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D Wolfe

Publications and source records attributed to D Wolfe.

At least 19 recordsLinked to original sources
PubMed2005

HSV vector-mediated transduction and GDNF secretion from adipose cells.

The accessibility of adipose tissue and its ability to secrete various bioactive molecules suggest that adipose cells may be attractive targets for gene therapy applications. Here, we report the use of highly defective herpes simplex virus (HSV) vectors as suitable gene transfer agents for adipose cells in culture and fat tissue in animals. Using an in vitro model of human adipose differentiation, we first demonstrated that mature adipocytes and their precursor cells express the two principal HSV viral entry receptors HveA and HveC (nectin-1) and are efficiently transduced at a low multiplicity of infection by HSV-lacZ reporter gene and glial cell line-derived neurotrophic factor (GDNF) gene vectors. Extended expression of beta-galactosidase and secretion of GDNF occurred in transduced fat tissue explants from rabbits. In vivo gene transfer to rabbit subcutaneous adipose tissue resulted in local GDNF expression for at least 2 months. These experiments establish the efficient transduction of adipose cells by HSV vectors and suggest that fat tissue may represent a useful site for HSV-mediated gene delivery with potential for therapeutic applications.

Adipocytes↗
PubMed2005

HSV-mediated gene transfer of vascular endothelial growth factor to dorsal root ganglia prevents diabetic neuropathy.

We examined the utility of herpes simplex virus (HSV) vector-mediated gene transfer of vascular endothelial growth factor (VEGF) in a mouse model of diabetic neuropathy. A replication-incompetent HSV vector with VEGF under the control of the HSV ICP0 promoter (vector T0VEGF) was constructed. T0VEGF expressed and released VEGF from primary dorsal root ganglion (DRG) neurons in vitro, and following subcutaneous inoculation in the foot, expressed VEGF in DRG and nerve in vivo. At 2 weeks after induction of diabetes, subcutaneous inoculation of T0VEGF prevented the reduction in sensory nerve amplitude characteristic of diabetic neuropathy measured 4 weeks later, preserved autonomic function measured by pilocarpine-induced sweating, and prevented the loss of nerve fibers in the skin and reduction of neuropeptide calcitonin gene-related peptide and substance P in DRG neurons of the diabetic mice. HSV-mediated transfer of VEGF to DRG may prove useful in treatment of diabetic neuropathy.

Animals↗
PubMed2004

Safety and biodistribution studies of an HSV multigene vector following intracranial delivery to non-human primates.

Malignant glioma is a fatal human cancer in which surgery, chemo- and radiation therapies are ineffective. Therapeutic gene transfer used in combination with current treatment methods may augment their effectiveness with improved clinical outcome. We have shown that NUREL-C2, a replication-defective multigene HSV-based vector, is effective in treating animal models of glioma. Here, we report safety and biodistribution studies of NUREL-C2 using rhesus macaques as a model host. Increasing total doses (1 x 10(7) to 1 x 10(9) plaque forming units (PFU)) of NUREL-C2 were delivered into the cortex with concomitant delivery of ganciclovir (GCV). The animals were evaluated for changes in behavior, alterations in blood cell counts and chemistry. The results showed that animal behavior was generally unchanged, although the chronic intermediate dose animal became slightly ataxic on day 12 postinjection, a condition resolved by treatment with aspirin. The blood chemistries were unremarkable for all doses. At 4 days following vector injections, magnetic resonance imaging showed inflammatory changes at sites of vector injections concomitant with HSV-TK and TNFalpha expression. The inflammatory response was reduced at 14 days, resolving by 1 month postinjection, a time point when transgene expression also became undetectable. Immunohistochemical staining following animal killing showed the presence of a diffuse low-grade gliosis with infiltrating macrophages localized to the injection site, which also resolved by 1 month postinoculation. Viral antigens were not detected and injected animals did not develop HSV-neutralizing antibodies. Biodistribution studies revealed that vector genomes remained at the site of injection and were not detected in other tissues including contralateral brain. We concluded that intracranial delivery of 1 x 10(9) PFU NUREL-C2, the highest anticipated patient dose, was well tolerated and should be suitable for safety testing in humans.

