PubMed Health⌕ Search

Biomedical subjects

M G Tovey

Publications and source records attributed to M G Tovey.

At least 19 recordsLinked to original sources
PubMed1992

Specific interferon genes are expressed in individual cells in the peritoneum and bone marrow of normal mice.

The use of a highly sensitive method of in situ hybridization capable of detecting one copy of IFN mRNA per cell showed that from 20-50% of the cells from the peritoneum and bone marrow of both normal pathogen-free and axenic mice exhibited grain counts significantly greater than background levels following hybridization with riboprobes specific for the mouse interferon-alpha (IFN-alpha), IFN-beta, or IFN-gamma genes. Labeling was shown to be specific, as the labeled probe was displaced by a 200-fold excess of the specific unlabeled probe but not by a 200-fold excess of an unrelated probe. Grain counts were reduced to background levels when cells were pretreated with ribonuclease prior to in situ hybridization. The extent of labeling with either IFN-alpha or IFN-beta-specific probes increased following i.v. inoculation of mice with the IFN-inducer Newcastle disease virus (NDV) whereas the degree of labeling observed with a probe specific for beta-actin remained unchanged. No significant differences were observed in the number of bone marrow or peritoneal cells that expressed IFN-alpha or IFN-beta mRNA from either high (C57B1/6) or low (BALB/c) IFN-producing strains of mice. The majority of IFN-alpha and IFN-beta-containing cells from both the bone marrow and peritoneum of normal pathogen-free and axenic mice resembled monocytes morphologically, whereas the majority of IFN-gamma mRNA-containing cells resembled small lymphocytes. In addition, in the bone marrow a number of large cells which resembled megacaryocytes were found to express high levels of IFN-alpha mRNA. Nuclear run-on assays showed that IFN-alpha and IFN-beta genes were actively transcribed in both bone marrow and peritoneal cells from normal and axenic mice. Low levels of de novo IFN-gamma RNA synthesis were detected in the nuclei of peritoneal cells only. The expression of IFN genes in individual cells in the tissues of normal animals may constitute a basis for the regulation of both homeostasis and host defense against virus infection and neoplastic cells.

Animals↗
PubMed1992

Tandem repeat polymers of a critical region of the human interferon-beta promoter exhibit a marked constitutive activity and enhanced responsiveness to transcriptional regulators in transfected HeLa cells.

Multiple copy tandem repeats polymers of an authentic 30-bp region of the human interferon-beta (IFN-beta) promoter between positions-91 to -62 relative to the cap site or the hexanucleotide GAAAGT derived from this region, both acted as strong constitutive regulatory elements in transfected HeLa cells. Such polymers were unresponsive to treatment with IFN-alpha despite their considerable homology with the IFN-responsive elements of other genes but were highly responsive to treatment of HeLa cells with IFN-gamma. Virus induction of HeLa cells transfected with polymers of the 30-bp region linked to a CAT gene increased the activity of the reporter gene 500- to 2,000-fold over baseline levels. Treatment with IFN-alpha prior to virus induction did not increase further CAT activity. Cotransfection of HeLa cells with the CAT gene under the control of a 12-element tandem repeat polymer of the human IFN-beta promoter and an expression vector for the IRF-1 transcriptional activator markedly increased CAT activity while cotransfection of HeLa cells with the IFN-beta construct together with an expression vector for the transcriptional regulator IRF-2 markedly decreased CAT activity relative to cells transfected with the IFN-beta polymer alone.

Base Sequence↗
PubMed1991

Differential in situ expression of cytokines in renal allograft rejection.

