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PubMed1985

In vivo recombinant interleukin 2 administration enhances survival against a lethal challenge with Toxoplasma gondii.

Administration of recombinant interleukin 2 (rIL 2) resulted in a significant (p less than 0.01) decrease in mortality in mice infected with a dose of Toxoplasma gondii that killed 100% of untreated mice. Mice treated with rIL 2 had a significantly (less than 0.005) lower numbers of cysts in the brains. The protection afforded by rIL 2 could not be correlated with increased antibody synthesis or be explained by increased macrophage killing in the treated mice. Mice treated with rIL 2 after Toxoplasma infection demonstrated increased natural killer (NK) cell activity compared with either Toxoplasma-infected or rIL 2-treated mice. rIL 2 failed to reverse the suppressed proliferative response of lymphocytes to concanavalin A and lipopolysaccharide in mice acutely infected with a virulent strain of T. gondii. These results reveal that rIL 2 may have a remarkably protective effect against intracellular parasites.

Animals↗
PubMed1979

Fundamental studies on macrophage migration inhibitory factor(s) in the supernatant from spleen cells in mice infected with Toxoplasma gondii.

When migration inhibitory factor (MIF) assay in vitro was conducted on the lymphokines (LKs), it was observed that the percentage of MIF activity was greatly increased from the 3rd to the 4th week postinfection. On the succeeding weeks there was a noticeable decrease in the MIF activity noted on the 8th week postinfection of Toxoplasma. MIF activity was examined at 1, 6, 12, 18, 24 and 48 hours in non-immune spleen cells as well as Toxoplasma immune spleen cells in the 2nd week after the final challenge inoculation. MIF activity in Toxoplasma immune spleen cells were 2, 21, 29, 54, 70 and 93 percentage, respectively. The MIF activity of hyperimmunized spleen cells produced an activity of approximately 50% at 18 hours as compared to the non-immune spleen cells. Characterization of the MIF was performed using Sephadex G-100 and DEAE Sephadex A 50. Two distinct peaks of MIF were identified and separated by Sephadex G-100 gel filtration, calculating molecular weights between 30,000 to 40,000 and 3,000 to 5,000, respectively. When the fast peak by Sephadex G-100 was eluted again in DEAE Sephadex A 50, the peak was separated into 4 units, all units showing MIF activity.

Animals↗
PubMedDate not supplied

[Detection of toxoplasma-specific IgM antibodies--comparison with the ISAGA (immunosorbent agglutination assay) and immunofluorescence results].

Serum samples from 702 persons were examined for Toxoplasma-specific IgM-antibodies using the immunosorbent agglutination assay (ISAGA) and the immunofluorescence-test (IIFT). 250 samples showed a positive reaction in the Sabin-Feldman-test (SFT) with titers greater than or equal to 1: 1 024, 58% were positive in ISAGA and 36% in the IIFT. Samples of persons with acute Toxoplasma-infection, showed high titers in the ISAGA and SFT as well, even in cases where the IgM-IIFT was negative. SFT-negative sera and others with weakly-positive reactions and also rheuma-positive samples were negative in the ISAGA. It is discussed how far it is possible to determine the duration of the Toxoplasma infection by applying the ISAGA in combination with other antibody tests.

Adolescent↗
PubMed1981

Latex agglutination microtiter test for diagnosis of toxoplasma infection in animals.

The assay kit of Toxoplasma latex agglutination (LA) microtiters (Toxotest and Toxocheck "Eiken") was applied for the diagnosis of Toxoplasma infection in pig, cat, dog and some rodents. Frequency distribution of the LA titers of pigs and cats showed a through at 1:32. The starting point of the reaction for positive diagnosis was 1:64 dilution in these animals and the kit was found to be practical and useful. Application of the kit to the diagnosis of dogs was not yet successful. The LA titer of experimentally infected mice and rabbits with Toxoplasma was detected from 1--2 weeks after infection. In wild rats caught in Sapporo City 26.1% had LA titers of 1:4 or more.

Animals↗
PubMed1994

Use of thallium-201 brain SPECT to differentiate cerebral lymphoma from toxoplasma encephalitis in AIDS patients.