Animals↗
PubMed2002

Measurement of analyzing power for proton-carbon elastic scattering in the coulomb-nuclear interference region with a 22-GeV/c polarized proton beam.

The analyzing power for proton-carbon elastic scattering in the Coulomb-nuclear interference region of momentum transfer, 9.0x10(-3)<-t<4.1x10(-2) (GeV/c)(2), was measured with a 21.7 GeV/c polarized proton beam at the Alternating Gradient Synchrotron of Brookhaven National Laboratory. The ratio of hadronic spin-flip to nonflip amplitude, r(5), was obtained from the analyzing power to be Rer(5)=0.088+/-0.058 and Imr(5)=-0.161+/-0.226.

Journal Article↗
PubMed2002

Double suicide gene therapy using a replication defective herpes simplex virus vector reveals reciprocal interference in a malignant glioma model.

Herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli cytosine deaminase (CD) are non-mammalian enzymes capable of converting innocuous prodrugs into cytotoxic metabolites. Both enzymes have been utilized independently, as well as together in 'suicide' gene therapy protocols to eliminate tumor cells in vitro and in vivo. We have used a set of replication defective HSV vectors expressing either or both enzymes to compare the efficacies of single and double suicide gene therapies in the 9L gliosarcoma model in vitro and in vivo. In cell culture experiments at high and low multiplicities of infection, combined expression of the two genes by vector TOCD/TK along with exposure to the matching prodrugs (ganciclovir and 5-fluorocytosine) showed increased cytotoxicity compared with exposure to either prodrug alone. However, the two gene combination was inferior to single gene treatments, suggesting that HSVtk and CD are mutually counteractive in the prodrug-dependent killing of glioma cells. In animal experiments, survival was not significantly prolonged by administration of both prodrugs to TOCD/TK-treated animals, while each single gene/prodrug pair resulted in increased survival. These results indicate that single suicide gene systems employing HSVtk or CD may be preferable over combinations of the two.

Animals↗
PubMed2001

Bcl-2 and GDNF delivered by HSV-mediated gene transfer act additively to protect dopaminergic neurons from 6-OHDA-induced degeneration.

Previous studies have demonstrated that either the neurotrophin glial-derived neurotrophic factor (GDNF) or the antiapoptotic peptide Bcl-2 delivered into striatum by a viral vector protects dopaminergic neurons of the substantia nigra in vivo from degeneration induced by the administration of the neurotoxin 6-hydroxydopamine (6-OHDA). In this study we used recombinant, replication-incompetent, genomic herpes simplex virus-based vectors to deliver the genes coding for Bcl-2 and GDNF into rat substantia nigra (SN) 1 week prior to 6-OHDA injection into the striatum. Vector-mediated expression of either Bcl-2 or GDNF alone each resulted in a doubling in cell survival as measured by retrograde labeling with fluorogold (FG) and a 50% increase in tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the lesioned SN compared to the unlesioned side. Gene transfer of Bcl-2 and GDNF were equivalent in this effect. Coadministration of the Bcl-2-expressing vector with the GDNF-expressing vector improved the survival of lesioned SN neurons as measured by FG labeling by 33% and by the expression of TH-IR by 15%. These results suggest that the two factors delivered together act in an additive fashion to improve DA cell survival in the face of 6-OHDA toxicity.

Animals↗
PubMed2001

Herpesvirus-mediated systemic delivery of nerve growth factor.

