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Biomedical subjects

D J Fink

Publications and source records attributed to D J Fink.

At least 19 recordsLinked to original sources
PubMed1999

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo.

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson's disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson's disease.

Animals↗
PubMed1999

Herpes simplex virus type 1 vector-mediated expression of nerve growth factor protects dorsal root ganglion neurons from peroxide toxicity.

Nerve growth factor beta subunit (beta-NGF) transgene delivery and expression by herpes simplex virus type 1 (HSV-1) vectors was examined in a cell culture model of neuroprotection from hydrogen peroxide toxicity. Replication-competent (tk- K mutant background) and replication-defective (ICP4(-);tk- S mutant background) vectors were engineered to contain the murine beta-NGF cDNA under transcriptional control of either the human cytomegalovirus immediate-early gene promoter (HCMV IEp) (e.g., KHN and SHN) or the latency-active promoter 2 (LAP2) (e.g., KLN and SLN) within the viral thymidine kinase (tk) locus. Infection of rat B103 and mouse N2A neuronal cell lines, 9L rat glioma cells, and Vero cells with the KHN or SHN vectors resulted in the production of beta-NGF-specific transcripts and beta-NGF protein reaching a maximum at 3 days postinfection (p.i.). NGF protein was released into the culture media in amounts ranging from 10.83 to 352.86 ng/ml, with the highest levels being achieved in B103 cells, and was capable of inducing neurite sprouting of PC-12 cells. The same vectors produced high levels of NGF in primary dorsal root ganglion (DRG) cultures at 3 days. In contrast to HCMV IEp-mediated expression, the LAP2-NGF vectors showed robust expression in primary DRG neurons at 14 days. The neuroprotective effect of vector produced NGF was assessed by its ability to inhibit hydrogen peroxide-induced neuron toxicity in primary DRG cultures. Consistent with the kinetics of vector-mediated NGF expression, HCMV-NGF vectors were effective in abrogating the toxic effects of peroxide at 3 but not 14 days p.i. whereas LAP2-NGF vector transduction inhibited apoptosis in DRG neurons at 14 days p.i. but was ineffective at 3 days p.i. Similar kinetics of NGF expression were observed with the KHN and KLN vectors in latently infected mouse trigeminal ganglia, where high levels of beta-NGF protein expression were detected at 4 wks p.i. only from the LAP2; HCMV-NGF-driven expression peaked at 3 days but could not be detected during HSV latency at 4 weeks. Together, these results indicate that (i) NGF vector-infected cells produce and secrete mature, biologically active beta-NGF; (ii) vector-synthesized NGF was capable of blocking peroxide-induced apoptosis in primary DRG cultures; and (iii) the HCMV-IEp functioned to produce high levels of NGF for several days; but (iv) only the native LAP2 was capable of long-term expression of a therapeutic gene product in latently infected neurons in vivo.

Animals↗
PubMed1999

Multicenter clinical and analytical evaluation of the AxSYM troponin-I immunoassay to assist in the diagnosis of myocardial infarction.

We evaluated the AxSYM troponin I (cTnI) immunoassay for assisting in the detection of acute myocardial infarction (AMI). At four sites, the total imprecision (CV) over 20 days was 6.3-10.2%. The minimum detectable concentration was 0.14 +/- 0.05 microgram/L. Comparison of cTnI measurements between the AxSYM and Stratus (n = 406) over the dynamic range of the AxSYM assay demonstrated good correlation, r = 0.881, with a proportional bias: AxSYM cTnI = 3.50(Stratus cTnI) - 1. 10. The confidence intervals (95%) for the slope and intercept were 3.39-3.64 and -1.32 to -0.95, respectively. The expected cTnI concentration in healthy individuals was </=0.5 microgram/L, whereas the ROC curve-determined cutoff for AMI was 2.0 microgram/L. This gave a diagnostic sensitivity of 91.8% and specificity of 92.4% when tested in serial samples collected within 24 h of admission in 633 patients presenting with chest pain, of which 122 had an AMI. The concordances of the AxSYM cTnI with the Stratus cTnI, OPUS cTnI, and Access cTnI were 95.3%, 95.1%, and 94.3%, respectively, from patients with suspected AMI. The AxSYM cTnI demonstrated excellent clinical specificity, >/=96%, in skeletal muscle injury, chronic renal disease, and same-day noncardiac surgery patients.