PURPOSE: To determine whether thallium-201 brain single-photon emission CT could be used to make the distinction between central nervous system lymphoma and toxoplasma encephalitis, which may not be possible by routine MR and CT. METHODS: A total of 37 patients with acquired immunodeficiency syndrome who had intracranial mass lesions found during a 9-month prospective study by either MR or CT underwent further evaluation with Tl-201 brain single-photon emission CT. RESULTS: Twelve patients had increased intense focal Tl-201 uptake. All of these patients had either biopsy- or autopsy-proven lymphoma. Twenty-five of the patients studied had no Tl-201 brain uptake in the lesion(s); 24 of these patients had toxoplasma encephalitis on clinical follow-up. One patient with no Tl-201 uptake was found by cerebrospinal fluid analysis to have mycobacterium tuberculosis abscess. CONCLUSION: Patients with acquired immunodeficiency syndrome who have intracranial mass lesions on MR or CT may benefit from Tl-201 brain single-photon emission CT because it can help distinguish between lymphoma and infectious lesions such as toxoplasma encephalitis.

AIDS Dementia Complex↗
PubMed1996

[Retrospective evaluation of the detection of Toxoplasma gondii by polymerase chain reaction in AIDS patients].

OBJECTIVES: We retrospectively evaluated routine detection of Toxoplasma gondii by polymerase chain reaction (PCR) amplification of the B1 gene and the TGR 1E sequence in blood and CSF samples from patients with acquired immune deficiency syndrome (AIDS) and suspected toxoplasmosis. METHODS: From January 1993 to February 1994, 93 blood, 33 cerebrospinal fluid (CSF) and 1 aqueous humor samples were obtained from 83 HIV-positive patients with CD4 counts under 200/mm3 and suspected toxoplasmosis. RESULTS: Authentic cerebral toxoplasmosis was confirmed by response to specific treatment in 18/29 patients with typical focal brain lesions. Blood samples were available in 17/18 and CSF in 6. PCR was positive for both B1 and TGR (23.5% sensitivity, 100% specificity) in 4/17 blood samples and for either B1 or TGF (58.9% sensitivity, 72.7% specificity) in 10/18. PCR of CSF was positive in only 1/6 patients with cerebral toxoplasmosis. PCR (TGR 1E only) was positive in 3/11 blood samples and in one CSF sample from patients without cerebral toxoplasmosis. Five out of 21 patients with diffuse neurologic symptoms and presumed HIV encephalitis had positive Toxoplasma detection in blood or CSF. However, no clinical improvement was obtained after specific antitoxoplasmic therapy. Two out of 38 patients with unexplained fever with or without pneumonia had a proven disseminated toxoplasmosis. These two patients had PCR-positive blood samples. One of them was cured by a specific anti-toxoplasmic treatment and the other died two days later. Seven other patients had a positive PCR result in blood or CSF. Three of them improved with specific treatment and 2 died before treatment could be initiated. The one patient with toxoplasmic retinitis was PCR-positive both in blood and aqueous humor. CONCLUSION: PCR detection of Toxoplasma gondii appears to add little to the diagnosis of cerebral toxoplasmosis but could be helpful in the diagnosis of cerebral toxoplasmosis associated with extracerebral reactivation and in disseminated toxoplasmosis.

AIDS-Related Opportunistic Infections↗
PubMed1964

MULTIPLICATION AND THERAPY OF TOXOPLASMA GONDII IN TISSUE CULTURE.

Maloney, Emily D. (University of Florida College of Medicine, Gainesville), and Herbert E. Kaufman. Multiplication and therapy of Toxoplasma gondii in tissue culture. J. Bacteriol. 88: 319-321. 1964.-The effect of temperature on the rate of multiplication of the RH strain of Toxoplasma gondii and the results of treatment with pyrimethamine, an antifolic, at various temperatures were studied in tissue culture. At 37 C incubation, the organism was found to have a lag time of 10.5 hr and a mean generation time thereafter of 4.85 hr for binary multiplication during the exponential phase of growth. At 34 C, the lag time was 12 hr and generation time was 5.26 hr. When the organisms were incubated at 31 C, the lag time was 12.5 hr and generation time was 11.1 hr. Treatment of infected monkey kidney tissue cultures with 0.05 mg per 100 ml of pyrimethamine at 37 C for 4 days killed all organisms and permitted survival of the mice which were injected with the cultures. When cultures were incubated at 34 C, a dose of 0.6 mg per 100 ml was required to spare any mice, and with a 31 C incubation even this dose was only slightly effective when therapy was continued for 4 days. If the treatment was prolonged for 7 days before injection of the cultures into mice, however, no difference in therapy with different rates of multiplication was found. Since the rates of multiplication at 34 and 31 C are similar to the rates of multiplication of some naturally occurring strains, these data confirm the fact that prolonged therapy, as used by many, is rational.