Sustained systemic dissemination of therapeutic proteins from peripheral sites is an attractive prospect for gene therapy applications. Replication-defective genomic herpes simplex virus type 1 (HSV-1) vectors were evaluated for their ability to express nerve growth factor (NGF) as a model gene product both locally and systemically. Intra-articular inoculation of NGF expression vectors in rabbits resulted in significant increases in joint lavage and blood plasma NGF that persisted for 1 year. A rhesus macaque injected intra-articularly displayed a comparable increase in plasma NGF for at least 6 months, at which time the serum NGF levels of this animal were sufficient to cause differentiation of PC12 cells in culture, but not to increase footpad epidermis innervation. Long-term reporter transgene expression was observed primarily in ligaments, a finding confirmed by direct inoculation of patellar ligament. Patellar ligament inoculation with a NGF vector resulted in elevated levels of circulating NGF similar to those observed following intra-articular vector delivery. These results represent the first demonstration of sustained systemic release of a transgene product using HSV vectors, raising the prospect of new applications for HSV-1 vectors in the treatment of systemic disease.

Animals↗
PubMed2001

Cerebral effects of cold reperfusion after hypothermic circulatory arrest.

OBJECTIVES: This study was undertaken to explore whether an interval of cold reperfusion can improve cerebral outcome after prolonged hypothermic circulatory arrest. METHODS: Sixteen pigs (27-30 kg) underwent 90 minutes of circulatory arrest at a brain temperature of 20 degrees C. Eight animals were rewarmed immediately after hypothermic circulatory arrest (controls), and 8 were reperfused for 20 minutes at 20 degrees C and then rewarmed (cold reperfusion). Electrophysiologic recordings, fluorescent microsphere determinations of cerebral blood flow, calculations of cerebral oxygen consumption, and direct measurements of intracranial pressure (millimeters of mercury) were obtained at baseline (37 degrees C), before hypothermic circulatory arrest, after discontinuing circulatory arrest at 37 degrees C deep brain temperature, and at 2, 4, and 6 hours thereafter. Histopathologic features and percent brain water were determined after the animals were sacrificed. RESULTS: Cerebral blood flow and oxygen consumption decreased during cooling: cerebral oxygen consumption returned to baseline levels after 4 hours, but cerebral blood flow remained depressed until 6 hours in both groups. Cold reperfusion failed to improve electrophysiologic recovery or to reduce brain weight, but median intracranial pressure increased significantly less after cold reperfusion than in controls (P =.02). Although no significant difference in the incidence of histopathologic abnormalities between groups was found, all 3 animals with an intracranial pressure of more than 15 mm Hg after immediate rewarming had histopathologic lesions, and high intracranial pressure was more prevalent among all animals with subsequent histopathologic lesions (P =.03). CONCLUSIONS: Cold reperfusion significantly inhibited the rise in intracranial pressure seen in control pigs after 90 minutes of circulatory arrest at 20 degrees C, suggesting that cold reperfusion may decrease cerebral edema and thereby improve outcome after prolonged hypothermic circulatory arrest.

Animals↗
PubMed2001

Multiple maltreatment, attribution of blame, and adjustment among adolescents.

The study examined the predictive utility of blame attributions for maltreatment. Integrating theory and research on blame attribution, it was predicted that self-blame would mediate or moderate internalizing problems, whereas other-blame would mediate or moderate externalizing problems. Mediator and moderator models were tested separately. Adolescents (N = 160, ages 11-17 years) were randomly selected from the open caseload of a child protection agency. Participants made global maltreatment severity ratings for each of physical abuse, psychological abuse, neglect. sexual abuse, and exposure to family violence. Participants also completed the Attribution for Maltreatment Interview (AFMI), a structured clinical interview that assessed self- and perpetrator blame for each type of maltreatment they experienced. The AFMI yielded five subscales: self-blaming cognition, self-blaming affect, self-excusing. perpetrator blame, and perpetrator excusing. Caretaker-reported (Child Behavior Checklist) and self-reported (Youth Self Report) internalizing and externalizing were the adjustment criteria. Controlling for maltreatment severity, the AFMI subscales explained significant variance in self-reported adjustment. Self-blaming affect was the most potent attribution, particularly among females. Attributions mediated maltreatment severity for self-reported adjustment but moderated it for caretaker-reported adjustment. The sophistication and relevance of blame attributions to adjustment are discussed, and implications for research and clinical practice are identified.

