Biomedical subjects

D R Hinton

Publications and source records attributed to D R Hinton.

At least 55 records · Page 3Linked to original sources

PubMed1998

Expression of hemagglutinin/esterase by a mouse hepatitis virus coronavirus defective-interfering RNA alters viral pathogenesis.

A defective-interfering (DI) RNA of mouse hepatitis virus (MHV) was developed as a vector for expressing MHV hemagglutinin/esterase (HE) protein. The virus containing an expressed HE protein (A59-DE-HE) was generated by infecting cells with MHV-A59, which does not express HE, and transfecting the in vitro-transcribed DI RNA containing the HE gene. A similar virus (A59-DE-CAT) expressing the chloramphenicol acetyltransferase (CAT) was used as a control. These viruses were inoculated intracerebrally into mice, and the role of the HE protein in viral pathogenesis was evaluated. Results showed that all mice infected with parental A59 or A59-DE-CAT succumbed to infection by 9 days postinfection (p.i.), demonstrating that inclusion of the DI did not by itself alter pathogenesis. In contrast, 60% of mice infected with A59-DE-HE survived infection. HE- or CAT-specific subgenomic mRNAs were detected in the brains at days 1 and 2 p.i. but not later, indicating that the genes in the DI vector were expressed only in the early stage of viral infection. No significant difference in virus titer or viral antigen expression in brains was observed between A59-DE-HE- and A59-DE-CAT-infected mice, suggesting that virus replication in brain was not affected by the expression of HE. However, at day 3 p.i. there was a slight increase in the extent of inflammatory cell infiltration in the brains of the A59-DE-HE-infected mice. Surprisingly, virus titers in the livers of A59-DE-HE-infected mice were 3 log10 lower than that of the A59-DE-CAT-infected mice at day 6 p.i. Also, substantially less necrosis and viral antigen were detected in the livers of the A59-DE-HE-infected mice. This may account for the reduced mortality of these mice. The possible contribution of the host immune system to this difference in pathogenesis was analyzed by comparing the expression of four cytokines. Results showed that both tumor necrosis factor-alpha and interleukin-6 mRNAs increased in the brains of the A59-DE-HE-infected mice at day 2 p.i., whereas interferon-gamma and interleukin-1 alpha mRNAs were similar between A59-DE-HE- and A59-DE-CAT-infected mice. These data suggest that the transient expression of HE protein enhances an early innate immune response, possibly contributing to the eventual clearance of virus from the liver. This study indicates the feasibility of the DI expression system for studying roles of viral proteins during MHV infection.

Animals↗

PubMed1998

Mitogen-activated protein kinase activation mediates PDGF-directed migration of RPE cells.

Growth factor-directed migration is a critical component of the wound healing response although little is known about the signaling pathways involved. We examined the effect of inhibiting the mitogen-activated protein kinase (MAPK) pathway on platelet-derived growth factor (PDGF) and fibronectin-induced cell migration of human retinal pigment epithelial (RPE) cells. Using transwell cell-culture chambers, the effect of PDGF-BB (10-50 ng/ml) and fibronectin on components of migration was measured with or without the MAPK pathway inhibitor PD98059 (10-30 microM) MAPK activation of serum-starved cells by PDGF-BB was demonstrated by an immunoprecipitation/kinase assay and by immunohistochemistry using antibody specific for phosphorylated MAPK. PDGF-BB (10 ng/ml) stimulated MAPK activity in RPE (10 min) and its nuclear localization (1 h). PD98059 inhibited the activation of MAPK by PDGF-BB or serum. PDGF-BB stimulated RPE chemokinesis, chemotaxis, and haptotaxis; chemokinesis was additively increased and chemotaxis synergistically increased by the presence of a fibronectin substratum. PD98059 potently inhibited fibronectin-induced haptotaxis and PDGF-BB-induced chemotaxis but inhibited chemokinesis only at higher PDGF-BB (50 ng/ml) concentrations in the presence of fibronectin substratum. These results demonstrate that MAPK is critically involved in multiple components of RPE migration in vitro and suggest the potential of targeting MAPK to inhibit RPE migration in vivo.

