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M G Tovey

Publications and source records attributed to M G Tovey.

At least 19 recordsLinked to original sources
PubMed2006

Single-stranded RNA viruses inactivate the transcriptional activity of p53 but induce NOXA-dependent apoptosis via post-translational modifications of IRF-1, IRF-3 and CREB.

To characterize the mechanisms underlying apoptosis induced by viral infection, transcriptional activation of genes encoding members of the 'BH3-only' family of proteins was analysed during the course of virus infection. Among these genes, only NOXA is transcriptionally activated by vesicular stomatitis virus (VSV), sendai virus (SV), measles virus, herpes simplex virus, or dsRNA and required for efficient apoptosis of cells. Transcriptional activation of NOXA by VSV or SV is independent of p53, but requires the presence of interferon regulatory factor 1 (IRF-1), IRF-3 and cAMP-responsive element binding protein (CREB). Binding to and transactivation of the NOXA promoter by each of these transcription factors is governed by post-translational modification involving different pathways for each factor. Thus, SV infection activates IRF-3 and CREB by phosphorylation triggered by Toll like receptor 3 signalling, and a pathway involving calcium-independent phopholipase A2, respectively. In addition transactivation induced by IRF-1 during viral infection correlates with a 10 kDa increase in its molecular weight, suggesting a covalent linkage with a previously unknown regulatory polypeptide.

Apoptosis↗
PubMed2003

Do retroviruses preferentially integrate within highly plastic regions of the human genome?

Whether retroviral integration is a phenomenon specific to properties of the surrounding genomic region is a widely debated question. In this paper we attempt to enlight the involvement of genomic regions prone to DNA double strand breaks in such process, as well as the more general concept of genome plasticity concerning repair, recombination, transposition events. While performing a differential display analysis of the promonocytic cell line U937 and clone U42 HIV infected counterpart, we found, out of about 15 highly dysregulated genes, expected according to our previous proteomic analysis, two dysregulated cellular transcripts that are shown in the present study to colocalize on band 22q11. The LB14 transcript maps within the DiGeorge critical region. Whereas the AG46 transcript encodes the immunoglobulin-lambda like polypeptide 1 (IGLL1) 4.7Mb apart from LB14. The 22q11 band is remarkable for its high plasticity involving DNA double strand breaks, that may lead to translocations, large deletions, and immunoglobulin rearrangements, frequently observed in this region. We suggest that provirus integration preferentially occurs in such genomic regions and that the subsequent insertional mutagenesis leads to the present observations. Finally, we stress out the possibility that the small size of chromosome 22 is associated with this physical property of the genome.

Chromosomes, Human, Pair 22↗
PubMed2002

Direct signal transduction via functional interferon-alphabeta receptors in CD34+ hematopoietic stem cells.

Affinity purified, freshly isolated CD34+ progenitors were shown to express low levels of type I interferon (IFN) receptors (740 +/- 60 binding sites/cell, K(d) 0.7 +/- 0.04 nM) determined by Scatchard's analysis using a radiolabelled, neutralizing, monoclonal antibody directed against the IFNAR1 chain of the human type I IFN receptor. Treatment of freshly isolated (day 0), highly purified (>95% pure) CD34+ cells with recombinant IFN-alpha resulted in rapid tyrosine phosphorylation and activation of STAT1, Tyk2 and JAK1 as shown by Western immunoblotting. Similarly, IFN treatment was shown by confocal microscopy to result in rapid nuclear localization of the transcription factors IRF1 and STAT2, demonstrating the presence of functional IFN receptors on freshly isolated (day 0) CD34+ cells. The number of specific type I IFN receptor binding sites expressed on hematopoietic progenitor cells increased to some 1440 +/- 40 per cell after 11 days of cultivation of CD34+ cells in vitrosuggesting that receptor expression increases with cell differentiation. IFN-mediated signal transduction and the inhibitory effect of IFN-alpha on 7 or 14 days CFU-GM and BFU-E colony formation was abrogated in the presence of the anti-IFNAR1 mAb, indicating that IFN-alpha acts directly on the proliferation of human hematopoietic progenitor cells via receptor activated signal transduction without excluding the induction of other cytokines or growth factors by residual accessory cells.

Active Transport, Cell Nucleus↗
PubMed2001

Induction of tolerance to recombinant therapeutic proteins.

