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E R Pfefferkorn

Publications and source records attributed to E R Pfefferkorn.

At least 37 records · Page 2Linked to original sources
PubMed1988

Toxoplasma gondii: in vivo and in vitro studies of a mutant resistant to arprinocid-N-oxide.

The anticoccidial drug arprinocid and arprinocid-N-oxide, a metabolite produced in vivo, blocked the growth of Toxoplasma gondii in human fibroblasts. The more potent arprinocid-N-oxide inhibited growth by 50% at 20 ng/ml while arprinocid inhibited at 2 micrograms/ml. For both drugs, the host cell was less sensitive than was the parasite. Hypoxanthine did not reverse the antitoxoplasma activity of either drug. We isolated a parasite mutant, R-AnoR-1 that was 16- to 20-fold more resistant to arprinocid-N-oxide than was the wild type RH T. gondii. This mutant was not resistant to arprinocid in vitro. Arprinocid in a daily oral dose of 136 micrograms regularly protected mice against an otherwise fatal infection with RH T. gondii and 55 micrograms had some protective effect. However, all mice infected with R-AnoR-1 and treated with 360 micrograms arprinocid per day died. Since this mutant is fully sensitive to arprinocid, the form of the drug that is therapeutically active in vivo cannot be arprinocid and is likely to be arprinocid-N-oxide.

Adenine↗
PubMed1988

Production of gamma interferon by cultured human lymphocytes stimulated with a purified membrane protein (P30) from Toxoplasma gondii.

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.

Animals↗
PubMed1986

Interferon-gamma suppresses the growth of Toxoplasma gondii in human fibroblasts through starvation for tryptophan.

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.

Animals↗
PubMed1986

Characterization of an indoleamine 2,3-dioxygenase induced by gamma-interferon in cultured human fibroblasts.

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.

Arylformamidase↗
PubMed1984

Interferon gamma blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan.

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.

Cells, Cultured↗
PubMed1984

Characterization of a mutant of Toxoplasma gondii resistant to aphidicolin.

Aphidicolin, a mycotoxin that inhibits eucaryotic DNA polymerase alpha, blocked the growth of Toxoplasma gondii in confluent cultured human fibroblasts. Aphidicolin immediately inhibited DNA synthesis by T. gondii while it had a delayed and less dramatic effect on RNA synthesis. A mutant of T. gondii resistant to aphidicolin was isolated with the aid of mutagenesis by ethylnitrosourea. Parasite growth measured three days after drug treatment and parasite DNA synthesis measured immediately after drug treatment were, respectively, five- and four-fold more resistant to aphidicolin in the mutant as compared with the wild type parasite. The mutant had a three-fold greater capacity than the wild type to incorporate uracil into its deoxycytidine triphosphate pool. This increased deoxycytidine triphosphate pool is the probable explanation for the mutant's resistance because this deoxynucleotide is known, in mammalian cells, to reverse the inhibition of DNA synthesis by aphidicolin in a competitive manner.

Animals↗
PubMed1984

Inhibition of growth of Toxoplasma gondii in cultured fibroblasts by human recombinant gamma interferon.

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.

Animals↗
PubMed1984

Identification of stage-specific sporozoite antigens of Toxoplasma gondii by monoclonal antibodies.

Stage-specific antigens of Toxoplasma gondii have not been reported. We developed a panel of monoclonal antibodies that identified unique surface membrane antigens on the sporozoite and oocyst wall of T. gondii. These monoclonal antibodies failed to react with T. gondii tachyzoite surface membrane antigens in several immunologic assays. A comparison of surface membrane radioiodinated tachyzoites and sporozoites by one- and two-dimensional electrophoresis showed that T. gondii sporozoites have two major membrane proteins of approximate m.w. 67,000 and 25,000, not present in the tachyzoite stage. Two of the stage-specific monoclonal antibodies (2E4 and 5A4) were directed against the sporozoite 67,000 m.w. protein. In addition, T. gondii sporozoites are deficient in the major radioiodinated tachyzoite protein P30, which is identified by both human and mouse convalescent antitoxoplasma antisera. A convalescent antitoxoplasma serum failed to recognize sporozoite antigens by both immunoprecipitation and reaction with nitrocellulose transfer blots.

Animals↗
PubMed1983

Toxoplasma gondii: use of mutants to study the host-parasite relationship.