Adaptation, Psychological↗
PubMed2000

An indoleamine 2,3-dioxygenase-negative mutant is defective in stat1 DNA binding: differential response to IFN-gamma and IFN-alpha.

We have previously reported the isolation of mutant cell lines from the human carcinoma line ME180 that are resistant to the antiproliferative effect of interferon-gamma (IFN-gamma). These cell lines were defective in the induction of indoleamine 2,3-dioxygenase (IDO), a key enzyme of tryptophan catabolism. One of these cell lines, 3B6A, was chosen for further study. This cell line was also defective in the ability of IFN-gamma to protect against vesicular stomatitis virus (VSV) infection. However it maintained a normal antiviral response to IFN-alpha. A promoter-chloramphenicol acetyltransferase (CAT) construct containing the promoter region of IDO, which includes IFN-gamma activation site (GAS), IFN-stimulated response element-1 (ISRE-1), and ISRE-2 regions, was not expressed in 3B6A in the presence of IFN-gamma, indicating that the defect was likely to be in either Stat1 or IFN regulatory factor-1 (IRF-1), transcription factors known to bind to these cis-acting sequences. The induction of other IFN-gamma-inducible genes, such as tryptophanyl-tRNA synthetase (hWRS), was also affected. Electrophoretic mobility shift assays (EMSA) comparing nuclear extracts from parental and mutant cells indicated that Stat1 from the mutant did not bind to GAS sequences. However, Western blot analysis indicated that Stat1 protein was present. This IDO-negative phenotype can be reversed by transfection with a Stat1 expression vector. DNA sequencing of the Stat1 cDNA from wild-type and 3B6A cells indicated that an amino acid change occurred in the Stat1 protein of the mutant at W573, a tryptophan conserved in all known Stat proteins. We hypothesize that a change in this region of the Stat protein affects the response to IFN-gamma but not to IFN-alpha.

Chloramphenicol O-Acetyltransferase↗
PubMed2000

Herpes simplex virus type 1 ICP0 protein does not accumulate in the nucleus of primary neurons in culture.

Infected-cell protein 0 (ICP0), the product of the herpes simplex virus (HSV) immediate-early (IE) alpha0 gene, is a promiscuous transactivator of viral early (E) and late (L) gene expression. HSV mutants lacking ICP0 function are severely deficient in viral growth and protein synthesis, particularly at low multiplicities of infection. Early in the infectious process in vitro, ICP0 protein accumulates in distinct domains within the nucleus to form characteristic structures active in the transcription of viral genes. However, following infection of primary trigeminal ganglion cells in vitro with a recombinant HSV mutant that expresses only ICP0, we observed that ICP0 protein accumulated in the characteristic intranuclear distribution only in the nuclei of Schwann cells; neurons in the culture did not accumulate ICP0 despite expression of ICP0 RNA in those cells. The same phenomenon was observed in PC12 cells differentiated to assume a neuronal phenotype. In primary neurons in culture, the amount of ICP0 protein could be increased by pharmacologic inhibition of calcium-activated protease (calpain) activity or by inhibition of protein phosphatase 2B (calcineurin). The failure of ICP0 protein to accumulate in the nucleus of neurons suggests that one mechanism which may impair efficient replication of the virus in neurons, and thus favor the establishment of viral latency in those cells, may be found in the cell-specific processing of that IE gene product.

Animals↗
PubMed1999

Intra-articular delivery of a herpes simplex virus IL-1Ra gene vector reduces inflammation in a rabbit model of arthritis.