The specific IgM and IgG antibody responses to subcutaneous (s.c.) treatment of mice with recombinant human IFN-alpha2a (rHuIFN-alpha2a) or IFN-beta were inhibited in a dose-dependent manner by prior oromucosal (o.m.) administration of rHuIFN-alpha2a or IFN-beta, respectively. Pretreatment of animals once a day for 7 days by the o.m. route with the highest dose of IFN-alpha2a tested (10(7) IU) resulted in complete inhibition of the peak IFN-alpha2a-specific IgG antibody response detected 28 days after subsequent s.c. injection of IFN-alpha2a (p < 0.001). Similarly, prior o.m. administration of 1-10 microg rHuGM-CSF per day for 7 days resulted in a statistically significant (p < 0.001) inhibition of the peak GM-CSF-specific IgG antibody response detected 28 days after s.c. administration of GM-CSF. In contrast, prior o.m. treatment with a quantity of bovine serum albumin (BSA) (100 microg) or human serum albumin (HSA) (10 microg) equivalent, respectively, to the protein content of the highest dose of IFN-alpha2a or GM-CSF administered by the o.m. route, did not affect significantly the IFN-alpha2a-specific or GM-CSF-specific IgG antibody responses detected on subsequent s.c. administration of IFN-alpha2a or GM-CSF. Oromucosal administration of IFN-alpha2a, IFN-beta, or GM-CSF alone did not induce detectable IFN-alpha2a-specific, IFN-beta-specific, or GM-CSF-specific IgM or IgG antibody responses at any of the time points tested. These results suggest that short-term o.m. administration of a recombinant protein is an effective means of inducing peripheral tolerance to subsequent parenteral administration of a therapeutic protein.

Administration, Intranasal↗
PubMed2001

Oromucosal interferon therapy: relationship between antiviral activity and viral load.

Intraperitoneal (i.p.) administration of 20,000 IU recombinant murine IFN-alpha (rMuIFN-alpha) was highly effective in protecting mice challenged i.p. with doses of encephalomyocarditis virus (EMCV) ranging from 44 to 440 LD(50) (p<0.001). Oromucosal (o.m.) IFN therapy was also found to be effective in protecting mice challenged with a lethal dose of EMCV. Thus, 40% of animals infected with 44 LD(50) of EMCV and treated o.m. with 20,000 IU rMuIFN-alpha survived infection with a mean survival time of 12.0 +/- 2.46 days relative to a mean of 6.11 +/- 0.38 days in the control group (p<0.05). Oromucosal IFN therapy was found to be ineffective, however, in animals infected with higher doses of EMCV (88-440 LD(50)), even though intraperitoneal administration of the same dose of rMuIFN-alpha resulted in the survival of 90%, 50%, and 60% of animals infected with 88, 220, and 440 LD(50) of EMCV, respectively. These results suggest that oromucosal IFN therapy is effective at relatively low viral load only and that the mechanism of action of oromucosal IFN therapy may be different from that of parenterally administered IFN. Our results suggest that oromucosal IFN therapy may be most effective in chronic viral infections as an alternative to parenterally administered IFN, which is clinically effective but poorly tolerated.

Administration, Intranasal↗
PubMed2001

Effect of oromucosal administration of IFN-alpha on allergic sensitization and the hypersensitive inflammatory response in animals sensitized to ragweed pollen.

Oromucosal (o.m.) administration of interferon-alpha (IFN-alpha) during either allergic sensitization (days 0-6) or the hypersensitive response (days 11 and 12) or both periods caused a dose-dependent reduction in allergen-specific IgE production and allergen-induced eosinophil recruitment in mice sensitized to ragweed pollen, a common allergen in humans. Treatment during the hypersensitive response period alone appeared to be most effective. Oromucosal treatment was as effective as intraperitoneal (i.p.) treatment, with maximum inhibition of both allergen-specific IgE production and allergen-induced eosinophil recruitment observed at a dose of a 1000 IU IFN-alpha. Treatment of animals with up to 10(5) IU murine IFN-alpha/beta (MuIFN-alpha/beta) by either the om. or i.p. route did not inhibit significantly allergen-specific IgG production, which may even have been increased at certain doses of IFN. Treatment of animals with up to 10(5) IU MuIFN-alpha/beta by either the o.m. or i.p. route did not affect significantly total serum IgE or IgG levels. Oromucosal administration of IFN-alpha reduced allergen-specific IgE production and allergen-induced eosinophil recruitment in the absence of detectable toxicity, the induction of H(2) antigen expression, and 2',5'-oligoadenylate synthetase activity associated with parenteral administration of IFN-alpha and thus may find application for the treatment of asthma and associated viral infections.