Becaplermin↗

PubMed1998

Advanced glycation end products in age-related macular degeneration.

OBJECTIVE: To investigate the localization of N epsilon-(carboxymethyl)lysine (CML), a component and major immunologic epitope of advanced glycation end products, in aged eyes and choroidal neovascular membranes (CNVMs) surgically excised from eyes with age-related macular degeneration. METHODS: Immunohistochemistry for CML was performed using 8 snap-frozen, surgically excised CNVMs. Twelve eyes from patients aged 69 to 82 years and 2 donor eyes, 1 each from a 23-week-old fetus and 21-year-old patient, without age-related macular degeneration or diabetic retinopathy were also examined. To determine if retinal pigment epithelial cells in CNVMs accumulate advanced glycation end products, cytokeratin and CML were stained in paired serial sections. RESULTS: Soft, macular drusen and/or basal laminar and basal linear deposits were observed in 8 of 12 aged eyes. Each case showed CML accumulation, while overlying retinal pigment epithelial cells showed no accumulation in all 12 eyes. In CNVMs, however, retinal pigment epithelial cells showed CML accumulation in their cytoplasm. CONCLUSION: The additional accumulation of advanced glycation end products in soft, macular drusen and/or retinal pigment epithelial cells may play a role in the pathogenesis of CNVM formation in age-related macular degeneration. CLINICAL RELEVANCE: Recently, advanced glycation end products have been found to play a role both in aging changes and neovascularization. Localization of advanced glycation end products in the above-mentioned tissue may lead to a better understanding of the pathogenesis of age-related macular degeneration.

Adult↗

PubMed1998

Apoptosis in surgically excised choroidal neovascular membranes in age-related macular degeneration.

BACKGROUND: Choroidal neovascular membranes (CNVMs) in age-related macular degeneration show progressive histologic changes from active, cellular, highly vascularized membranes to inactive paucicellular scars. The purpose of this study was to determine whether apoptosis was involved in the evolution of these changes, what cell types are involved, and whether there was an association with the Fas antigen (Fas or CD95) and Fas ligand (FasL). METHODS: Serial frozen sections from 10 surgically excised CNVMs were stained by the TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine 5-triphosphate nick-end labeling) method for detection of DNA strand breaks and by propidium iodide staining for morphologic detection of apoptosis. Immunoperoxidase staining was used for detection of Fas, FasL, and cell-type specific antigens. RESULTS: Highly vascularized membranes contained cells with TUNEL-positive nuclei, particularly in the regions of neovascularization, while fibrotic membranes showed few, if any, TUNEL-positive cells. Many of the TUNEL-positive cells were stromal retinal pigment epithelial cells, although smaller numbers were identified as endothelial cells and macrophages. Confocal microscopy of propidium idoide-stained sections confirmed the presence of apoptotic nuclei. The extent of Fas antigen expression correlated with extent of apoptosis. FasL expression was found in all specimens but was most intense in the highly vascularized membranes. CONCLUSIONS: Highly vascularized CNVMs related to age-related macular degeneration show apoptosis in stromal retinal pigment epithelial cells, endothelial cells, and occasional macrophages. Apoptosis is associated with prominent Fas and FasL expression.

Aged↗

PubMed1998

Verapamil inhibits proliferation, migration and protein kinase C activity in human retinal pigment epithelial cells.