Toxoplasma gondii, an obligate intracellular parasite, is readily grown in nearly all cultured cells. The host-parasite relationship in these cultures can often be explored by using mutant host cells or mutant parasites. Host cells incapable of incorporating uracil or hypoxanthine, which were excellent precursors for T. gondii, allowed the demonstration that the host cell had no access to the purine or pyrimidine nucleotide pools of the parasite. Conversely, a T. gondii mutant that was defective in the principal pyrimidine salvage pathway allowed the demonstration that the parasite had no access to pyrimidine nucleotide or deoxynucleotide pools of the host cell. One metabolite that must pass from the host cytoplasm to T. gondii is a purine. An absolute defect in purine biosynthesis by the parasite was disclosed by growing T. gondii in a mutant host cell that was, itself, incapable of purine synthesis. T. gondii grew normally at 40 degrees C in a mutant host cell that was incapable of protein synthesis at that temperature. Thus, the parasite did not depend on concomitant protein synthesis in the host cell. An antigenic mutant of T. gondii was isolated with the aid of parasiticidal monoclonal antibody. This mutant lacked a major parasite surface protein, of relative molecular mass (Mr) 22 000. The antibody used to select this mutant immunoprecipitated a protein of this Mr from the wild-type parasite.

Animals↗
PubMed1983

Toxoplasma gondii: genetic crosses reveal phenotypic suppression of hydroxyurea resistance by fluorodeoxyuridine resistance.

Mutants resistant to sinefungin (SF) and hydroxyurea (HU) were isolated from an oocyst-producing strain of Toxoplasma gondii with the aid of mutagenesis with ethylnitrosourea. These mutants were used with previously described mutants resistant to adenine arabinoside (araA) and fluorodeoxyuridine (FUDR) in genetic crosses in cats. In order to interpret the data from crosses in which all four mutants were used to infect the same cat, it was necessary to devise a mathematical expression to predict the recombination frequency for unlinked markers. This frequency was shown in theory to be half of the product of the two parental phenotype frequencies. A series of crosses in which the parental frequencies were systematically varied yielded frequencies of recombination that were in accord with this calculation. The four-way crosses in the same cat showed unlinked recombination between all markers except HU and FUDR. This pair of markers yielded no doubly resistant recombinants, suggesting complete linkage. However, linkage was excluded when a binary cross between the HU- and FUDR-resistant mutants resulted in the normal number of doubly sensitive recombinants. The lack of doubly resistant recombinants was shown to be a consequence of phenotypic suppression of HU resistance by FUDR resistance. This suppression was first demonstrated by showing that an FUDR-resistant mutant selected from an HU-resistant parasite lost the HU resistance. The phenotypically suppressed HU-resistant gene was revealed by genetic crosses with wild type T. gondii. Although both parental stains were sensitive to HU, some of the progeny parasites were resistant.

Animals↗
PubMed1983

Immunofluorescent localization of myosin at the anterior pole of the coccidian, Toxoplasma gondii.

Indirect immunofluorescence using anti-myosin rabbit sera showed myosin localized in a characteristic pattern at the anterior pole of Toxoplasma gondii. This polar fluorescent staining was abolished by pre-absorption of the anti-sera with myosin extracted from avian muscle. Both intracellular and extracellular T. gondii showed similar patterns when formaldehyde-fixed, but neither showed polar fluorescence when acetone was used as the sole fixative. Immunofluorescent staining of live T. gondii revealed no polar fluorescence, suggesting that myosin is not present on the outer parasite membrane. Anti-myosin serum did not prevent host cell invasion and plaque formation in the presence of human complement. Inhibition of contractile proteins with cytochalasin D inhibited T. gondii motility and infectivity in a plaque formation assay. The pattern of polar fluorescence described here resembles the IgM-associated polar staining frequently detected in human sera, but we believe it is a different phenomenon because human sera that showed such staining retained their activity after pre-absorption with avian myosin. The unusual localization of myosin at the anterior pole of T. gondii tachyzoites may play a role in the function of anterior organelles, which are thought to facilitate the invasion of host cells.

Animals↗
PubMed1983

Temperature-sensitive mutants of Toxoplasma gondii: pathogenicity and persistence in mice.

Certain temperature-sensitive mutants of the RH strain of Toxoplasma gondii and a "back mutant," all maintained in human fibroblasts, were studied in mice. Most mutants gave rise to acute, fatal infections, and after sulfonamide prophylaxis rarely persisted as chronic infections in mice. However, the ts-4 strain was nonfatal in doses up to 10(3) to 10(5) tachyzoites, elicited high titers of antibody, and did not persist beyond 2 mo. No Toxoplasma cysts were found. There was no evidence that a febrile reaction of the mice was restrictive, because the highest temperatures, 37.9 to 38.4 C, occurred 3 days after infection, whereas the organisms were recoverable for 16 to 32 days. Because doses of Toxoplasma, survived by 11 of 12 normal BALB/c mice, and by one of five thymic transplanted athymic mice, killed six of six athymic mice, it appears that the limited persistence of ts-4 is related to the immunologic response.

