7 results on '"Pfefferkorn, E R"'
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2. Interferon gamma blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan.
- Author
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Pfefferkorn ER
- Subjects
- Cells, Cultured, Fibroblasts drug effects, Fibroblasts physiology, Humans, Kinetics, Toxoplasma drug effects, Tritium, Tryptophan pharmacology, Interferon-gamma toxicity, Toxoplasma growth & development, Tryptophan metabolism
- Abstract
Treatment of human fibroblasts with human recombinant gamma interferon blocked the growth of Toxoplasma gondii, an obligate intracellular protozoan parasite. Growth of the parasite was measured by a plaque assay 7 days after infection or by the incorporation of [3H]uracil 1 or 2 days after infection. The antitoxoplasma activity induced in the host cells by gamma interferon was strongly dependent upon the tryptophan concentration of the medium. Progressively higher minimal inhibitory concentrations of gamma interferon were observed as the tryptophan concentration in the culture medium was increased. Treatment with gamma interferon did not make the cells impermeable to tryptophan. The kinetics of [3H]tryptophan uptake into the acid-soluble pools of control and gamma interferon-treated cultures were identical during the first 48 sec. Thereafter uptake of [3H]tryptophan into the acid-soluble pool of control fibroblasts reached the expected plateau after 96 sec. In contrast, uptake of [3H]tryptophan continued for at least 12 min in the gamma interferon-treated cultures. At that time, the acid-soluble pool of the gamma interferon-treated cultures contained 8 times the radioactivity of the control cultures. This continued accumulation was the result of rapid intracellular degradation of [3H]tryptophan into kynurenine and N-formylkynurenine that leaked slowly from the cells. These two metabolites were also recovered from the medium of cultures treated for 1 or 2 days with gamma interferon. Human recombinant alpha and beta interferons, which have no antitoxoplasma activity, did not induce any detectable degradation of tryptophan. Several hypotheses are presented to explain how the intracellular degradation of tryptophan induced by gamma interferon could restrict the growth of an obligate intracellular parasite.
- Published
- 1984
- Full Text
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3. Characterization of an indoleamine 2,3-dioxygenase induced by gamma-interferon in cultured human fibroblasts.
- Author
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Pfefferkorn ER, Rebhun S, and Eckel M
- Subjects
- Arylformamidase biosynthesis, Cells, Cultured, Enzyme Induction drug effects, Fibroblasts drug effects, Fibroblasts metabolism, Humans, Kinetics, Protein Biosynthesis, RNA biosynthesis, Tryptophan metabolism, Tryptophan Oxygenase, Virus Replication drug effects, Interferon-gamma pharmacology, Oxygenases biosynthesis
- Abstract
We have previously observed that gamma-interferon (IFN-gamma) inhibited the growth of the intracellular protozoan parasite Toxoplasma gondii in cultured human fibroblasts and that this inhibition was related to the disappearance of tryptophan from the medium with the concomitant appearance of kynurenine and N-formylkynurenine. In this report, we show that IFN-gamma induced an indoleamine 2,3-dioxygenase in human fibroblasts that converts tryptophan to N-formylkynurenine. The induction of this enzyme was a function of IFN-gamma concentration over the range of 1 to at least 32 NIH reference units/ml. The induction was also a function of time, with the greatest increase in indoleamine 2,3-dioxygenase seen 8-24 h after treatment of cultures with IFN-gamma. The induction of indoleamine 2,3-dioxygenase by IFN-gamma was inhibited by treatment of the cultures with either actinomycin D or cycloheximide, and thus was dependent on both RNA and protein synthesis. The indoleamine 2,3-dioxygenase induced by IFN-gamma appeared to differ from other mammalian enzymes that degrade tryptophan. It had a Km for tryptophan that was 100-fold lower than that for rat liver tryptophan 2,3-dioxygenase and its substrate specificity was narrower than that of rabbit intestine indoleamine 2,3-dioxygenase. N-Formylkynurenine formamidase, the enzyme that produces kynurenine, was a constitutive enzyme and its activity was not further increased by treatment of human fibroblasts with IFN-gamma. The indoleamine 2,3-dioxygenase induced by IFN-gamma did not appear to play a major role in the antiviral activity of IFN-gamma in human fibroblasts.
- Published
- 1986
- Full Text
- View/download PDF
4. Production of gamma interferon by cultured human lymphocytes stimulated with a purified membrane protein (P30) from Toxoplasma gondii.
- Author
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Khan IA, Eckel ME, Pfefferkorn ER, and Kasper LH
- Subjects
- Animals, Antibodies, Protozoan analysis, Antigens, Protozoan immunology, Antigens, Surface immunology, Cells, Cultured, Cytopathogenic Effect, Viral, Enzyme-Linked Immunosorbent Assay, Humans, Lymphocyte Activation, Oxygenases metabolism, T-Lymphocytes metabolism, Toxoplasma immunology, Tryptophan metabolism, Tryptophan Oxygenase, Viral Plaque Assay, Interferon-gamma biosynthesis, Membrane Glycoproteins immunology, Protozoan Proteins, T-Lymphocytes immunology
- Abstract
Purified P30, the principal iodinatable membrane protein of Toxoplasma gondii, induced proliferation of peripheral blood mononuclear cells from seropositive individuals but not from seronegative individuals. Culture supernatants from stimulated cells of seropositive individuals blocked the growth of T. gondii in human fibroblasts, whereas those from antibody-negative individuals failed to do so. The anti-toxoplasmic effect of culture supernatants correlated with the induction of indoleamine 2,3-dioxygenase and the destruction of tryptophan, as previously described for fibroblasts treated with recombinant gamma interferon (IFN-gamma). The anti-toxoplasmic effect was blocked by monoclonal antibody to IFN-gamma. The protective effect correlated with the amount of IFN-gamma in the culture supernatant, as measured by inhibition of viral CPE. Thus, the level of IFN-gamma appears to be an important immune factor in protection against toxoplasmosis in humans.