The effects of three calcium channel blockers, verapamil, diltiazem and nifedipine, were examined on in vitro proliferation and migration of human retinal pigment epithelial cells. Human retinal pigment epithelial cells were seeded in Dulbecco's modified essential medium with 10% fetal bovine serum and different concentrations of the three calcium channel blockers. After 3 days of treatment, cell proliferation was determined by cell counting and by [3H]-thymidine uptake. Cell viability was determined with trypan blue exclusion. For determination of cell migration, retinal pigment epithelial cells were grown to confluence and then growth-inhibited with mitomycin C. After a 3 mm zone was denuded, the cells were treated with different concentrations of the calcium channel antagonists. After 24 hr, the cells that had migrated over the wound edge were counted. To determine the involvement of protein kinase C in the verapamil effect, its activity was measured in both verapamil-treated and untreated cells. Verapamil dose dependently inhibited serum-induced proliferation of retinal pigment epithelial cells, when measured by cell number (IC50 14.6 microM) or [3H]-thymidine incorporation (IC50 11.3 microM). At concentrations of 15 microM and below, there was no effect on cell viability, as determined by morphology and trypan blue exclusion. Diltiazem inhibited cell proliferation at a concentration of 100 microM; however, 100 microM nifedipine had no effect. Verapamil showed a significant inhibition of serum-induced migration in the range of 10 microM to 0.1 microM. The IC50 of the inhibition of retinal pigment epithelial cell proliferation and migration by verapamil is significantly higher than that seen for effects on calcium channel blockage. Eight micromolar verapamil reversibly inhibited total protein kinase-C activity in retinal pigment epithelial cells suggesting the possibility that the drug may act by inhibiting the protein kinase-C pathway. These data suggest the potential of the calcium channel blocker verapamil as a pharmacological modulator of disorders such as proliferative vitreoretinopathy in which there is increased retinal pigment epithelial cell proliferation and migration.

Calcium Channel Blockers↗

PubMed1998

Apoptosis of JHMV-specific CTL in the CNS in the absence of CD4+ T cells.

The role of CD4+ T cells in altering the activity of cytotoxic T lymphocytes (CTL) during infection of the central nervous system (CNS) by the neuroptropic JHMV strain of mouse hepatitis virus was examined. Adoptive transfer of in vitro activated CTL into CD4-depleted and control recipients showed that CTL were not effective in reducing JHMV replication within the CNS. The distribution of CD4+ and CD8+ T cells within the CNS during JHMV infection showed that the CD4+ T cells remained in perivascular and subarachnoid spaces and few entered the parenchyma. By contrast approximately half of the CD8+ T cells entered the parenchyma. In CD4-depleted mice the trafficking of CD8+ T cells was not inhibited; however, the majority of the cells were found to be apoptotic. These data suggested that CD4+ T cells were not required for CTL induction but were required for the maintenance of CTL viability. The limited role of CD4+ T cells in CTL induction was confirmed by comparison of CTL activity from CD4-depleted and control mice.

Adoptive Transfer↗

PubMed1998

Mechanisms of viral clearance in perforin-deficient mice.

The roles of CD4+ T cells, IFN-gamma and TNF-alpha in viral clearance from the central nervous system (CNS) were examined in perforin gene deficient (PKO) mice. Depletion of CD4+ T cells from the PKO mice resulted in a significant 1 log10 PFU/gm increase in viral titer over control-treated PKO mice. PKO mice treated with anti-IFN-gamma mAb also had a significant 1 log10 increase in infectious virus whereas inhibition of TNF-alpha did not alter viral clearance or clinical disease in the PKO mice. These data suggest, in addition to perforin-mediated cytolysis, CD4+ T cells and IFN-gamma, but not TNF-alpha could contribute to JHMV clearance from the CNS.

Animals↗

PubMed1998

Retrovirus-mediated gene transfer targeted to retinal photocoagulation sites.

Diabetic retinopathy is a major cause of acquired blindness due to the development of retinal neovascularization and associated traction retinal detachment. It is commonly treated with retinal photocoagulation therapy; however, progression to blindness remains a significant problem. To determine the feasibility of adjunctive anti-angiogenic gene therapy, we evaluated the capability of retroviral vectors, which transfer exogenous genes only into dividing cells, to transfer and express a beta-galactosidase gene selectively into photocoagulation sites. Thirty-five rabbits received 30 retinal photocoagulation burns in the right eye followed 2 days later by beta-galactosidase (G1nBgSvNa) or control (G1XSvNa) vector injection into the subretinal space. Beta-galactosidase expression was observed in the photocoagulation sites from 5 days after vector administration (31.7+/-7.0%) to 12 weeks (6.7+/-3.4%). Immunohistochemical studies of the treated retinas using antibody Ber-MAC3 and anti-cytokeratin antibodies revealed that transduced cells were macrophages and retinal pigment epithelial cells. To determine feasibility in a primate, two monkeys received 10 laser burns in the macula superior to the fovea followed 2 days later by G1nBgSvNa vector. beta-galactosidase expression was found in photocoagulation sites and foveal retina was well preserved. We conclude that gene transfer to retinal photocoagulation sites provides stable expression of the transduced gene with relatively high efficiency. This feasibility study suggests the possibility of transferring genes encoding for anti-angiogenic factors into photocoagulation sites to improve the efficacy of laser photocoagulation therapy.

