in culture and to the central nervous system in vivo. Gene delivery in vivo is further enhanced by the retrograde transport capabilities of HSV. Here the vector is efficiently transported from the site of inoculation to connected sites within the nervous system. This is demonstrated by gene delivery to both the striatum and substantia nigra following striatal inoculation; to the spinal cord, spinal ganglia, and brainstem following injection into the spinal cord; and to retinal ganglion neurons following injection into the superior colliculus and thalamus.

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PubMed2001

Effect of aging on the ability of preconditioning to protect rat hearts from ischemia-reperfusion injury.

Ischemic preconditioning (IP) reduces infarct size in young animals; however, its impact on aging is underinvestigated. The effect of variations in IP stimuli was studied in young, middle-aged, and aged rat hearts. Isolated hearts underwent 35 min of regional ischemia and 120 min of reperfusion. Hearts with IP were subjected to either one ischemia-reperfusion cycle (5 min of ischemia and 5 min of reperfusion per cycle) or three successive cycles before 35 min of regional ischemia. Additional studies investigated the effects of pharmacological preconditioning in aged hearts using the adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine, the protein kinase C analog 1,2-dioctanoyl-sn-glycerol, and the mitochondrial ATP-sensitive potassium (K(ATP))-channel opener diazoxide. Infarct sizes indicated that the aged rat heart could not be preconditioned via ischemic or pharmacological means. The middle-aged rat heart had a blunted IP response compared with the young adult (only an increased IP stimulus caused a significant reduction in infarct size). These results suggest that there are defects within the IP signaling cascade of the aged heart. Clinical relevance is important if we are to use any IP-like mimetics to the benefit of an aging population.

Adenosine↗
PubMed2001

Gene therapy with herpes simplex virus vectors: progress and prospects for clinical neuroscience.

Gene delivery to the nervous system represents perhaps the ultimate challenge of gene therapy in view of the complexity of this system, the wide variety of intractable neurological diseases, and the need to deliver the gene to nondividing cells. Although a variety of systems for such gene delivery are under development, herpes simplex virus has unique advantages in terms of its large genome size and its ability to naturally enter a latent state in neuronal cells. Considerable progress has been made in the effective disablement of this virus while retaining its ability to deliver genes and in producing long-term expression of the foreign gene. It is likely that these viruses may ultimately be of use in human gene therapy procedures for otherwise intractable neurological diseases such as Parkinson's disease.

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PubMed2001

Viral vectors for gene therapy in Parkinson's disease.

The ability of transplanted neurons from aborted foetuses to produce some therapeutic benefit in Parkinson's disease makes this disease an obvious target for the development of gene therapy procedures which involve delivering the same factors as are provided by the foetal neurons but using a reagent which could be produced in large amounts in a standardised manner. This approach could involve both the delivery of the gene encoding tyrosine hydroxylase to boost dopamine production or the delivery of genes encoding neurotrophic factors such as GDNF to promote the survival of dopaminergic neurons. A variety of different viral and non-viral methods for achieving such gene delivery has been described. These are discussed together with the particular advantages of herpes simplex virus-based vectors which have the potential to deliver multiple therapeutic genes in a single virus vector.

Animals↗
PubMed2000

Molecular cloning and expression of the Fabs of human autoantibodies in Escherichia coli. Determination of the heavy or light chain contribution to the anti-DNA/-cardiolipin activity of the Fab.

The Fabs of three human autoantibodies (B3/33H11, anti-DNA; UK4, anti-phospholipid) and six related hybrids have been cloned, expressed in Escherichia coli, and purified to homogeneity. SDS-polyacrylamide gel electrophoresis and Western blot analysis of the recombinant Fab demonstrated the purified Fab to be of correct size and in assembled form. Protein expression levels of up to 5-9 mg per liter of culture were achievable. A sensitive and reliable comparative anti-DNA enzyme-linked immunosorbent assay, involving a defined biotinylated 35-mer oligonucleotide in its single- or double-stranded form, is also described. Crithidia assay and anti-DNA or anti-cardiolipin antibody enzyme-linked immunosorbent assay analyses demonstrated convincing binding of the recombinant Fab proteins to DNA/cardiolipin, confirming the expression of functional molecule. The comparative DNA/cardiolipin binding analyses of the nine Fabs revealed that the anti-DNA (light, B3/33H11) or anti-cardiolipin (heavy, UK4) activity lies predominantly on one of the two chains. However, a compatible partner chain is necessary for optimum antigen binding activity of the antibody.

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PubMed2000

The BRN-3A transcription factor protects sensory but not sympathetic neurons from programmed cell death/apoptosis.