The expression of the interleukin 6, tumor necrosis factor alpha, and interferon gamma (IFN-gamma) genes was studied in human renal biopsies from individuals without evidence of kidney disease and from patients undergoing acute renal allograft rejection using a method of in situ hybridization capable of detecting 1-5 copies of a specific cellular messenger RNA in individual cells. IL-6, TNF-alpha, and IFN-gamma RNA transcripts were not detected in any of the sections of normal human kidneys. Elevated levels of IL-6 mRNA but not IFN-gamma were, however, detected in the sections of the renal biopsies from six of eight patients exhibiting acute rejection. A uniform level of expression of IL-6 mRNA was observed in all the cells examined, including glomerular cells, tubular epithelia, smooth muscle cells, and vascular endothelia, as well as the interstitial mononuclear infiltrate. Juxtatabular clusters of TNF-alpha mRNA were detected in the absence of IL-6 mRNA in one patient exhibiting acute rejection. Only a small number of grains (1-5 per high-power field) was detected in the urinary space or in the tubular or vascular lumen following hybridization with the IL-6 or TNF-alpha probes. In contrast, in kidney transplant patients with stable renal function no significant labeling was observed with the IL-6, TNF-alpha, or IFN-gamma probes. A similar level of expression of actin mRNA was observed in all the sections of normal and transplanted kidneys studied, suggesting that the overall level of RNA synthesis was similar in both groups. These results suggest that cytokines such as IL-6 play a role in acute allograft rejection.

Cytokines↗
PubMed1991

Genes for interleukin-1, interleukin-6, and tumor necrosis factor are expressed at markedly reduced levels in the livers of patients with severe liver disease.

The genes for interferon (IFN) alpha, IFN gamma, IL-1 beta, IL-6, and TNF alpha were transcribed at readily detectable levels both in liver biopsies from individuals with normal liver function and in samples of normal viable liver taken for transplantation. These results provided evidence for the concept that such multifunctional cytokines play a role in homeostasis in normal human tissues. In normal human liver, in situ hybridization studies showed that, in the absence of a detectable inflammatory response, both hepatocytes and mononuclear cells exhibited a similar degree of expression of IL-6 mRNA in keeping with the finding that IL-6 is produced by cells of different lineages. The levels of IL-1, IL-6, and TNF mRNA were found to be markedly reduced in extracts of the livers of patients with primary biliary cirrhosis and other forms of autoimmune liver disease at a time when extensive liver lesions were apparent, compared to the levels of expression of these cytokines in the livers of normal individuals. The reduced expression of IL-1, IL-6, and TNF mRNAs appeared to be a specific effect and not due to a general reduction in RNA synthesis as the IFN alpha, IFN gamma and actin mRNAs were expressed at similar levels in both normal and diseased livers. The levels of IL-1 beta, IL-6, and TNF mRNAs were also reduced in samples of liver from a patient with a drug induced fulminant hepatitis suggesting that this specific pattern of altered cytokine gene expression was characteristic of the advanced stage of severe liver disease.

Autoimmune Diseases↗
PubMed1991

Expression of the genes of class I interferons and interleukin-6 in individual cells.

The use of a highly sensitive method of in situ hybridization capable of detecting one copy of interferon (IFN) mRNA per cell coupled with quantitative analysis of cytokine mRNA showed that the number of copies of mRNA per cell was directly proportional to the logarithm of the number of silver grains formed over that cell. More than 90% of both virus-induced human Namalwa and mouse C243 cells exhibited grain counts significantly greater than background values following in situ hybridization with riboprobes complementary to human IFN- alpha and mouse IFN- beta mRNA, respectively. Labeling was shown to be specific, as the labeled probe was displaced by a 200-fold excess of the specific unlabeled probe but not by a 200-fold excess of an unrelated probe. Although the large majority of cells within a population responded to induction, considerable variation was observed, however, in the content of IFN mRNA per cell: 24% of induced C243 cells contained more than 50 copies of IFN-beta mRNA per cell while 60% of the cells contained 10 copies or less. Low levels of IFN mRNA were also detected in both uninduced C243 cells and uninduced Namalwa cells. Five to 10% of peripheral blood mononuclear cells from normal donors expressed INF-alpha mRNA following induction in vitro. Approximately 1% of untreated peripheral blood mononuclear cells also exhibited low levels of IFN-alpha mRNA. Analysis of interleukin-6 (IL-6) mRNA showed that 97% of TNF-induced human MG63 cells contained IL-6 mRNA, although, again, the amount varied considerably from cell to cell.