To evaluate the use of HSV-based vectors for arthritis gene therapy we have constructed a first-generation, ICP4 deficient, replication defective herpes simplex virus (HSV) vector (S/0-) and a second-generation HSV vector derivative (T/0-) deficient for the immediate-early genes ICP4, 22 and 27, each carrying a soluble TNF receptor or IL-1 receptor antagonist transgene cassette. A rabbit synovial-fibroblast line in culture, infected by either vector enabled high-level expression of the transgene product. However, following a single intra-articular injection of the vectors into rabbit knee joints, only the second-generation, HSV T/0- vector expressed detectable levels of soluble TNFR in synovial fluid. Synovial lavage fluid from inoculated joints con- tained up to 12 ng/ml of soluble receptor that persisted at detectable, but reduced levels for at least 7 days. When tested in an experimental model of arthritis generated by intra-articular overexpression of interleukin-1beta using retrovirus transduced synovial cells, the HSV T/0- vector expressing the interleukin-1 receptor antagonist was found to inhibit leukocytosis and synovitis significantly. The improved levels and duration of intra-articular transgene expression achieved via HSV-mediated gene delivery suggest that an HSV vector system could be used for therapeutic applications in patients with rheumatoid arthritis (RA) and other joint-related inflammatory diseases.

Adoptive Transfer↗
PubMed1999

Ubiquitin metabolism affects cellular response to volatile anesthetics in yeast.

To investigate the mechanism of action of volatile anesthetics, we are studying mutants of the yeast Saccharomyces cerevisiae that have altered sensitivity to isoflurane, a widely used clinical anesthetic. Several lines of evidence from these studies implicate a role for ubiquitin metabolism in cellular response to volatile anesthetics: (i) mutations in the ZZZ1 gene render cells resistant to isoflurane, and the ZZZ1 gene is identical to BUL1 (binds ubiquitin ligase), which appears to be involved in the ubiquitination pathway; (ii) ZZZ4, which we previously found is involved in anesthetic response, is identical to the DOA1/UFD3 gene, which was identified based on altered degradation of ubiquitinated proteins; (iii) analysis of zzz1Delta zzz4Delta double mutants suggests that these genes encode products involved in the same pathway for anesthetic response since the double mutant is no more resistant to anesthetic than either of the single mutant parents; (iv) ubiquitin ligase (MDP1/RSP5) mutants are altered in their response to isoflurane; and (v) mutants with decreased proteasome activity are resistant to isoflurane. The ZZZ1 and MDP1/RSP5 gene products appear to play important roles in determining effective anesthetic dose in yeast since increased levels of either gene increases isoflurane sensitivity whereas decreased activity decreases sensitivity. Like zzz4 strains, zzz1 mutants are resistant to all five volatile anesthetics tested, suggesting there are similarities in the mechanisms of action of a variety of volatile anesthetics in yeast and that ubiquitin metabolism affects response to all the agents examined.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Retrograde cerebral perfusion enhances cerebral protection during prolonged hypothermic circulatory arrest: a study in a chronic porcine model.