Evaluation Studies as Topic↗
PubMed1998

Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair.

This investigation tested the hypothesis that delivering mesenchymal stem cell-seeded implants to a tendon gap model results in significantly improved repair biomechanics. Cultured, autologous, marrow-derived mesenchymal stem cells were suspended in a collagen gel delivery vehicle; the cell-gel composite was subsequently contracted onto a pretensioned suture. The resulting tissue prosthesis was then implanted into a 1-cm-long gap defect in the rabbit Achilles tendon. Identical procedures were performed on the contralateral tendon, but only the suture material was implanted. The tendon-implant constructs were evaluated 4, 8, and 12 weeks later by biomechanical and histological criteria. Significantly greater load-related structural and material properties were seen at all time intervals in the mesenchymal stem cell-treated tendons than in the contralateral, treated control repairs (p < 0.05), which contained suture alone with natural cell recruitment. The values were typically twice those for the control tissues at each time interval. Load-related material properties for the treated tissues also increased significantly over time (p < 0.05). The treated tissues had a significantly larger cross-sectional area (p < 0.05), and their collagen fibers appeared to be better aligned than those in the matched controls. The results indicate that delivering mesenchymal stem cell-contracted, organized collagen implants to large tendon defects can significantly improve the biomechanics, structure, and probably the function of the tendon after injury.

Achilles Tendon↗
PubMed1998

Drug inducible transgene expression in brain using a herpes simplex virus vector.

The ability to regulate transgene expression is likely to be important in the use of gene transfer to treat diseases of the central nervous system (CNS). In order to achieve regulatable gene expression we created a replication-incompetent genomic herpes simplex vector containing a RU486-inducible transactivator and a lacZ reporter gene under transcriptional control of a minimal promoter. Reporter gene expression from the vector was regulated by administration of RU486 in vitro and in vivo. In cell culture half maximal expression was achieved with 10(-8) M RU486, and maximal expression was achieved by 24 h. Following stereotactic inoculation of the vector into rat hippocampus, expression was increased 150-fold by i.p. administration of RU486. This demonstrates that the RU486 system functions as a tight on/off switch for regulating expression of a transgene delivered to the brain via an HSV vector.

Animals↗
PubMed1998

Development of herpes simplex virus replication-defective multigene vectors for combination gene therapy applications.

Some gene therapy applications will require simultaneous expression of multiple gene products to achieve a therapeutic effect. In this study we describe the generation and characterization of replication incompetent herpes simplex virus type 1 (HSV-1) vectors (HX86Z or HX86G) carrying distinct and independently regulated expression cassettes for five transgenes (hIL-2, hGM-CSF, hB7.1, HSV-tk and lacZ or hIFN gamma). The transgenes, representing 12 kb of DNA sequence, were recombined into separate loci of a single mutant virus vector deleted for 11.6 kb of vector sequences representing portions of nine viral genes, ICP4, ICP22, ICP27, ICP47, UL24, UL41, UL44, US10 and US11. Deletion of the immediate--early genes ICP4, ICP22 and ICP27 substantially reduced vector cytotoxicity, prevented early and late viral gene expression and left intact MHC class I antigen expression. Simultaneous expression of multiple transgenes was obtained for up to 7 days in primary human melanoma cells with peak expression at 2-3 days after infection. The transgenes were chosen for their potential to function synergistically in tumor destruction and vaccine gene therapy applications, but the method and vector employed could be applied to other multigene therapy strategies. This study demonstrates the potential for engineering large transgene capacity DNA viruses such as HSV-1 for expression of multiple transgenes.

Defective Viruses↗
PubMed1998

Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons.