Animals↗
PubMed1990

Priming affects the activity of a specific region of the promoter of the human beta interferon gene.

Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter.

Cell Line↗
PubMed1990

Genomic footprinting: detection of putative regulatory proteins in the promoter region of the interferon alpha-1 gene in normal human tissues.

Dimethyl sulfate (DMS) genomic footprinting revealed the presence of putative regulatory proteins attached to specific sequences of the promoter region of the interferon (IFN) alpha-1 gene in normal human tissue. The pattern of protein-DNA interactions observed for the human alpha-1 promoter in freshly isolated human spleen cells was identical to that seen in DNA from the B-cell line Namalwa. The protein interactions involving the human IFN alpha-1 promoter spanned a region from positions -38 to -174 relative to the cap site which encompasses that part of the IFN alpha-1 promoter previously shown by deletion analysis to confer virus inducibility on the IFN alpha-1 gene. DNase I footprinting performed on isolated nuclei revealed a pattern of protein-DNA interactions for the promoter region of the IFN alpha-1 gene similar to that obtained with DMS footprinting performed on whole cells, with the appearance or disappearance of only a few additional protected nucleotides outside the region identified by the use of DMS. These results provide the first direct evidence for the presence of proteins bound in vivo to those parts of the IFN alpha-1 promoter between positions -64 and -109 previously shown by deletion analysis to confer virus inducibility on the IFN alpha-1 gene. The pattern of protein-DNA interactions observed for the IFN alpha-1 promoter after virus induction was identical to that seen before induction, in keeping with the finding that many transcriptional activators are present in both induced and uninduced cells.

Base Sequence↗
PubMed1989

Expression of the genes of interferons and other cytokines in normal and diseased tissues of man.

Specific interferon genes are transcribed at low levels in the spleen, liver, and peripheral blood leukocytes of normal individuals in the apparent absence of virus infection while other interferon genes remain unexpressed in the same tissues. In contrast, the genes of cytokines such as IL-1, IL-6 and TNF are expressed at relatively high levels in the organs of normal individuals. The level of expression of the IL-1, IL-6 and TNF genes is markedly reduced in the livers of patients with autoimmune liver disease compared to the level of expression in the liver of normal individuals, whereas the expression of interferon genes is similar in both normal and diseased liver, suggesting that a defect in the expression of specific cytokines is associated with severe liver disease.

Biological Factors↗
PubMed1988

Genes for IFN-beta-2 (IL-6), tumor necrosis factor, and IL-1 are expressed at high levels in the organs of normal individuals.

The gene of a cytokine designated IFN-beta-2, or IL-6, and recently identified as identical to the B cell-stimulatory factor 2, is transcribed at high levels in the spleen, liver, kidney, and peripheral blood leukocytes of normal individuals. The number of IFN-beta-2/IL-6 transcripts present endogenously in normal human tissues (0.6 to 16 copies/cell) is comparable to that present in normal cells induced in vitro with human rTNF. This is in marked contrast to the absence of detectable IFN-beta-1 transcripts (less than 0.0003 copy/cell) in the same samples of human tissue. The expression of the IFN-beta-2/IL-6 gene is closely associated with that of two other cytokines TNF, and IL-1. Thus, significant levels of IFN-beta-2/IL-6, TNF, IL-1 alpha, and IL-1 beta, mRNA were detected in all the samples of normal tissue tested and those samples which contained high levels of IFN-beta/IL-6 mRNA also contained high levels of TNF, and IL-1 beta mRNA. These results suggest that these cytokines may function in consort as regulators of cellular growth and function in normal tissues.

Gene Expression Regulation↗
PubMed1988

Antibody to mouse interferon alpha/beta abrogates resistance to the multiplication of Friend erythroleukemia cells in the livers of allogeneic mice.