Inactivation of the gene encoding the POU domain transcription factor BRN-3A results in the absence of specific neurons in knockout mice. Here we demonstrate for the first time a direct effect of BRN-3A on the survival of neuronal cells. Specifically, overexpression of BRN-3A in cultured trigeminal ganglion or dorsal root ganglion sensory neurons enhanced their survival following the withdrawal of nerve growth factor. Moreover, reduction of BRN-3A levels impaired the survival of these neurons. The survival of sympathetic neurons was not affected by either approach. Similarly, overexpression of BRN-3A activated the endogenous Bcl-2 gene in trigeminal neurons, but not in sympathetic neurons. The protective effect of BRN-3A on trigeminal neuron survival following nerve growth factor withdrawal significantly increased during embryonic development. In contrast, overexpression of the related factor BRN-3B enhanced survival of trigeminal neurons only at an early stage of embryonic development. Thus, BRN-3A (and in some circumstances, BRN-3B) can promote the survival of nerve growth factor-dependent sensory but not sympathetic neurons, allowing it to play a direct role in the survival of some (but not all) neuronal populations in the developing and adult nervous systems.

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PubMed2000

IGF-1 regulates cardiac fibroblast apoptosis induced by osmotic stress.

In this study we have determined the ability of IGF-1 to protect cardiac fibroblasts against osmotic-induced apoptosis and investigated the potential mechanism(s) underlying this protection. Treatment with IGF-1 (1-100 ng/ml) promoted a dose dependent increase in cell survival against osmotic cell death. Both Akt and ERK1/2 were rapidly phosphorylated by IGF-1 and blocked by wortmannin and PD98059, inhibitors of their upstream activators respectively. However, IGF-1-induced protection was mediated via a wortmannin-dependent but PD98059-independent pathway as determined by cell survival assay suggesting a role of PI3-K/Akt. Furthermore, IGF-1 appeared to reduce the activation of a number of early components in the apoptotic pathway in a wortmannin dependent manner including the osmotic stress-induced perturbation in mitochondrial membrane potential, cleavage and activation of caspase-3 and DNA fragmentation. Thus, the results suggest that IGF-1 regulates osmotic stress-induced apoptosis via the activation of the PI3-K/Akt pathway at a point upstream of the mitochondria and caspase-3.

Animals↗
PubMed2000

Ischemia-induced STAT-1 expression and activation play a critical role in cardiomyocyte apoptosis.

We show here that exposure of cardiac cells to simulated ischemia results in apoptosis and is accompanied by phosphorylation and increased expression and transcriptional activity of STAT-1. Similarly, interferon-gamma, which is known to induce STAT-1 activation, also induced apoptosis in cardiac cells. STAT-1-transfected cells were more susceptible to ischemia-induced cell death than cells transfected with a control plasmid lacking the STAT-1 coding sequence. Furthermore, an antisense STAT-1 vector reduced both ischemia- and overexpressed STAT-1-induced cell death in cardiac cells. Both STAT-1 overexpression and interferon-gamma treatment or exposure to ischemia activated the promoter of the pro-apoptotic caspase-1 gene in cardiomyocytes. Finally, ischemia/reperfusion also induced STAT-1 activation and caspase-1 processing in ventricular myocytes in the intact heart ex vivo. Immunofluorescent staining demonstrated an increase in STAT-1-positive staining in cardiomyocytes in response to ischemia/reperfusion that co-localized with terminal deoxynucleotidyl transferase dVTP nick end-labeling-positive apoptotic cells. These results suggest that STAT-1 plays a critical role in the regulation of ischemia/reperfusion-induced apoptosis in cardiac cells, acting at least in part via a caspase-1 activation-dependent pathway.

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PubMed2000

Urocortin protects against ischemic and reperfusion injury via a MAPK-dependent pathway.

Urocortin (UCN) is a peptide related to hypothalamic corticotrophin-releasing hormone and binds with high affinity to corticotrophin-releasing hormone receptor-2beta, which is expressed in the heart. In this study, we report that UCN prevented cell death when administered to primary cardiac myocyte cultures both prior to simulated hypoxia/ischemia and at the point of reoxygenation after simulated hypoxia/ischemia. UCN-mediated cell survival was measured by trypan blue exclusion, 3'-OH end labeling of DNA (TUNEL), annexin V, and fluorescence-activated cell sorting. To explore the mechanisms that could be responsible for this effect, we investigated the involvement of MAPK-dependent pathways. UCN caused rapid phosphorylation of ERK1/2-p42/44, and PD98059, which blocks the MEK1-ERK1/2-p42/44 cascade, also inhibited the survival-promoting effect of UCN. Most important, UCN reduced damage in isolated rat hearts ex vivo subjected to regional ischemia/reperfusion, with the protective effect being observed when UCN was given either prior to ischemia or at the time of reperfusion after ischemia. This suggests a novel function of UCN as a cardioprotective agent that could act when given after ischemia, at reperfusion.

