Toxoplasma Gondii Growth Research Articles

Beta interferon inhibits Toxoplasma gondii growth in human monocyte-derived macrophages

Gamma interferon (IFN-gamma) previously has been shown to block the replication of Toxoplasma gondii in fibroblasts by the induction of indoleamine 2,3-dioxygenase (IDO) activity. IFN-beta also is known to induce IDO activity in monocyte-derived macrophages, but its ability to block the growth of T. gondii has not been demonstrated. We found not only that the combination of IFN-beta and lipopolysaccharide induced greater IDO activity in monocyte-derived macrophages than did IFN-beta alone but that this combination also was effective in inhibiting the growth of T. gondii. In addition, the inhibition was reversed by the addition of exogenous tryptophan, thus demonstrating that a mechanism by which IFN-beta inhibited T. gondii replication was by the induction of IDO.

Toxoplasma gondii: The biochemical basis of resistance to emimycin

Emimycin was a potent and selective inhibitor of the growth and nucleic acid synthesis of Toxoplasma gondii in human fibroblasts. An emimycin-resistant mutant of T. gondii lost the pyrimidine salvage enzyme uracil phosphoribosyltransferase, the same enzyme absent in parasites resistant to fluorodeoxyuridine. The mutant resistant to emimycin was completely cross-resistant to fluorodeoxyuridine. Emimycin was as good a substrate as uracil for the uracil phosphoribosyltransferase of T. gondii. [ 3H]Emimycin supplied in the medium of cultures with actively growing intracellular parasites was converted to emimycin riboside-5′-phosphate in the soluble pool of T. gondii. All other emimycin analogs of uracil-containing nucleotides were also formed but little emimycin riboside diphosphate- N-acetylhexosamine was found. [ 3H]Emimycin was not converted to analogs of the cytidine nucleotides. When intracellular T. gondii were treated with a concentration of [ 3H]emimycin that partially inhibited parasite RNA synthesis, much less [ 3H]emimycin was incorporated into RNA than would be predicted by the amount of intracellular [ 3H]emimycin riboside triphosphate.

Incorporation of recombinant gamma interferon into liposomes enhances its ability to induce peritoneal macrophage antitoxoplasma activity

In this study we compared the ability of free- and liposome-incorporated murine recombinant gamma interferon (rIFN-gamma) to enhance peritoneal macrophage H2O2 release and antitoxoplasma activity in vitro. rIFN-gamma was efficiently (37 to 47%) incorporated into multilamellar vesicles composed of phosphatidylglycerol/cholesterol in a 2:1 molar ratio. The amount of rIFN-gamma incorporated into multilamellar vesicles and added to macrophages (0.1 to 1,000 U/ml) was quantitated with [3H]rIFN-gamma. The concentration of liposomal rIFN-gamma required to enhance macrophage H2O2 release (1 U/ml) and maximally inhibit Toxoplasma gondii growth (10 U/ml) was one-tenth the concentration required for free rIFN-gamma (10 and 100 U/ml, respectively). This increase in potency was observed in both thioglycolate-elicited and resident peritoneal macrophages. Control liposomes containing encapsulated buffer had no effect on the potency of free rIFN-gamma. The duration of macrophage activation induced by 24 h of liposomal rIFN-gamma treatment was also considerably longer than that induced by free rIFN-gamma (2 days versus less than 1 day). These data indicate that liposomal rIFN-gamma is more active than free rIFN-gamma as an inducer of macrophage microbicidal properties in vitro. This enhanced activity, combined with the potential for selective delivery of liposomal rIFN-gamma to phagocytic cells in vivo, may improve the therapeutic efficacy of rIFN-gamma in infections characterized by parasitization of phagocytes.

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 μg/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-Ano R-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 μg regularly protected mice against an otherwise fatal infection with RH T. gondii and 55 μg had some protective effect. However, all mice infected with R-Ano R-1 and treated with 360 μg 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.

Defective activation of granuloma macrophages from Mycobacterium leprae-infected nude mice.

We have previously demonstrated that granuloma macrophages from the foot pads of Mycobacterium leprae-infected nude mice are functionally normal despite heavy intracellular burdens of bacilli. However, unlike peritoneal macrophages, these macrophages fail to restrict the intracellular growth of Toxoplasma gondii when stimulated with recombinant murine gamma interferon (Mu IFN-gamma) and thus appear defective in their response to macrophage-activating factor(s). In further characterizing this defect we have examined tumoristatic capacity, superoxide radical formation, and expression of la antigens on granuloma macrophages before and after treatment with Mu IFN-gamma. By all three criteria, M. leprae-burdened granuloma macrophages failed to become activated by doses of Mu IFN-gamma that readily activate peritoneal macrophages from M. leprae-infected nude mice or normal Balb/c mice. M. leprae-infected granuloma macrophages produced elevated levels of prostaglandin E2 (PGE2) in vitro, which was suppressed by indomethacin. However, the inhibition of PGE2 production for 48 hr in vitro did not restore normal responses to Mu IFN-gamma.