Animals↗
PubMed1994

Cytologic identification of Toxoplasma gondii from vitreous fluid.

A 75-year-old man presented with a diffuse necrotizing retinitis in his left eye. Although his condition was initially diagnosed as acute retinal necrosis syndrome, the serologic test results were interpreted as indicating recurrent toxoplasmosis retinitis. Cytopathologic examination of the vitreous following pars plana vitrectomy for repair of a retinal detachment revealed Toxoplasma organisms. The observation of Toxoplasma gondii in the vitreous specimen confirmed our clinical diagnosis and helped guide treatment of the patient.

Aged↗
PubMed2001

IL-4 and IL-13 regulate the induction of indoleamine 2,3-dioxygenase activity and the control of Toxoplasma gondii replication in human fibroblasts activated with IFN-gamma.

The ability of up-regulatory [recombinant (r) IFN-gamma, rIFN-beta and rTNF-alpha] and down-regulatory (rIL-4, rIL-10 and rIL-13) cytokines to control the expression of indoleamine 2,3-dioxygenase (INDO) and anti-Toxoplasma activity in the human fibrosarcoma cell line 2C4 was evaluated. Activation of fibroblasts with rIFN-gamma, rIFN-beta and rTNF-alpha resulted in augmentation of INDO expression and activity leading to 40.0, 25.0 and 27.0 % inhibition of tachyzoite growth, respectively. An additive effect was observed when host cells were incubated with rIFN-gamma plus rTNF-alpha. With regard to the down-regulatory cytokines we observed that IL-4 as well as IL-13, but not IL-10, induced significant inhibition of IFN-gamma-induced control of parasite replication, INDO mRNA expression and tryptophan catabolism. Similarly, IL-4 but not IL-10 inhibited the cell surface expression of HLA-DR and CD2 induced by IFN-gamma. Consistent with these findings we were able to detect by reverse transcription-PCR the expression of mRNA for different chains of IL-4 and IL-13 receptors (IL-4Ralpha, IL-13Ralpha1 and IL-13Ralpha2) but not for IL-10 receptor in the 2C4 and other human lung fibroblast cell lines (LL24 and MRC5). Together our results indicate that IL-4 and IL-13, but not IL-10, are implicated in the negative regulation of IFN-gamma-induced anti-Toxoplasma activity in human cells from fibroblast lineage.

Animals↗
PubMed1993

Rat intestinal epithelial cell line IEC-6 is activated by recombinant interferon-gamma to inhibit replication of the coccidian Toxoplasma gondii.

Toxoplasma gondii tachyzoites invaded and replicated in cultured rat small intestine cells of the line IEC-6. Treatment of rat small intestine cells with murine recombinant interferon-gamma resulted in a dose-dependent inhibition of intracellular T. gondii replication. This could be of relevance as a first line defense against Toxoplasma infection.

Animals↗
PubMed1993

Subcellular calcium localization in Toxoplasma gondii by electron microscopy and by X-ray and electron energy loss spectroscopies.

The localization of calcium in Toxoplasma gondii tachyzoites was studied at the ultrastructural level, with a cytochemical pyroantimonate precipitation method (PA) and controlled by EGTA chelating and EDX and EELS microanalyses. Appropriate conditions for material preparation, fixation and embedding, were defined. The proportion of precipitates that were either free or inside vacuoles and their distribution inside Toxoplasma appeared to be PA dose-dependent. Precipitation mainly occurred in the anterior pole of the Epon-embedded tachyzoites. EDX and EELS analyses showed that out of 30 PA precipitates inside tachyzoites, 78% contained Ca. In Melamine sections, 96% of the tachyzoites had intracellular precipitates and the membrane complex was stained; 25% of the tachyzoites inside host cells contained PA-Ca precipitates, but most of them were retained in the reticular network of the parasitophorous vacuole. Melamine embedding appeared to improve the preservation of calcium pyroantimonate precipitates.

Animals↗
PubMed1998

Characterization of the gene encoding the catalytic subunit of the DNA polymerase alpha from Toxoplasma gondii.

The gene encoding the catalytic subunit of the Toxoplasma gondii DNA polymerase alpha enzyme has been isolated. The coding region is 6487 bp in length, containing three introns, and specifies a protein of 1690 aa. The seven conserved regions which characterize the polalpha polypeptide, as well as four of the five polalpha-specific aa domains, were found in the T. gondii gene. The absence of one of these domains, as well as the presence of a unique cysteine cluster between domains IV and B in the T. gondii polalpha, may result in a slight difference in the secondary or even tertiary structure compared with the human homologue and thus may be suitable for designing anti-Toxoplasma drugs. A number of amino acid differences within the seven conserved regions between the human and T. gondii polalpha, as well as variations in the spacings of these regions, were also observed.