Herpes simplex virus type 1 (HSV-1) has many attractive features that suggest its utility for gene transfer to neurons. However, viral cytotoxicity and transient transgene expression limit practical applications even in the absence of viral replication. Mutant viruses deleted for the immediate early (IE) gene, ICP4, an essential transcriptional transactivator, are toxic to many cell types in culture in which only the remaining IE genes are expressed. In order to test directly the toxicity of other IE gene products in neurons and develop a mutant background capable of longterm transgene expression, we generated mutants deleted for multiple IE genes in various combinations and tested their relative cytotoxicity in 9L rat gliosarcoma cells, Vero monkey kidney cells, and primary rat cortical and dorsal root neurons in culture. Viral mutants deleted simultaneously for the IE genes encoding ICP4, ICP22 and ICP27 showed substantially reduced cytotoxicity compared with viruses deleted for ICP4 alone or ICP4 in combination with either ICP22, ICP27 or ICP47. Infection of neurons in culture with these triple IE deletion mutants substantially enhanced cell survival and permitted transgene expression for over 21 days. Such mutants may prove useful for efficient gene transfer and extended transgene expression in neurons in vitro and in vivo.

Animals↗
PubMed1997

Development of an HSV-based vector for the treatment of Parkinson's disease.

The restricted pattern of neurodegeneration seen in Parkinson's disease, and the identification of trophic factors that prevent toxin-induced degeneration of dopaminergic neurons, has spurred research into potential gene therapy for this disease. Herpes simplex virus (HSV-1) is a neurotrophic virus which naturally establishes latency in neurons. HSV-based vectors have been demonstrated to transfer and transiently express transgenes in neurons in brain in vivo. Recent experiment have shown that deletion of multiple immediate-early HSV genes reduces the potential cytotoxicity of these vectors, and in addition results in altered patterns of transgene expression that may allow for long-term expression required for human gene therapy applications.

Gene Expression↗
PubMed1997

Modulation of phosphorylation of neuronal cytoskeletal proteins by neuronal depolarization.

1. The neuronal cytoskeletal protein tau and the carboxy tails of cytoskeletal proteins neurofilament-M (NF-M) and neurofilament-H (NF-H) are phosphorylated on serine residues by the cyclin-dependent kinase cdk-5. 2. In aggregating neuronal-glial cultures we show that veratridine-mediated cation influx causes dephosphorylation of tau, NF-M and NF-H. Dephosphorylation was blocked specifically by cyclosporine A but not by okadiac acid at concentrations up to 200 nM. 3. These results suggest that veratridine-triggered cation influx causes activation of PP-2B (calcineurin) leading to dephosphorylation of these cytoskeletal proteins.

Animals↗
PubMed1997

Engineering herpes simplex virus vectors for gene transfer to neurons.

HSV has a unique relationship with neurons which the virus naturally utilizes for efficient gene transfer and long-term gene expression. Progress in developing defective viral mutants with reduced cytotoxicity, and increasing insight into the state of the viral genome during latency and the functional elements of the latency promoter system, suggest that HSV vectors may be developed in which these natural features are exploited for effective transgene expression in brain.

Animals↗
PubMed1997

Rapid method for construction of recombinant HSV gene transfer vectors.

Herpes simplex virus type 1 (HSV-1) is a neurotrophic human pathogen that naturally persists in neurons in a latent state and carries a large number of viral functions which can be replaced by foreign genes to create a vector for gene therapy applications. In this report we describe a two-step method for insertion/deletion mutagenesis of HSV genes and the efficient insertion of transgenes into these locations in the viral genome. The first step is the insertion of a reporter gene (lacZ) cassette flanked by Pacl restriction enzyme sites not otherwise found in the viral genome, using standard marker transfer procedures to interrupt a portion of the target HSV gene. The second step is substitution of the reporter gene with other foreign cDNAs by digestion of the vector DNA with Pacl to remove the lacZ gene and subsequent repair of the vector genome by homologous recombination with a transgene expression plasmid. Potential recombinants identified by a 'clear plaque' phenotype after X-gal staining arose at high frequency (80-100%). Of these recombinants containing the transgene in place of the lacZ gene ranged from 19-65%. Insertion of the transgene expression construct into the viral genome eliminates the Pacl sites, allowing this method to be used repeatedly for the sequential deletion of multiple HSV genes while inserting multiple transgenes. This procedure was repeated in succession to produce a vector carrying two independent expression cassettes at distinct viral loci.

DNA, Viral↗
PubMed1997

Deletion of the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5 from the herpes simplex virus type 1 genome substantially impairs productive viral infection in cell culture and pathogenesis in the rat central nervous system.