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma.

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.

Activation of murine macrophages and a bovine monocyte cell line by bovine lymphokines to kill the intracellular pathogens Eimeria bovis and Toxoplasma gondii.

Macrophage (M phi)-activating lymphokines present in concanavalin A-stimulated bovine T-lymphocyte cultures (ConAS) were studied by assessing their effects on Eimeria bovis and Toxoplasma gondii growth in cultured bovine monocytes (BM) and mouse M phi. The in vitro development of both parasites was assessed by incorporation of [3H]uracil and by microscopic examination of parallel cultures. Incorporation of [3H]uracil into infected cultures was an accurate indicator of growth of both E. bovis and T. gondii in BM and mouse M phi. Sporozoites of E. bovis underwent merogony in untreated BM but not in mouse M phi, whereas T. gondii developed in both cell types. Inhibition of T. gondii growth was greatest in ConAS-treated BM, whereas preincubation of mouse M phi with ConAS resulted in about 80% growth inhibition. There was no significant difference between the inhibition of either T. gondii sporozoite- or tachyzoite-induced growth in ConAS-treated cells, showing that activation pathways are equally effective against both stages. Treatment of ConAS with glycine-hydrochloride buffer (pH 2) resulted in a total loss of antiviral activity mediated by gamma interferon (IFN-gamma). When pH 2 dialyzed ConAS was used to activate BM, inhibition of T. gondii growth was only partially affected. Because bovine IFN-gamma does not activate mouse M phi and due to the partial effects of pH 2 on ConAS-induced growth inhibition, the major component(s) of ConAS responsible for T. gondii growth inhibition is distinct from IFN-gamma. Furthermore, IFN-gamma may act synergistically rather than being part of a priming sequence for M phi responsiveness to other lymphokines. Murine recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) was tested for any microbistatic activity against T. gondii sporozoites and tachyzoites. There was no significant difference in either colony formation or [3H]uracil incorporation between rGM-CSF-treated and control cultures, regardless of host cell type. Thus, rGM-CSF does not induce adequate M phi activation to kill T. gondii and is not a major microbistatic component of ConAS. rGM-CSF also had no effect on T. gondii infection in vivo.

Mycobacterium leprae-burdened macrophages are refractory to activation by gamma interferon.

Mycobacterium leprae grows to enormous numbers in the nu/nu mouse footpad, producing granulomas resembling those of lepromatous leprosy in humans. Footpad granuloma cells gorged with M. leprae were established in primary cell culture to examine their functional capabilities. These cells were classified as macrophages by the following criteria: positive staining for nonspecific esterase, reduction of Nitro Blue Tetrazolium during phagocytosis of Candida albicans, possession of Fc receptors, and possession of Mac-1 antigen. Footpad macrophages also phagocytized and supported the intracellular growth of Toxoplasma gondii. However, unlike peritoneal macrophages, footpad macrophages could not be activated to kill or inhibit T. gondii by macrophage-activating factor produced by mitogen-stimulated spleen cells or by recombinant gamma interferon. Thus, although the lepromatous macrophages appeared to be normal in many of their functions, they were defective in response to macrophage-activating signals.

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

The effect of human recombinant interferon-γ (IFN-γ) 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-γ 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-γ for 24 h has no effect on the growth of T. gondii, when transferred to fresh cultures provided that the residual IFN-γ is first removed by ultrafiltration or neutralized with a specific monoclonal antibody. Thus minor metabolites produced from tryptophan in response to IFN-γ and excreted into the medium are not parasitostatic. When cultures treated with IFN-γ 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-γ is most likely to result from the starvation of T. gondii for tryptophan.

In vitro culture of Toxoplasma gondii in primary and established cell lines

The growth of Toxoplasma gondii (RH strain) was compared in HEp2, VERO, MRC-5 and African green monkey primary kidney (AGMPK) cell lines. Optimal growth conditions with regard to parasite yield, purity and antigen secretion (assessed by immunoelectrophoresis) occurred when AGMPK were the host cells. The kinetics of antigen release from infected AGMPK cultures were studied, and although antigen recognized by ELISA was detectable after l h of culture, supernatants were only able to induce antigen-specific mitogenesis after 60 h culture, suggesting that certain components of secreted antigen may be functionally distinct. Continuous culture of T. gondii in AGMPK cells did not result in a change in virulence of the parasite. Toxoplasma from this cell line could also be used in the dye test for serodiagnosis, with no significant difference between culture- or mouse-derived parasites ( P > 0.05).