2',5'-Oligoadenylate Synthetase↗
PubMed2000

Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain.

The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine proteinase inhibitor spi 2, an analogue of the human alpha-1 antichymotrypsin gene, was overexpressed to a greater extent in the brains of scrapie-infected animals than in animals infected with BSE, reflecting either an agent specific or a mouse strain specific response. The levels of spi 2 mRNA were increased during the course of scrapie prior to the onset of clinical signs of the disease and the increase reached 11 to 45 fold relative to uninfected controls in terminally ill mice. Spi 2, in common with four of the other scrapie responsive genes studied, is known to be associated with pro-inflammatory processes. These observations underline the importance of cell reactivity in TSE. In addition, scrg2 mRNA the level of which is enhanced in TSE-infected mouse brain, was identified as a previously unrecognized long transcript of the murine aldolase C gene. However, the level of the principal aldolase C mRNA is unaffected in TSE. The increased representation of the longer transcript in the late stage of the disease may reflect changes in mRNA processing and/or stability in reactive astrocytes or in damaged Purkinje cells.

Animals↗
PubMed2000

Localization of a receptor nonapeptide with a possible role in the binding of the type I interferons.

Interferons (IFNs) in common with other cytokines activate Janus tyrosine kinases and latent STAT transcription factors upon binding to their cell surface receptor. Type I IFNs bind to a receptor composed of two transmembrane polypeptides, IFNAR1 and IFNAR2, which belong to the class II cytokine receptor family that also includes the cellular receptors for IFN-gamma, interleukin-10 and coagulation protease factor VII (tissue factor). The extracellular domain of the type I IFN receptor chain IFNAR1, has four fibronectin type-III sub-domains. Human IFNAR1 has intrinsic weak affinity for type I IFNs and plays an essential role in transmembrane signaling, formation of a high affinity complex with IFN and the modulation of ligand specificity. In order to characterise the ligand binding site on IFNAR1 we analysed the epitope recognized by the anti-IFNAR1 mAb, 64G12, which inhibits the binding and biological activities of both IFN-alpha and IFN-beta. The target peptide recognized by the 64G12 mAb was determined by screening a set of 48 overlapping peptides covering the first two subdomains (residues 23-229) of the extracellular region of IFNAR1. The results of this study show that the peptide (FSSLKLNVY), localized within the first sub-domain (residues 89-97) of IFNAR1, which is recognized by the 64G12 mAb, most likely overlaps a site to which both IFN-alpha and IFN-beta bind in the ligand-receptor complex. Thus, since the 64G12 mAb can neutralize the biological activities of all the type I IFNs tested, we suggest that the target peptide recognized by the 64G12 mAb, is a possible anchorage point on IFNAR1, common to binding of both IFN-alpha and IFN-beta.

Amino Acid Sequence↗
PubMed1999

Cloning of a long HIV-1 readthrough transcript and detection of an increased level of early growth response protein-1 (Egr-1) mRNA in chronically infected U937 cells.

To identify the pathways involved in HIV-1 modification of cellular gene expression, chronically infected U937 cells were screened by mRNA differential display. A chimeric transcript consisting of the 3' end of the LTR of a HIV-1 provirus, followed by 3.7 kb of cellular RNA was identified suggesting that long readthrough transcription might be one of the mechanisms by which gene expression could be modified in individual infected cells. Such a phenomenon may also be the first step towards the potential transduction of cellular sequences. Furthermore, the mRNA encoding for the transcription factor Egr-1 was detected as an over-represented transcript in infected cells. Northern blot analysis confirmed the increase of Egr-1 mRNA content in both HIV-1 infected promonocytic U937 cells and T cell lines such as Jurkat and CEM. Interestingly a similar increase of Egr-1 mRNA has previously been reported to occur in HTLV-1 and HTLV-2 infected T cell lines. Despite the consistent increase in the level of Egr-1 mRNA, the amount of the encoded protein did not appear to be modified in HIV-1 infected cells, suggesting an increased turn over of the protein in chronically infected cells.

Cloning, Molecular↗
PubMed1999

Mucosal cytokine therapy: marked antiviral and antitumor activity.