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PubMed2000

Distinct signalling pathways mediate the cAMP response element (CRE)-dependent activation of the calcitonin gene-related peptide gene promoter by cAMP and nerve growth factor.

The gene encoding the calcitonin gene-related peptide (CGRP) is activated in neuronal cells by treatment with cAMP and nerve growth factor (NGF). Both stimuli induce the phosphorylation of the cAMP response element (CRE)-binding protein (CREB) transcription factor on Ser-133 and require the CRE in the CGRP promoter to stimulate transcription. However, whereas the CRE is necessary and sufficient for promoter activation by cAMP, it is necessary but not sufficient for activation by NGF. We show that this difference is paralleled by a difference in the signalling pathways which are required for each stimulus to activate the CGRP promoter. Thus whilst cAMP-mediated activation requires the protein kinase A pathway, NGF-mediated stimulation requires the Ras/Raf mitogen-activated protein kinase kinase-1 (MEK-1)/p42/p44 mitogen-activated protein kinase (MAPK) pathway. Although NGF can activate the protein kinase C, p38 MAPK and c-Jun N-terminal kinase (JNK) pathways, these pathways are not involved in its effect on the CGRP promoter. The effect of the p42/p44 MAPK pathway on CREB and associated transcription factors, and the manner in which this results in activation of the CGRP promoter is discussed.

Animals↗
PubMed2000

Viral vectors in the treatment of Parkinson's disease.

Parkinson's disease is an obvious target for the development of gene therapy procedures which could involve both the delivery of the gene encoding tyrosine hydroxylase to boost dopamine production or the delivery of genes encoding neurotrophic factors such as GDNF to promote the survival of dopaminergic neurons. A variety of different viral and nonviral methods for achieving such gene delivery are described together with the particular advantages of herpes simplex virus-based vectors which have the potential to deliver multiple therapeutic genes in a single virus vector.

Animals↗
PubMed2000

Insulin administered at reoxygenation exerts a cardioprotective effect in myocytes by a possible anti-apoptotic mechanism.

The metabolic cocktail of glucose-insulin-potassium (GIK) has been shown to reduce mortality in humans and reduce infarct size in the rat when administered from the onset of reperfusion following an ischemic insult. The mechanisms underlying GIK mediated cardioprotection are, however, still unclear. Recent data implicates insulin "alone" as the major protagonist of cardioprotection when administered at the time of reperfusion. We have therefore begun to investigate an insulin activated signalling pathway and the putative role of apoptosis in this insulin-induced cardioprotection. Simulated ischemia and reoxygenation were induced in rat neonatal cardiocyte experiments. The administration of insulin [0.3 mU/ml] at the moment of reoxygenation (Ins(R)) enhanced myocardial cell viablility as assessed by trypan blue exclusion compared to vehicle alone treated control myocytes (Ins(R)50+/-2%v controls 70+/-1%, P<0.001). This insulin-mediated cardioprotection was due, in part to a reduction in myocyte apoptosis as measured by TUNEL (Ins(R)29+/-2%v controls 49+/-3%, P<0.001) and Annexin V staining (Ins(R)34+/-2%v controls 65+/-3%, P<0.001). These cardioprotective and anti-apoptotic effects of insulin were completely abolished by the tyrosine kinase inhibitor lavendustin A and by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin. Thus, we conclude that the early administration of insulin appears to be an effective modality to reduce reoxgygenation injury in cardiocytes, in part, via the attenuation of ischemia/reoxygenation-induced apoptosis. Moreover, the cardioprotective and anti-apoptotic effects of insulin are mediated via tyrosine kinase and PI3-kinase signalling pathways.

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PubMed2000

Cardiotrophin-1: a novel cytokine and its effects in the heart and other tissues.