Interferon gamma and the growth of Toxoplasma gondii in fibroblasts

Interferon gamma and the growth of Toxoplasma gondii in fibroblasts

Enzyme immunoassay for evaluation of Toxoplasma gondii growth in tissue culture.

An enzyme-linked immunosorbent assay (ELISA) for the evaluation of growth kinetics of Toxoplasma gondii in tissue cultures was developed. Tissue culture microplates (96 wells) were seeded with Vero cells, infected with a virulent strain of T. gondii, and incubated for different time periods. The ELISA was performed with anti-T. gondii antibodies on the infected cells. The method was simple, rapid, and accurate, and very good correlations between the ELISA results and the percentage of T. gondii rosettes in infected cells, the number of free T. gondii in the supernatant, and the amount of T. gondii antigen in the supernatant were observed.

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.

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.

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.

Kinetics of the growth of toxoplasma gondii (RH strain) in mice

A method is described for obtaining maximum yield of Toxoplasma tachyzoites from the peritoneal cavity of infected mice. Mice injected with 10 2 parasites contain more Toxoplasma in this site at the time of death than mice injected with larger numbers. The host does not mount a detectable humoral response to the parasite.

Toxoplasma gondii: Characterization of a mutant resistant to 5-fluorodeoxyuridine

An analog of deoxyuridine, 5-fluorodeoxyuridine (FUDR), inhibited the growth of intracellular wild-type Toxoplasma gondii in human fibroblast cultures. FUDR inhibited both RNA and DNA synthesis by the parasite. A single-step FUDR-resistant mutant of T. gondii was isolated in human fibroblast cultures. This mutant was about 100-fold more resistant to FUDR than was the wild-type parasite. To understand the resistance to FUDR we traced the metabolism of deoxyuridine in the host cell and in the parasite. In the host cell, deoxyuridine was specifically incorporated into DNA. In the wild-type parasite deoxyuridine proved to be a general precursor for both RNA and DNA. This pattern of incorporation in T. gondii can be explained if deoxyuridine is converted to uracil, then uridine, and finally uridylic acid in the parasite. The FUDR-resistant but not the wild-type T. gondii was also resistant to fluorouracil and fluorouridine. Autoradiographic analysis showed that intracellular wild-type T. gondii was labeled by deoxyuridine, uracil, or uridine while the FUDR-resistant mutant failed to incorporate any of these precursors. The single genetic defect in salvage or biosynthetic enzymes that is consistent with this pattern of incorporation and resistance is a lack of uridine kinase. However, a general defect in the pyrimidine permeability of the mutant parasite cannot be excluded.

Toxoplasma gondii: Specific labeling of nucleic acids of intracellular parasites in Lesch-Nyhan cells

Lesch-Nyhan cells, which are incapable of incorporating hypoxanthine and guanine, supported the growth of Toxoplasma gondii. Massive infection of these cultured mutant human fibroblast cells resulted in a marked increase in the incorporation of radioactive hypoxanthine or guanine supplied in the medium. Autoradiography established that these radioactive purines supplied to infected Lesch-Nyhan cells were incorporated specifically into the intracellular parasites. No such specificity was seen in infected wild-type human fibroblast cells in which the nucleic acids of both the parasite and the host cell were labeled. Purine nucleotides within the intracellular parasite were not available for the synthesis of host nucleic acids. Toxoplasma gondii was less active than wild-type human fibroblast cells in the interconversion of purine nucleotides.

Growth of Toxoplasma gondii in various tissue cultures treated with In-Cn or interferon.

SummaryThe present study was undertaken to determine whether interferon or an interferon inducer, polyinosinic-polycytidylic acid (In·Cn) could modify the growth of T. gondii (RH and Beverley strain) in several different types of cell culture. Pretreatment of human WI38, conjunctiva, rabbit kidney or mouse L cells with In·Cn induced interferon within 24 hr in all but the L cells. Inhibition of toxoplasma multiplication occurred in all the treated cell types including the L cells, but the inhibition did not begin until 72 hr. These findings suggest a noninterferon mechanism for inhibition of toxoplasma by In·Cn. In other experiments a crude rabbit interferon preparation inhibited toxoplasma replication in rabbit kidney cells, but a similar mouse interferon preparation failed to inhibit toxoplasma multiplication in mouse L cells. In both cell types the interferon exerted a strong inhibitory effect on virus replication. Thus interferon preparations did not consistently inhibit T. gondii.

Further Observations on the Growth of Toxoplasma gondii in Roller Tube Cultures of Mouse and Primate Tissues

Further Observations on the Growth of Toxoplasma gondii in Roller Tube Cultures of Mouse and Primate Tissues