BACKGROUND: This study was undertaken to confirm earlier findings that retrograde cerebral perfusion (RCP) can improve cerebral outcome after prolonged hypothermic circulatory arrest (HCA), and to determine whether RCP with inferior vena caval occlusion, which is more effective in removing particulate emboli, is superior to conventional RCP in enhancing cerebral protection. METHODS: Sixty-two pigs (27 to 30 kg) were randomly assigned to undergo one of the following for 90 minutes at 20 degrees C: antegrade cerebral perfusion (ACP); conventional RCP (RCP); RCP with occlusion of the inferior vena cava (RCP-O), or HCA with the head packed in ice. RCP flow was regulated to a sagittal sinus pressure of 20 mm Hg. Hemodynamic, electrophysiologic, and metabolic monitoring were carried out until 4 hours after rewarming, daily behavioral and neurologic assessments until elective sacrifice on day 7, and histologic analysis of the brain after death. RESULTS: Complete behavioral recovery was seen in all surviving animals by day 5 after ACP or RCP, but in only 83% after RCP-O and 50% after HCA (p = 0.001). A histopathologic score of 2 or more, indicating more than mild injury, was not found in any animal after ACP, in 27% after RCP, in 47% after HCA, and in 68% after RCP-O (p = 0.002). The median oxygen consumption was 6.66 mL/min after ACP, 1.37 mL/min with RCP, and 1.02 mL/min with RCP-O (p < 0.0001). The median amount of fluid sequestered was 2,450 mL after RCP-O, 760 mL after RCP, and -200 mL after ACP (p < 0.0001). CONCLUSIONS: Conventional RCP without inferior vena caval occlusion results in a significantly better outcome than RCP-O after prolonged HCA, despite more efficient cerebral perfusion during RCP-O, and also provides cerebral protection superior to prolonged HCA alone, but care must be taken during its implementation to minimize cerebral edema and other possible causes of retroperfusion-related cerebral injury.

Animals↗
PubMed1998

Can retrograde perfusion mitigate cerebral injury after particulate embolization? A study in a chronic porcine model.

OBJECTIVE: We assessed the impact on histologic and behavioral outcome of an interval of retrograde cerebral perfusion after arterial embolization, comparing retrograde cerebral perfusion with and without inferior vena caval occlusion with continued antegrade perfusion. METHODS: Sixty Yorkshire pigs (27 to 30 kg) were randomly assigned to the following groups: antegrade cerebral perfusion control; antegrade cerebral perfusion after embolization; retrograde cerebral perfusion control; retrograde cerebral perfusion after embolization; retrograde cerebral perfusion with inferior vena cava occlusion, retrograde cerebral perfusion with inferior vena cava occlusion control, and retrograde cerebral perfusion with inferior vena cava occlusion after embolization. After cooling to 20 degrees C, a bolus of 200 mg of polystyrene microspheres 250 to 750 (microm diameter (or saline solution) was injected into the isolated aortic arch. After 5 minutes of antegrade cerebral perfusion, 25 minutes of antegrade cerebral perfusion, retrograde cerebral perfusion, or retrograde cerebral perfusion with inferior vena cava occlusion was instituted. After the operation, all animals underwent daily assessment of neurologic status until the time of death on day 7. RESULTS: Aortic arch return, cerebral vascular resistance, and oxygen extraction data during retrograde cerebral perfusion showed differences, suggesting that more effective flow occurs during retrograde cerebral perfusion with inferior vena cava occlusion, which also resulted in more pronounced fluid sequestration. Microsphere recovery from the brain revealed significantly fewer emboli after retrograde cerebral perfusion with inferior vena cava occlusion. Behavioral scores showed full recovery in all but one control animal (after retrograde cerebral perfusion with inferior vena cava occlusion) by day 7 but were considerably lower after embolization, with no significant differences between groups. The extent of histopathologic injury was not significantly different among embolized groups. Although no histopathologic lesions were present in either the antegrade cerebral perfusion control group or the retrograde cerebral perfusion control group, mild significant ischemic damage occurred after retrograde cerebral perfusion with inferior vena cava occlusion even in control animals. CONCLUSIONS: Although effective washout of particulate emboli from the brain can be achieved with retrograde cerebral perfusion with inferior vena cava occlusion, no advantage of retrograde cerebral perfusion with inferior vena cava occlusion after embolization is seen from behavioral scores, electroencephalographic recovery, or histopathologic examination; retrograde cerebral perfusion with inferior vena cava occlusion results in greater fluid sequestration and mild histopathologic injury even in control animals. Retrograde cerebral perfusion with inferior vena cava occlusion shows clear promise in the management of embolization, but further refinements must be sought to address its still worrisome potential for harm.

Animals↗
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↗