Friend erythroleukemia cells (FLC) (H-2d) injected intravenously into adult syngeneic DBA/2 or allogeneic C57B1/6 (H-2b) or C3H (H-2k) mice lodge in the liver but only multiply in the liver of syngeneic mice. Our results indicated that endogenous IFN-alpha/beta was a crucial factor in preventing the multiplication of FLC in the liver of adult allogeneic mice. (a) Treatment of allogeneic adult C57B1/6 or C3H mice with polyclonal antibody to mouse IFN-alpha/beta (but not antibody to IFN-gamma) completely abrogated the resistance to the multiplication of FLC in the liver and 87% of tumor-injected, antibody-treated C57B1/6 mice died with extensive tumor involvement of the liver. In contrast, after intravenous inoculation FLC do not multiply at all (or very rarely) in the liver of adult C57B1/6 mice left untreated or treated with a variety of control globulins, and no deaths occurred. (b) 8 h after intravenous inoculation of FLC, poly(A)+ RNA hybridizable with specific DNA probes for mouse IFN-alpha or -beta (but not -gamma) was present in the liver of injected C57B1/6 mice. Using the expression of the Mx protein as an indicator of the presence of IFN-alpha/beta, we showed that Mx+ congenic C57B1/6 mice injected with FLC exhibited a marked increase in the expression of the Mx protein in the liver, spleen, kidney and lung, and this increase was blocked by treatment of mice with antibody to IFN-alpha/beta. The possibility that different host mechanisms are elicited depending on the site of tumor growth in allogeneic mice is discussed. IFN-alpha/beta appears to be of particular importance in determining the resistance of the liver to FLC in allogeneic mice.

Age Factors↗
PubMed1987

Interferon messenger RNA is produced constitutively in the organs of normal individuals.

The use of RNA blot hybridization with DNA or RNA probes of high specific activity has shown that interferon (IFN)-alpha mRNA is present constitutively in the spleen, kidney, liver, and peripheral blood leukocytes of normal individuals. A single band (approximately equal to 1.2 kilobases) was detected in poly(A)+ RNA isolated from human organs. This RNA hybridized specifically to human IFN-alpha 1 DNA and comigrated with mature IFN-alpha mRNA from virus-induced human peripheral blood leukocytes. No IFN-beta RNA transcripts were detected in any of the tissues tested. IFN-gamma mRNA was detected in only one sample of normal human spleen, which also contained an unusually high level of IFN-alpha mRNA. The use of a modified S1 mapping technique revealed the presence of IFN-alpha 1 and -alpha 2 transcripts only. No IFN-alpha 4, -alpha 5, -alpha 6, -alpha 7, -alpha 8, or -alpha 14 transcripts were detected in the same sample. The detection, in all the samples tested, of a characteristic pattern of expression of IFN genes, different from that obtained following induction, together with the low number of transcripts present (less than or equal to 0.03 copy per cell) suggest that specific IFN genes are transcribed constitutively in vivo.

Gene Expression Regulation↗
PubMed1987

Relationship between inhibition of cell growth and of transferrin receptor expression by interferon (IFN) alpha: studies in IFN-sensitive and IFN-resistant Daudi cells.

We previously showed that treatment of different cell lines with interferon-alpha (IFN-alpha) concurrently inhibited both cell growth and the rise observed in 125I-labelled transferrin binding when cells are exposed to culture conditions that stimulate proliferation. To gain insight into the relationship between these two IFN-induced inhibitory processes, we investigated the effect of IFN-alpha on the binding of 125I-labelled transferrin to Daudi cells sensitive or resistant to its antiproliferative action. We found a close correlation between the ability of IFN-alpha to inhibit cell growth and to inhibit transferrin receptor expression. Since growth inhibition induced by other agents is not always accompanied by an inhibition of transferrin receptor expression, the previous and present observations suggest that the inhibitory effect of IFN on this expression is at least one of the mechanisms by which IFN inhibits cell proliferation. We also observed that IFN-alpha did not modify transferrin receptor biosynthesis in IFN-sensitive Daudi cells, suggesting that IFN-alpha may change the processing of the transferrin receptor molecules, making them unable to bind transferrin.

Cell Cycle↗
PubMed1986

Anti-tumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant friend leukemia cells. IV. Definition of optimal treatment regimens.