Angiography↗

PubMed1998

Ability of retroviral transduction to modify the angiogenic characteristics of RPE cells.

BACKGROUND: Retinal pigment epithelial (RPE) cells play an important role in the modulation of ocular angiogenesis. Transduction of RPE cells with retroviral vectors bearing modulating genes can result in long-term transgene expression and may alter the angiogenic characteristics of RPE cells. This study was designed to determine whether changes in angiogenic characteristics of RPE cells result from transduction with retroviral vectors bearing modulating genes, using in vitro angiogenic assays, including analysis of endothelial proliferation and wound healing. METHODS: Human RPE cells were transduced with retroviral vectors bearing either a urokinase-type plasminogen activator (u-PA) or a tissue-type plasminogen activator (t-PA) cDNA. Ten weeks after gene transfer, RPE cells transduced with the u-PA (u-PA-RPE cells) or the t-PA cDNA (t-PA-RPE cells), or untransduced (control) RPE cells, were cocultured with human umbilical vein endothelial cells (HUVECs) by contacting and non-contacting coculture methods. The effects of these cells on proliferation and in vitro "wound healing" of HUVECs were evaluated. RESULTS: Over 18 weeks, u-PA-RPE cells released large amounts of biologically active u-PA (total amount, 50.2 +/- 9.7 ng/10(6) cells/24 h), while t-PA-RPE cells released large amounts of functional t-PA (15.4 +/- 3.2 ng/10(6) cells/24 h). Control RPE cells did not release any detectable t-PA or u-PA. In the proliferation assay, u-PA-RPE cells stimulated HUVEC proliferation in contacting cell cultures, but not in non-contacting cell cultures. In contrast, t-PA-RPE cells, normal RPE cells or exogenous u-PA had no effect on HUVEC proliferation. In the wound healing assay, u-PA-RPE cells in contacting coculture and exogenous u-PA stimulated wound healing of HUVECs, while non-contacting u-PA-RPE cells, t-PA-RPE cells and normal RPE cells had no effect on HUVEC wound healing. RPE cells transduced with u-PA secreted large amounts of u-PA for as long as 18 weeks, and these cells stimulate HUVEC proliferation and in vitro wound healing. As a result, the angiogenic characteristics of RPE cells can undergo long-term changes. CONCLUSIONS: These results suggest that genetically modified RPE cells can be used to modulate ocular angiogenesis and may have potential for gene therapy of ocular diseases.

Cell Division↗

PubMed1998

Rapid isolation of choriocapillary endothelial cells by Lycopersicon esculentum-coated Dynabeads.

In vitro studies of choroidal endothelial cells may be critical for understanding the pathogenesis of neovascularization in age-related macular degeneration, since endothelial cells from different sites are highly heterogeneous in their morphology and behavior. Isolation of choroidal endothelial cells is complicated and labor intensive because of the small size of the choroid and the difficulty of excluding contaminating cells. We describe a rapid, simplified method for the isolation of bovine choroidal endothelial cells using microdissection followed by the use of superparamagnetic beads (Dynabeads) coated with the endothelial cell-specific lectin Lycopersicon esculentum, which selectively binds to fucose residues on the endothelial cell surface. Cells bound to beads are isolated using a magnetic particle concentrator. Isolated cells grew to confluence in a monolayer with a cobblestone morphology and were shown to be endothelial cells by their greater than 95% immunoreactivity to von Willebrand factor and phagocytosis of dil-acetylated LDL. Isolated cells grew as tubes in three-dimensional cultures. This method markedly reduces the time needed for pure culture of cells and makes the in vitro study of choroidal endothelial cells practical and reproducible.