Animals↗
PubMed1983

Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody.

The principal iodinatable surface protein (P30) of our cloned RH strain of Toxoplasma gondii has an apparent molecular weight of 30,000, as measured by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Monoclonal antibody B specifically immunoprecipitated protein P30 from a detergent extract of surface radioiodinated T. gondii. Monoclonal antibody B in the presence of complement was also parasiticidal for T. gondii, and this parasiticidal effect could be blocked by protein P30. Monoclonal antibody B was purified from mouse ascitic fluid and linked to cyanogen bromide-activated Sepharose. The resulting immunoabsorbent was used to purify 1.7 mg of protein P30 from a large number of parasites. The efficiency of recovery of protein P30 was measured by assays of radioactivity and of parasiticidal blocking activity. Protein P30 represented 3 to 5% of the total protein. It is also present in a recently isolated strain of T. gondii. A convalescent human antitoxoplasma serum immunoprecipitated radiolabeled protein P30. Three convalescent antisera when quantitated by an ELISA test had a high anti-protein P30 titer. Charge shift electrophoresis showed that protein P30 has an extensive hydrophobic region and thus is probably an integral membrane protein. Electrophoresis under nonreducing conditions showed no evidence that protein P30 exists as a disulfide linked homo- or heterodimer, although it probably has intramolecular disulfide bonds.

Absorption↗
PubMed1982

Hydroxyurea inhibition of growth and DNA synthesis in Toxoplasma gondii: characterization of a resistant mutant.

Hydroxyurea inhibited the growth and DNA synthesis of Toxoplasma gondii growing in human fibroblast cells. A concentration of 18 micrograms/ml totally suppressed plaque formation. The synthesis of T. gondii RNA was not acutely inhibited. The parasite was equally sensitive to hydroxyurea when grown in wild type or hydroxyurea resistant host cells. With the aid of chemical mutagenesis, we isolated a stable hydroxyurea resistant mutant of T. gondii. This mutant showed no increased ability to incorporate [3H]uracil into its pyrimidine deoxynucleotide pool. Hydroxyurea depressed the [3H]uracil labeling of the pyrimidine deoxynucleotide pool in the wild type parasite but not in the mutant, suggesting that the mutant ribonucleotide reductase was resistant to the inhibitory effect of the drug.

Animals↗
PubMed1982

Isolation and characterization of a monoclonal antibody-resistant antigenic mutant of Toxoplasma gondii.

Several monoclonal antibodies against Toxoplasma gondii were parasiticidal in the presence of normal human serum as measured by reduction in plaque titer or in assays that detected lysis. One monoclonal antibody, G, was used to select a resistant mutant from a large population of chemically mutagenized wild-type parasites. This mutant retained the wild-type sensitivity to other monoclonal antibodies and to polyclonal antisera. A comparison of surface radioiodinated wild-type and mutant parasites by using one and two-dimensional electrophoresis showed that the mutant lacked a protein (or proteins) of approximate m.w. 22,000. An immunoprecipitation procedure using monoclonal antibody G yielded a protein(s) of this m.w. from surface radioiodinated wild-type T. gondii but not from surface radioiodinated mutant parasites.

Animals↗
PubMed1981

Pyrimidine synthesis by intracellular Toxoplasma gondii.

Pyrimidine biosynthesis was studied in actively dividing, intracellular Toxoplasma gondii, in mutant Chinese hamster ovary cells blocked in pyrimidine biosynthesis to eliminate any contribution by the host cell. The parasite grew normally in these cells even though pyrimidines were not supplied in the medium. Uninfected, mutant cultures showed negligible pyrimidine synthesis. However, mutant cultures infected wit T. gondii efficiently incorporated 14C from glucose or aspartic acid into the pyrimidine bases of nucleic acids. Thus T. gondii is capable of de novo pyrimidine biosynthesis. The parasite may also be able to use pyrimidines of the host cell, because of pyrazofurin, an antimetabolite that blocks pyrimidine biosynthesis, markedly inhibited only a mutant parasite defective in the salvage of pyrimidines.

Amides↗