A distinctive feature of the genetic make-up of herpes simplex virus type 1 (HSV-1), a human neurotropic virus, is that approximately half of the 81 known viral genes are not absolutely required for productive infection in Vero cells, and most can be individually deleted without substantially impairing viral replication in cell culture. If large blocks of contiguous viral genes could be replaced with foreign DNA sequences, it would be possible to engineer highly attenuated recombinant HSV-1 gene transfer vectors capable of carrying large cellular genes or multiple genes having related functions. We report the isolation and characterization of an HSV-1 mutant, designated d311, containing a 12 kb deletion of viral DNA located between the L-S Junction a sequence and the U(S)6 gene, spanning the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5. Replication of d311 was totally inhibited in rat B103 and mouse Neuro-2A neuroblastoma cell lines, and was reduced by over three orders of magnitude in human SK-N-SH neuroblastoma cells compared to wild-type (wt) HSV-1 KOS. This suggested that the deleted genes, while nonessential for replication in Vero cells, play an important role in HSV replication in neuronal cells, particularly those of rodent origin. Unlike wt KOS which replicated locally and spread to other regions of brain following stereotactic inoculation into rat hippocampus, d311 was unable to replicate and spread within the brain, and did not cause any apparent local neuronal cell damage. These results demonstrate that d311 is highly attenuated for the rat central nervous system. d311 and other mutants of HSV containing major deletions of the nonessential genes within U(S) have the potential to serve as useful tools for gene transfer applications to brain.

Animals↗
PubMed1997

Herpes simplex virus vectors for gene transfer to the nervous system.

Herpes simplex virus (HSV) represents a candidate gene transfer vector for the treatment of nervous system disease. It has many natural biological features which make it attractive for gene delivery to a variety of tissues. The virus naturally establishes a latency in sensory neurons of the peripheral nervous system, wherein the virus in maintained as an extrachromosomal DNA element in the absence of viral lytic gene expression without altering the metabolism of the host neuron. The virus possesses a neuronal latency-specific promoter system which remains active long-term, while other viral and cellular promoters are repressed. Replication defective virus recombinants have been engineered to delete multiple essential immediate early gene functions rendering these new mutants significantly less cytotoxic to neurons and other cells in culture. Further developments in regulating transgene expression and reducing virus toxicity will continue to aid the design and use of these vectors for therapeutic applications for the nervous system.

Animals↗
PubMed1996

cis-acting elements involved in transcriptional regulation of the herpes simplex virus type 1 latency-associated promoter 1 (LAP1) in vitro and in vivo.

Latency-associated promoter 1 (LAP1) of herpes simplex virus type 1 is required to generate a series of latency-associated transcripts (LATs) in sensory neurons of latently infected animals. Sequence analysis and DNA binding studies have suggested the existence of several cis-acting elements within LAP1 that are potentially important for promoter function, although their role in LAT gene expression during latency is largely unexplored. In this report, we present evidence that the LAP1 TATA box is essential for transcription initiation in vitro. A reduction in LAT synthesis measured by in situ hybridization and reverse transcription-PCR (RT-PCR) of rat brain tissue latently infected with a LAP1 TATA substitution virus demonstrated that this sequence was required for full LAP1 activity in vivo. Analysis of additional site-directed and 5'-deletion mutants of LAP1 by in vitro transcription-primer extension assays showed that upstream elements including the USF and cyclic AMP response element (CRE) site specifically contributed to LAP1 function and that sequences beginning at position -620 relative to the transcription start site were essential for full promoter activity. The combination of deleting USF, CRE, and TATA completely abolished LAT expression in the brain, identifying these as essential elements for the neuron-specific functioning of LAP1 during latency. Mutation of the transcription start site did not abolish transcription, suggesting the absence of an initiator element. However, one of the most exciting findings from this study is that the region downstream of the TATA box appears to contain a true enhancer that is not only essential for transcription, but also functional when positioned 1.6 kb downstream of the start site of transcription. It was concluded that (i) the TATA box was essential for full transcriptional activity from LAP1 both in vitro and in vivo, (ii) the USF element and CRE contribute to LAP1 function during latency in combination with the TATA element, (iii) multiple trans-acting factors besides the USF- and CRE-binding proteins were required for full promoter activity in vitro, and (iv) sequences downstream of the TATA box enhanced promoter activity in vitro.

Animals↗