Animals↗

PubMed1998

Prolactinomas express human heparin-binding secretory transforming gene (hst) protein product: marker of tumour invasiveness.

BACKGROUND AND OBJECTIVE: We have shown previously that heparin-binding secretory transforming gene (hst) overexpression in rat pituitary cells mediates lactotroph tumour growth and stimulates PRL transcription, and that transforming sequences of the gene, which encode fibroblast growth factor-4 (FGF-4), are expressed in human prolactinomas. To further determine the role of hst in human PRL-secreting adenoma pathogenesis we studied the presence of hst protein in these tumours and other types of human pituitary adenoma. PATIENTS AND DESIGN: Pituitary adenoma tissue samples were obtained at surgery from 14 patients with PRL-secreting adenomas, 5 patients with GH-secreting tumours, 3 with ACTH-secreting, and 13 patients with nonfunctioning tumours. Two normal pituitary tissue specimens were also studied. Clinical data, including tumour invasiveness assessed by preoperative MRI studies, were available. For hst protein immunolocalization, tumour frozen sections were immunostained with antihuman FGF-4 antibody. Immunoperoxidase staining for the proliferation-related nuclear antigen Ki-67 was performed using MIB-1 monoclonal antibody. RESULTS: Normal anterior pituitary cells did not contain immunoreactive hst protein. Lactotrophs in five of 14 prolactinomas (36%) stained strongly for hst compared with immunoreactive pituicytes in only one of 21 nonfunctioning, GH-, and ACTH-secreting adenomas (P = 0.05). Immunoreactive hst in adenoma cells was detected in 3 of 5 invasive prolactinomas, and in 2 of 9 noninvasive PRL-cell adenomas. Immunostaining for the proliferation-related antigen Ki-67 showed a higher proliferation index in hst-positive adenomas (3.94 + 0.85%) as compared with those immunonegative for hst (1.98 + 0.7%; P = 0.05). CONCLUSIONS: hst protein may be directly involved in prolactinoma development or progression, particularly in invasive tumours, probably due to the growth promoting effects of FGF-4.

Adrenocorticotropic Hormone↗

PubMed1998

Mutation and expression analysis of the cyclin-dependent kinase inhibitor gene p27/Kip1 in pituitary tumors.

By regulating cyclin-cyclin-dependent kinase (CDK) complex activity, individual CDK inhibitors (CDKIs) are potential tumor suppressors. One of the CDKIs, p27/Kip1, binds to a variety of CDK-cyclin complexes. A link between loss of p27/Kip1 function and development of pituitary tumors was suggested by the formation of pituitary tumors in almost all mice with germline deletion of the p27/Kip1 gene. However, genetic aberrations in the p27/Kip1 locus have not been analyzed in human pituitary tumors. We investigated eighteen non-functioning and GH-secreting pituitary tumor samples for p27/Kip1 mutations by single-strand conformational polymorphism (SSCP) following PCR. We found five abnormally migrating samples on the PCR-SSCP analysis. The sequence of these samples revealed a polymorphism of codon 109 (Val-->Gly), which has been previously described. No other structural changes of p27/Kip1 were found in these pituitary tumors within the coding region. In addition, no difference in p27/Kip1 protein levels in pituitary tumor tissues compared with normal pituitary tissues was demonstrated by immunostaining. These data suggest that both p27/Kip1 mutations and decreases in p27/Kip1 protein levels are infrequent in the development of pituitary tumors.

Cell Cycle Proteins↗

PubMed1998

Excessively high prolactin level in a patient with a nonprolactin-secreting adenoma. Case report.