Cardiotrophin-1 (CT-1) originally was discovered as a factor that can induce hypertrophy of cardiac myocytes, both in vitro and in vivo. Subsequently, CT-1 has been shown to have a wide variety of different effects on cardiac and noncardiac, cells including the ability to stimulate the survival of both cardiac and neuronal cells. Like other members of the interleukin-6 family of cytokines, CT-1 stimulates both the p42/p44 mitogen-activated protein kinase pathway and the Janus-activated kinase/signal transducers and activators of transcription pathway. Interestingly, whilst activation of the p42/p44 mitogen-activated protein kinase pathway is necessary for the survival-promoting effects of CT-1 in cardiac cells, it is not required for its hypertrophic effect, which is likely to involve activation of the Janus-activated kinase/signal transducer and activator of transcription-3 pathway. CT-1, therefore, may be of use as a novel cardioprotective agent, particularly if its hypertrophic effect can be specifically inhibited.

Animals↗
PubMed2000

Development and optimization of herpes simplex virus vectors for multiple long-term gene delivery to the peripheral nervous system.

Herpes simplex virus (HSV) has often been suggested as a suitable vector for gene delivery to the peripheral nervous system as it naturally infects sensory nerve terminals before retrograde transport to the cell body in the spinal ganglia where latency is established. HSV vectors might therefore be particularly appropriate for the study and treatment of chronic pain following vector administration by relatively noninvasive peripheral routes. However parameters allowing safe and efficient gene delivery to spinal ganglia following peripheral vector inoculation, or the long-term expression of delivered genes, have not been comprehensively studied. We have identified combinations of deletions from the HSV genome which allow highly efficient gene delivery to spinal dorsal root ganglia (DRGs) following either footpad or sciatic nerve injection. These vectors have ICP34.5 deleted and have inactivating mutations in vmw65. We also report that peripheral replication is probably necessary for the efficient establishment of latency in vivo, as fully replication-incompetent HSV vectors allow efficient gene expression in DRGs only after peripheral inoculation at a high virus dose. Very low transduction efficiencies are otherwise achieved. In parallel, promoters have been developed that allow the long-term expression of individual or pairs of genes in DRGs by using elements from the latently active region of the virus to confer a long-term activity onto a number of promoters which otherwise function only in the short term. This work further defines elements and mechanisms within the latently active region that are necessary for long-term gene expression and for the first time allows multiple inserted genes to be expressed from HSV vectors during latency.

Animals↗
PubMed2000

PKC-dependent delayed metabolic preconditioning is independent of transient MAPK activation.

In this study we used an in vitro model of delayed preconditioning to investigate activation of mitogen-activated protein kinases (MAPKs) and their potential role in protection. Neonatal rat cardiomyocytes were preconditioned using a buffer containing glycolytic inhibitors and low pH (minimal metabolic preconditioning; MMPC) consisting of modified Krebs buffer, 10 mM 2-deoxyglucose, and 20 mM lactate, pH 6.8, for 2 h followed by 24 h of simulated reperfusion before lethal simulated ischemia (LSI). MAPK activation during the MMPC protocol was determined using phospho-specific antisera and the effect on protection determined following LSI. Rapid, transient phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 MAPK was observed during each of the MMPC, reperfusion, and LSI phases; an effect blocked by MAPK inhibitors PD-98059 and SB-203580, respectively, but not by the protein kinase C (PKC) inhibitor Ro31-8220. However, although MMPC was blocked by Ro31-8220, treatment with the MAPK inhibitors during the preconditioning protocol did not block delayed protection conferred by MMPC. Thus the data suggest that, in this model of delayed preconditioning, protection appears to be PKC dependent but independent of ERK1/2 or p38 MAPK activation.

Animals↗
PubMed2000

Gene therapy using herpes simplex virus-based vectors.

Gene therapy involves the use of specific genes to treat human diseases and is thus critically dependent on efficient gene delivery systems. Although a variety of systems for such gene delivery are under development, HSV has unique advantages in terms of its large genome size and for gene delivery in the nervous system because of its ability to enter a latent state in neuronal cells. Considerable progress has been made in the effective disablement of this virus whilst retaining its ability to deliver genes and in producing long term expression of the foreign gene. Although much remains to be achieved in the further disablement of the virus and its testing in rodent and primate models of human diseases, it is likely that these viruses may ultimately be of use in human gene therapy procedures particularly for otherwise intractable neurological diseases.

Animals↗
PubMed2000

Transcription factors as potential targets for therapeutic drugs.

Although drugs which target transcription are in wide therapeutic use, they were all identified on the basis of their effect on a specific biological process such as inflammation or hormone responses and were only subsequently shown to target transcription. Our recent progress in understanding the mechanism of action of these drugs and the mechanisms of transcriptional regulation in general offers hope for a new generation of drugs isolated on the basis of their ability to modulate either the synthesis of transcription factors, the regulation of their activity by ligands or phosphorylation events, their protein-protein interactions or their binding to DNA.

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