Mucosal administration of the Th1 stimulatory cytokines interleukin-2 (IL-2), IL-12, IL-15, IL-18, or granulocyte-macrophage colony-stimulating factor (GM-CSF) induced antiviral activity in mice challenged systemically with a lethal dose of encephalomyocarditis virus (EMCV) similar to that observed following parenteral administration. In contrast, mucosal administration of the Th2 stimulatory cytokines IL-4, IL-5, IL-10, or IL-13 did not affect significantly the survival of EMCV-infected animals. Mucosal administration of IL-2 or IL-12 also exerted a marked antitumor activity in mice inoculated intravenously with Friend erythroleukemia cells. Recombinant IL-2 and IL-18, but none of the other recombinant cytokines tested, induced low levels of IFN in vitro. Polyclonal antibodies to both mouse and human interferon-alpha/beta (IFN-alpha/beta) abrogated the antiviral activity of IL-2 in vivo, even though the anti-human IFN-alpha/beta antibody did not neutralize mouse IFN-alpha/beta, and neither antibody bound to IL-2. IL-15 did not exhibit antiviral activity in IFN-alpha/beta R-/- mice, which are deficient in natural killer (NK) cell activity. These results suggest that mucosal Th1 cytokine therapy induces a soluble factor or activates a specific cell population in the lymphoid or epithelial tissue of the oropharyngeal cavity, which potentiates elimination of virus-infected or neoplasic cells systemically.

Animals↗
PubMed1999

Oromucosal interferon therapy: marked antiviral and antitumor activity.

Oromucosal administration of murine interferon-alpha/beta (IFN-alpha/beta) or individual recombinant species of murine IFN-alpha, IFN-beta, or IFN-gamma or recombinant human IFN-alpha1-8, which is active in the mouse, exerted a marked antiviral activity in mice challenged systemically with a lethal dose of encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), or varicella zoster virus (VZV). The effects observed were dose dependent and similar in magnitude to those observed following parenteral administration of the same dose of IFN. No antiviral activity was observed after oromucosal administration of murine IFN-alpha/beta in animals in which the IFN receptor had been inactivated by homologous recombination. In contrast to parenteral treatment, oromucosal IFN therapy was found to be ineffective when IFNs were administered before virus infection. Oromucosal administration of IFN-alpha also exerted a marked antitumor activity in mice injected i.v. with highly malignant Friend erythroleukemia cells or other transplantable tumors, such as L1210 leukemia, which has no known viral etiology, the EL4 tumor, or the highly metastatic B16 melanoma. These results show that high doses of IFN can be administered by the oromucosal route apparently without ill effect, raising the possibility that the oromucosal route will prove to be an effective means of administering high doses of IFN that are clinically effective but poorly tolerated.

Administration, Oral↗
PubMed1999

Oromucosal interferon therapy: pharmacokinetics and pharmacodynamics.

Oromucosal administration of [125I]-labeled recombinant human interferon-alpha1-8 (IFN-alpha1-8), which is biologically active in the mouse, resulted in readily detectable levels of radioactivity in the serum of animals within 5 min. Biologically active IFN could not be detected in the serum at any time after oromucosal administration, however, and SDS-PAGE analysis showed that the material present in the serum was of low molecular weight and most probably reflected absorption of degradation products following digestion of IFN in the stomach and small intestine. Furthermore, oromucosal administration of murine IFN-alpha/beta (MuIFN-alpha/beta) had no significant effect on the expression of IFN-responsive genes in either peripheral blood mononuclear cells or splenic lymphocytes even though in the same animals IFN treatment activated gene transcription locally in the lymphoid tissue of the oropharyngeal cavity and caused a marked systemic antiviral activity. Oromucosal administration of MuIFN-alpha/beta had no significant effect on either the number of circulating peripheral blood leukocytes or the number of granulocyte-macrophage colonies recovered from the bone marrow of IFN-treated animals. These results suggest that the mechanism of action of oromucosal IFN therapy is distinct from that of parenterally administered IFN and may involve, in the abundant lymphoid or epithelial tissue of the oropharyngeal cavity, either production of a soluble factor or activation of a specific cell population that enters the circulation to mediate the elimination of virus-infected or neoplastic cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1998

Characterization of the human analogue of a Scrapie-responsive gene.