The authors report the case of a 48-year-old woman who presented with a nonprolactin-secreting adenoma and a preoperative prolactin level of 662 ng/ml. The patient's neoplasm subsequently enlarged despite normalization of her prolactin level with dopamine agonist therapy. Hyperprolactinemia, with levels of prolactin as high as 150 ng/ml, is commonly associated with sellar tumors and is attributed to disruption of the normal delivery of dopamine to the adenohypophysis. The prolactin level found in this patient represents the highest level attributed to the stalk-section effect reported in the literature and underscores the need for repeated radiographic assessment of patients who are undergoing treatment with bromocriptine and have prolactin levels in the 25 to 1000 ng/ml range.

Adenoma↗

PubMed1998

Up-regulation of the cAMP/PKA pathway inhibits proliferation, induces differentiation, and leads to apoptosis in malignant gliomas.

Manipulation of signal transduction pathways has been increasingly used to modulate tumor growth. We have investigated the effects of up-regulation of the cAMP/protein kinase A (PKA) pathway in cell lines and primary cultures of malignant gliomas. The malignant glioma cell line A-172 was treated with agonistic cAMP analogs dibutyryl cyclic AMP (dcAMP) and 8-bromo-cyclic AMP (8-Br-cAMP), an adenylate cyclase activator (forskolin), and a phosphodiesterase inhibitor (3-isobutyl-1-methyl-xanthene [IBMX]). Proliferation was determined by 3H-thymidine assay. Differentiation was measured by morphologic changes, glial fibrillary acidic protein (GFAP) content, and invasion potential. Apoptosis was measured quantitatively by the TUNEL method, which labels DNA fragments using terminal transferase. Agonistic cAMP analogs, forskolin, and IBMX were found to decrease proliferation in A-172 cells after 24 hours. Treatment with 8-Br-cAMP for 24 hours caused an increase in GFAP and decrease in invasion. Apoptosis was induced after 48 hours in the presence of synergistic cAMP analogs for the Type II PKA isozyme, but not Type I PKA isozyme. Activation of PKA by increasing cAMP levels (forskolin, IBMX) or directly by cAMP analogs correlated with decreased proliferation, increased differentiation, and induction of apoptosis in A-172 cells. Modulation of the cAMP/PKA pathway may thus represent a possible target site for treating malignant gliomas.

Apoptosis↗

PubMed1998

Retrovirus-mediated gene transfer to photocoagulation-induced choroidal neovascular membranes.

PURPOSE: To determine the feasibility of experimental gene transfer to laser-induced choroidal neovascular membrane (CNVM) in rats, with a retroviral vector containing the reporter construct beta-galactosidase (beta-gal). METHODS: Laser photocoagulation was used to induce CNVM in rats. To ascertain the duration of beta-gal expression in the CNVM, 23 rats received 10 burns (75 microm, 100 mW, 0.1 seconds) in their right eyes, and beta-gal expression was examined from day 3 to 4 months. In addition, 14 pigmented rats were treated with 3 photocoagulation burns in their right eyes. beta-gal vector was injected into the vitreous or subretinal space 2 days later. On day 14, fluorescein angiography was performed to detect choroidal neovascularization. Then, beta-gal expression in each photocoagulation-induced CNVM was examined by observing the exposed fundus of the eyes stained with the beta-gal substrate X-Gal. RESULTS: beta-gal expression was identified in the CNVM induced by photocoagulation from day 5 (16.2% +/- 6.8% of the lesions) to 4 months (3.7% +/- 2.4%). Histopathologic examination revealed beta-gal-transduced macrophages and spindle-shaped cells, which amounted to 1.12% +/- 0.58% (at 2 weeks) of the total cells in the CNVM. beta-gal expression was restricted to the CNVM, and there was no beta-gal transduction in surrounding normal retinochoroidal tissue. There was no correlation between choroidal neovascularization formation and beta-gal expression. CONCLUSIONS: The feasibility of gene transduction targeted to the photocoagulation-induced CNVM was demonstrated using retroviral vectors. By transducing functional genes, this model could be useful for investigating the possibility of gene therapy to inhibit formation of the CNVM in age-related macular degeneration.

Animals↗

PubMed1997

Inhibition of human malignant glioma cell motility and invasion in vitro by hypericin, a potent protein kinase C inhibitor.