- Published
- 1988
- Full Text
- View/download PDF
5. Interferon-gamma suppresses the growth of Toxoplasma gondii in human fibroblasts through starvation for tryptophan.
- Author
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Pfefferkorn ER, Eckel M, and Rebhun S
- Subjects
- Animals, Cells, Cultured, Fibroblasts, Humans, Kynurenine analogs & derivatives, Kynurenine metabolism, Kynurenine pharmacology, Recombinant Proteins pharmacology, Toxoplasma metabolism, Interferon-gamma pharmacology, Toxoplasma growth & development, Tryptophan metabolism
- Abstract
The effect of human recombinant interferon-gamma (IFN-gamma) on Toxoplasma gondii in cultured human fibroblasts is predominantly parasitostatic. This effect is dependent upon the induction in the host cell of a potent indoleamine 2,3-dioxygenase that converts tryptophan to N-formylkynurenine. This product is, in turn, degraded to kynurenine by a formamidase. Within 24 h of treatment with IFN-gamma most of the tryptophan originally present in the medium is converted to these products together with some minor metabolites. When added to the medium of infected cultures at concentrations equimolar to the tryptophan content, neither N-formylkynurenine nor kynurenine suppresses the growth of T. gondii, although at higher concentrations they are effective. The medium of uninfected cultures treated with IFN-gamma for 24 h has no effect on the growth of T. gondii, when transferred to fresh cultures provided that the residual IFN-gamma is first removed by ultrafiltration or neutralized with a specific monoclonal antibody. Thus minor metabolites produced from tryptophan in response to IFN-gamma and excreted into the medium are not parasitostatic. When cultures treated with IFN-gamma for 24 h are incubated with medium that contains [3H]tryptophan, the radioactive amino acid is converted to N-formylkynurenine and kynurenine as rapidly as it enters the cell. This degradation not only results in a very low intracellular concentration of tryptophan but also produces intracellular concentrations of tryptophan metabolites that are significantly higher than the tryptophan concentration in control cells. However, it is unlikely that either metabolite reaches intracellular concentrations that are sufficient to suppress the growth of the parasite. The parasitostatic effect of IFN-gamma is most likely to result from the starvation of T. gondii for tryptophan.
- Published
- 1986
- Full Text
- View/download PDF
6. Inhibition of growth of Toxoplasma gondii in cultured fibroblasts by human recombinant gamma interferon.
- Author
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Pfefferkorn ER and Guyre PM
- Subjects
- Animals, Cells, Cultured, DNA Replication drug effects, Dose-Response Relationship, Drug, Fibroblasts physiology, Humans, Interferon-gamma genetics, Kinetics, Protein Biosynthesis drug effects, Toxoplasma drug effects, Transcription, Genetic drug effects, Interferon-gamma toxicity, Toxoplasma growth & development
- Abstract
The growth of Toxoplasma gondii in cultured human fibroblasts was inhibited by recombinant human gamma interferon at concentrations of 8 to 16 U/ml. The interferon was titrated by observing a total inhibition of parasite plaque formation 7 days after infection. Inhibition of the growth of T. gondii in the early days after infection was measured by marked reductions in the incorporation of radioactive uracil, a precursor that can only be used by the parasites. This assay showed that when cells were pretreated with gamma interferon for 1 day and then infected, inhibition of T. gondii growth could be readily detected 1 or 2 days after infection. When the pretreatment was omitted and parasites and gamma interferon were added at the same time, no inhibition of parasite growth could be detected 1 day later, although it was apparent after 2 days. Cultures from which the gamma interferon had been removed by washing after a 1-day treatment showed inhibition of T. gondii growth. Gamma interferon had no effect on the viability of extracellular parasites, but it did inhibit the synthesis of host cell RNA and protein by ca. 50% 3 days after treatment. This degree of inhibition is unlikely, of itself, to compromise the growth of T. gondii. Recombinant alpha and beta interferons had no effect on the growth of T. gondii.
- Published
- 1984
- Full Text
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7. Interferon gamma and the growth of Toxoplasma gondii in fibroblasts.
- Author
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Pfefferkorn ER
- Subjects
- Amanitins pharmacology, Animals, Enzyme Induction drug effects, Humans, Interferon-gamma antagonists & inhibitors, Oxygenases analysis, Toxoplasma growth & development, Tryptophan Oxygenase, Fibroblasts parasitology, Interferon-gamma pharmacology, Toxoplasma drug effects
- Published
- 1986
- Full Text
- View/download PDF
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