Mouse interferon alpha/beta exerted a similar anti-tumor effect in DBA/2 mice injected i.p. with Friend erythroleukemia cells (FLC) either sensitive or resistant to interferon as determined by both in vitro and in vivo assays. Using this tumor system we attempted to define optimal treatment regimens for interferon administration. Interferon was most effective when injected at the site of tumor inoculation rather than at a distant site. Two factors seemed of especial importance: the amount of interferon injected and the frequency of interferon administration. Thus, for daily administration of interferon, the antitumor effect was directly related to the amount of interferon injected. For a given total dose of interferon, repeated administration of small doses of interferon was more effective than administration of a larger dose at more widely spaced intervals. The anti-tumor efficacy of interferon was independent of the number of FLC inoculated when 10(2) to 10(5) FLC were injected, but interferon treatment was less effective when 10(6) or 10(7) FLC were injected. The relevance of these results to the use of interferon in patients with cancer is discussed.

Animals↗
PubMed1986

Isolation of Daudi cells with reduced sensitivity to interferon. IV. Characterization of clones with altered binding of human interferon alpha subspecies.

Three clones of Daudi cells, DIF3, DIF8 and DIF9, have been isolated which possess specific interferon (IFN) receptors even though these cells are resistant to both the antiviral and antiproliferative actions of human IFN-alpha. Studies with 125I-labelled cloned human IFN-alpha subspecies showed that clone DIF3 bound IFN in a manner identical to that of the IFN-sensitive parental cells. The initial peak of binding observed 30 min after treatment of either Daudi or DIF3 cells at 37 degrees C with IFN-alpha 2 or IFN-alpha 8 was, however, either absent or much reduced when using DIF8 cells which are resistant to the antiproliferative action of IFN-alpha 2 and -alpha 8. Similarly, no initial peak of binding was observed after treatment of DIF9 cells with IFN-alpha 2 at 37 degrees C. In accord with its reduced biological activity, the binding of 125I-IFN-alpha 1 to either Daudi or DIF8 cells was considerably lower than that of either of the other two IFN-alpha subspecies and followed the form of a hyperbola without an initial transitory peak of binding. It is suggested that the phenotype of IFN resistance of DIF8 and DIF9 cells may be related to a defect in receptor activation whereas the same phenotype of DIF3 cells would appear to be unrelated to IFN binding.

Cell Division↗
PubMed1986

Biological activities and receptor binding of two human recombinant interferons and their hybrids.

Two human recombinant lymphoblastoid interferon-alpha subtypes, LyIFN-B (alpha 8) and LyIFN-D (alpha 1), and 10 hybrids generated therefrom were produced in Escherichia coli and purified. The antiviral and antiproliferative activities and the induction of (2'-5')oligoadenylate synthetase were compared to their receptor binding affinities. The IFN subtypes and their hybrids had similar specific antiviral activities on bovine cells. On human cells both the specific antiviral and antiproliferative activities of LyIFN-B were about 30-fold higher than those of LyIFN-D. This difference in activity could be attributed partly to the N-terminal amino acids 1 to 60 and partly to amino acids 61 to 92. A third domain affecting the biological activities was found within the carboxy-proximal segment from amino acids 93 to 150. The differences in these activities were found to correlate with their ability to bind the receptor, suggesting that the differences in activity might be due to altered binding of the IFNs to the cellular receptors. In contrast, the induction of (2'-5')oligoadenylate synthetase did not follow the same activity profile. On mouse cells, the efficiency of the hybrids was affected by at least four sites on the IFN protein. A hybrid with the N-terminal segment 1 to 60 from IFN-B and amino acids 61 to 166 from IFN-D had a specific antiviral activity on mouse cells as high as on human cells corresponding to a 500- and 5000-fold increase in specific activity compared to IFN-D and IFN-B, respectively. We suggest that on mouse cells the IFN activity may be more dependent on conformational differences than on human cells, which in turn might reflect a less precise fit to the mouse receptor than to the human receptor.

2',5'-Oligoadenylate Synthetase↗
PubMed1986

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.

Cell Line↗