Amino Acid Sequence↗
PubMed1993

Toxoplasma gondii: differential location of antigens secreted from encysted bradyzoites.

Since we have previously demonstrated the protective role against infection played by Toxoplasma excreted-secreted antigens (Darcy et al. Parasite Immunology, 10, 553-567, 1988), the aim of the present work was an attempt to precisely define the location of GRA1, GRA2, and GRA5 in both the tachyzoite and the bradyzoite stages from distinct strains, in order to explore the mechanisms of secretion by Toxoplasma gondii. Three monoclonal antibodies (Charif et al. 1990) and colloidal immunogold labeling were used to localize the 27-, 28.5-, and 21-kDa target antigens to the matrix of the dense granules of tachyzoites and bradyzoites. They were, moreover, detected in the parasitophorous vacuole and in the cyst ground substance after host cell invasion. Our data suggest that a selective sorting mechanism for dense-granule contents exists at least in encysted bradyzoites. GRA2 was found preferentially associated with the ground substance of the cyst wall and the tubular elements of the network of the modified host cell phagosome, whereas GRA5 was located on the delimiting membrane of both the cyst wall and the parasitophorous vacuole. These observations reveal the selective targeting of dense-granule molecules, which could have different functions and fates when exocytosed into the parasite-containing vacuole.

Animals↗
PubMed1994

Toxoplasma gondii: secretion of a potent nucleoside triphosphate hydrolase into the parasitophorous vacuole.

Toxoplasma gondii is an obligate intracellular parasite capable of invading a wide range of host cells where it residues in a specially modified compartment termed the parasitophorous vacuole. This compartment provides a protected environment for the parasite which enters a rapid growth phase shortly after invasion. To identify functional components of this interface, we have used immunolabeling and cell fractionation to localize the potent nucleoside triphosphate hydrolase (NTPase) produced by Toxoplasma. In extracellular tachyzoites, NTPase was not exposed on the cell surface but was localized in small vesicles, called dense granules, scattered throughout the cell cytoplasm. Shortly following invasion. NTPase was secreted from dense granules and occupied the lumen of the parasitophorous vacuole where it was often associated with the intravacuolar network. NTPase was exclusively found in the supernatant in cell fractionation studies of both extracellular tachyzoites lysed by freeze-thaw and following secretion into the parasitophorous vacuole. These studies provide evidence for the rapid secretion of NTPase into the parasitophorous vacuole, where it may play a key role in processing of nucleotides for purine salvage by the parasite.

Acid Anhydride Hydrolases↗
PubMed1999

Functional and quantitative analysis of splenic T cell immune responses following oral toxoplasma gondii infection in mice.

Functional and quantitative analysis of splenic T cell immune responses following oral Toxoplasma gondii infection in mice. Experimental Parasitology 91, 212-221. Immunity to Toxoplasma gondii is mediated primarily by the host T cell response. Although there is considerable information regarding host immunity following intraperitoneal infection with tachyzoites, little information is available regarding naturally acquired infection following peroral infection with bradyzoites. In this study, a sequential quantitative analysis of the cell-mediated immune response was performed at the single cell level. To assess the kinetics of this response and parasitic loads, inbred mice were orally infected with the 76K strain bradyzoites of T. gondii. Within 24 h of infection, follicular hyperplasia followed by infiltration with histiocytes, macrophages, and apoptotic bodies was observed in the spleens of infected mice. T. gondii were detected from day 1, and counts increased gradually during the experimental period. Splenocyte DNA synthesis to antigen and mitogen was severely suppressed at days 7 and 10. The percentages of NK1.1(+) or delta gamma T cells were increased from day 1, whereas CD4(+) and CD8alpha+ T cells were signficantly increased after day 7 postinfection. CD25 expression and intracellular IFN-gamma production increased in NK1.1(+) cells on day 1 and by all other T cell subsets after day 4. Intracellular IL-4 did not increase until day 7, and IL-10 production was increased in all T cell subsets after day 4. Together, these findings indicate that oral infection with T. gondii stimulates a strong cellular immune response that appears to polarize toward an early Th1 response. However, within 7 days, a strong immune Th2 regulatory response as well as high parasitic loads can be observed, with a reduction in lymphoproliferation to mitogen stimulation, increased production of IL-4 and IL-10, and evidence of T cell apoptosis in the splenic immune compartment.