We have recently described a novel mRNA denominated ScRG-1, the level of which is increased in the brains of Scrapie-infected mice (Dandoy-Dron, F., Guillo, F., Benboudjema, L., Deslys, J.-P., Lasmézas, C., Dormont, D., Tovey, M. G., and Dron, M. (1998) J. Biol. Chem. 273, 7691-7697). The increase in ScRG-1 mRNA in the brain follows the accumulation of PrPSc, the proteinase K-resistant form of the prion protein (PrP), and precedes the widespread neuronal death that occurs in late stage disease. In the present study, we have isolated a cDNA encoding the human counterpart of ScRG-1. Comparison of the human and mouse transcripts firmly established that both sequences encode a highly conserved protein of 98 amino acids that contains a signal peptide, suggesting that the protein may be secreted. Examination of the distribution of human ScRG-1 mRNA in adult and fetal tissues revealed that the gene was expressed primarily in the central nervous system as a 0.7-kilobase message and was under strict developmental control.

Adult↗
PubMed1998

Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts.

To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, beta2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and beta2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

Amino Acid Sequence↗
PubMed1998

Prolonged allograft survival in cynomolgus monkeys treated with a monoclonal antibody to the human type I interferon receptor and low doses of cyclosporine.

A monoclonal antibody (mAb) directed against the extracellular domain of the IFNAR1 chain of the human interferon-alpha (IFN-alpha) receptor (IFN-alphaR), which inhibits activation of the Jak-Stat signal transduction pathway, administered together with a subeffective dose of cyclosporine induced prolonged survival of skin allografts in major histocompatibility complex (MHC) divergent cynomolgus monkeys. Skin biopsies from animals treated with anti-IFN-alphaR mAb and cyclosporine revealed very low levels of MHC class I and class II antigen expression and the absence of histologic signs of rejection. Monkey antibodies (IgG) to the mouse antihuman IFN-alphaR mAb were not detected in the serum of any of the animals treated with the anti-IFN-alphaR mAb either alone or together with cyclosporine. The anti-IFN-alphaR mAb abrogated activation of the Jak-Stat signal transduction pathway in IFN-treated cells. These results, which show that selective and long-lasting immunosuppression can be obtained by short-term administration of an IFN-alpha antagonist together with a subeffective dose of cyclosporine, may have important implications for the therapy of human allograft rejection.

Animals↗
PubMed1997

Identification of a novel transcriptional regulatory element common to the p53 and interferon regulatory factor 1 genes.

The promoter regions of both the interferon regulatory factor (IRF1) and p53 antioncogenes contain a previously unidentified sequence denoted IRF1 p53 common sequence (IPCS), which markedly increases the transcriptional activity of a reporter gene placed under the control of an heterologous promoter in transfected U937 cells. In contrast, transfection of U937 cells with reporter vectors containing p53 and IRF1 promoters with mutated IPCS sites resulted in a 4-fold reduction in the constitutive expression of those two genes. The transcriptional activity of IPCS is strictly correlated with the binding of a novel nuclear factor, IPCS-binding factor (IPCS-BF). IPCS-BF, which is composed of a single polypeptide of 26 kDa, is present constitutively in nuclear extracts of both U937 cells and peripheral blood mononuclear cells from healthy donors. The finding that the pattern of binding of IPCS-BF to the IPCS is unlike that of any known transcription factor and that the IPCS sequence does not exhibit any significant homology with any known binding site present in the data base, strongly suggest that IPCS-BF is a novel transcription factor which, by virtue of this ability to regulate the expression of the p53 and IRF1 genes, could play a central role in the control of cell proliferation and/or apoptosis.

DNA-Binding Proteins↗
PubMed1997

Comparative study of the expression of cellular proteins in uninfected and HIV infected U937 cells using two dimensional SDS polyacrylamide gel electrophoresis.

The cellular response to HIV infection was determined by analysing the expression of cellular proteins in uninfected and HIV-1 infected U937 cells using two-dimensional protein electrophoresis. HIV infected U937 cells constitute a useful model for the study of the chronic productive infection of cells of the monocyte/macrophage lineage by the human immunodeficiency virus. Our data suggest that the expression of 70 proteins is modified following HIV infection: the expression of approximately half of these proteins was found to be increased, while that of the other half was repressed. We estimate that the expression of around fifteen of these proteins was markedly changed following HIV infection. These results suggest that HIV infection results in the modified expression of approximately 0.5% of total cellular proteins. To our knowledge, this study represents the first global quantitative analysis of the cellular response to HIV infection in a model of chronic infection of cells of the monocyte-macrophage lineage.

Cell Line↗