The effect of hypericin, an antiviral drug and a potent protein kinase C (PKC) inhibitor, on glioma cell invasion was investigated in vitro. Treatment of the established human glioblastoma cell line, T98G, with 1 microM hypericin for 24 h resulted in a significant inhibition of the cell invasion through an artificial basement membrane, but not cell attachment or proliferation. Furthermore, tamoxifen and staurosporine, both PKC inhibitors, also inhibited T98G cell invasion, suggesting that PKC may be the cellular target for hypericin-inhibited glioma cell migration. Similarly, hypericin decreased cell motility significantly in established lines, T98G and U87-MG, and also in a low-passage human malignant glioma cell line. Thus, hypericin may prove useful for studying mechanisms of glioma invasion, and may represent a new agent in malignant glioma therapy.

Anthracenes↗

PubMed1997

Retroviral vector-mediated transfer of an antisense cyclin G1 construct inhibits osteosarcoma tumor growth in nude mice.

Metastatic osteosarcoma is a potential target for gene therapy, because conventional therapies are only palliative and metastatic disease is invariably fatal. Overexpression of the cyclin G1 (CYCG1) gene is frequently observed in human osteosarcoma cells, and its continued expression is found to be essential for their survival. Previously, we reported that down-regulation of cyclin G1 protein expression induced cytostatic and cytocidal effects in human MG-63 osteosarcoma cells (Skotzko et al., Cancer Research, 1995). Here, we extend these findings in a tumorigenic MNNG/HOS cell line and report on the effective inhibition of tumor growth in vivo by an antisense cyclin G1 retroviral vector when delivered as concentrated high titer vector supernatants directly into rapidly growing subcutaneous tumors in athymic nude mice. Histologic sections from the antisense cyclin G1 vector-treated tumors showed decreased mitotic indices and increased stroma formation within the residual tumors. Furthermore, in situ analysis of the cell-cycle kinetics of residual tumor cells revealed a decrease in the number of cells in S and G2/M phases of the cell cycle concomittant with an accumulation of cells in the G1 phase. Taken together, these studies demonstrate in vivo efficacy of a high-titer antisense cyclin G1 retroviral vector in an animal model of osteosarcoma.

Animals↗

PubMed1997

Expression of interferon-gamma by a coronavirus defective-interfering RNA vector and its effect on viral replication, spread, and pathogenicity.

A defective-interfering (DI) RNA of the murine coronavirus mouse hepatitis virus (MHV) was developed as a vector for expressing interferon-gamma (IFN-gamma). The murine IFN-gamma gene was cloned into the DI vector under the control of an MHV transcriptional promoter and transfected into MHV-infected cells. IFN-gamma was secreted into culture medium as early as 6 hr posttransfection and reached a peak level (up to 180 U/ml) at 12 hr posttransfection. The DI-expressed IFN-gamma (DE-IFN-gamma) exhibited an antiviral activity comparable to that of recombinant IFN-gamma and was blocked by a neutralizing monoclonal antibody against IFN-gamma. Treatment of macrophages with DE-IFN-gamma selectively induced the expression of the cellular inducible nitric oxide synthase and the IFN-gamma-inducing factor (IGIF) but did not affect the amounts of the MHV receptor mRNA. Antiviral activity was detected only when cells were pretreated with IFN-gamma for 24 hr prior to infection; no inhibition of virus replication was detected when cells were treated with IFN-gamma during or after infection. Furthermore, addition of IFN-gamma together with MHV did not prevent infection, but appeared to prevent subsequent viral spread. MHV variants with different degrees of neurovirulence in mice had correspondingly different levels of sensitivities to IFN-gamma treatment in vitro, with the most virulent strain being most resistant to IFN-gamma treatment. Infection of susceptible mice with DE-IFN-gamma-containing virus caused significantly milder disease, accompanied by more pronounced mononuclear cell infiltrates into the CNS and less virus replication, than that caused by virus containing a control DI vector. This study thus demonstrates the feasibility and usefulness of this MHV DI vector for expressing cytokines and may provide a model for studying the role of cytokines in MHV pathogenesis.

Animals↗