Animals↗
PubMed1999

The expression and distribution of dense granule proteins in the enteric (Coccidian) forms of Toxoplasma gondii in the small intestine of the cat.

The expression and distribution of dense granule proteins in the enteric (coccidian) forms of Toxoplasma gondii in the small intestine of the cat. Experimental Parasitology 91, 203-211. The expression and location of the dense granule proteins (GRA1-6 and NTPase) in the merozoite and during asexual and sexual development of Toxoplasma gondii in the small intestine of the cat (definitive host) was examined by immuno-light and electron microscopy. This was compared with that of tachyzoites and bradyzoites present in the intermediate host. It was found that the merozoite contained the characteristic apical organelles plus a few large dense granules. By immunocytochemistry, dense granules in merozoites were negative for GRA proteins 1 to 6 in contrast to both tachyzoites and bradyzoites in which dense granules were positive for all six proteins. The GRA proteins were associated with the parasitophorous vacuole (PV) during tachyzoite and bradyzoite development but were absent from the PV of the enteric stages. However, the merozoite dense granules were positive for NTPase, which was similar to the tachyzoite while this antigen was down regulated in the bradyzoite. The apparent release of the NTPases into the PV formed by merozoites was also similar to that described for the tachyzoite, possibly reflecting the relative metabolic activity of the various stages. This study shows that the majority of GRA proteins have a similar stage-specific expression, which is independent of NTPases expression. These observations are consistent with T. gondii having a different host parasite relationship in the enteric forms, which does not involve the GRA proteins 1-6.

Acid Anhydride Hydrolases↗
PubMed2001

Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10.

Hoff, E. F., Cook, S. H., Sherman, G. D., Harper, J. M., Ferguson, D. J. P., Dubremetz, J. F., and Carruthers, V. B. 2001. Toxoplasma gondii: Molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Experimental Parasitology, 97, 77-88. During host cell invasion, Toxoplasma gondii secretes proteins from specialized organelles (micronemes and rhoptries) located at the apical end of the parasite. The contents of the micronemes appear to be crucial to T. gondii invasion, as inhibition of microneme secretion prevents parasite entry into host cells. Here we describe a new T. gondii microneme protein, TgMIC10. Molecular characterization of a full-length TgMIC10 cDNA revealed that TgMIC10 lacks homology to any previously characterized proteins, although a homologue, NcMIC10, was identified in a closely related parasite, Neospora caninum. TgMIC10 has an unusually long secretory leader sequence of 58 amino acids; the mature TgMIC10 is 18 kDa, possesses nine diglutamic acid repeats and an imperfect repeat sequence (RK(R/Y)HEEL), and is entirely devoid of cysteines. Antibodies raised against recombinant TgMIC10 recognized the native TgMIC10 and localized the protein to the micronemes in indirect immunofluorescence and immunoEM experiments. Comparison of immunofluorescence images indicates that TgMIC10 expression is higher in T. gondii tachyzoites, which are responsible for active infection, than in bradyzoites, which are responsible for latent infection.

Amino Acid Sequence↗
PubMed2001

The in vivo conformation of the plastid DNA of Toxoplasma gondii: implications for replication.

The Phylum Apicomplexa comprises thousands of obligate intracellular parasites, some of which cause serious disease in man and other animals. Though not photosynthetic, some of them, including the malaria parasites (Plasmodium spp.) and the causative organism of Toxoplasmosis, Toxoplasma gondii, possess a remnant plastid partially determined by a highly derived residual genome encoded in 35 kb DNA. The genetic maps of the plastid genomes of these two organisms are extremely similar in nucleotide sequence, gene function and gene order. However, a study using pulsed field gel electrophoresis and electron microscopy has shown that in contrast to the malarial version, only a minority of the plastid DNA of Toxoplasma occurs as circular 35 kb molecules. The majority consists of a precise oligomeric series of linear tandem arrays of the genome, each oligomer terminating at the same site in the genetic map, i.e. in the centre of a large inverted repeat (IR) which encodes duplicated tRNA and rRNA genes. This overall topology strongly suggests that replication occurs by a rolling circle mechanism initiating at the centre of the IR, which is also the site at which the linear tails of the rolling circles are processed to yield the oligomers. A model is proposed which accounts for the quantitative structure of the molecular population. It is relevant that a somewhat similar structure has been reported for at least three land plant chloroplast genomes.

Animals↗