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PubMed2003

Investigation of probable relationship between Toxoplasma gondii and cryptogenic epilepsy.

PURPOSE: Cryptogenic epilepsy, defines a group of epilepsy syndromes for which an aetiology is unknown but an underlying brain disease is suspected. We selected patients in this subgroup of epilepsy and investigated the sero-positivity rate for anti-Toxoplasma IgG antibodies by Enzyme Linked Immunosorbent Assay (ELISA). We investigated the probable relationship between Toxoplasma gondii and cryptogenic epilepsy. METHODS: We selected 50 patients with cryptogenic epilepsy, 50 patients with known cause epilepsy and 50 healthy volunteers and investigated the sero-positivity rate for anti-Toxoplasma IgG antibodies by ELISA. RESULTS: The sero-positivity rate for anti-Toxoplasma IgG antibodies among cryptogenic epilepsy patients (52%) was found to be higher than healthy volunteers (18%) and known cause epilepsy patients (22%) with statistical significance, (X(2)=18.095, P<0.01). CONCLUSION: There might be a causal relationship between chronic toxoplasmosis and the aetiology of cryptogenic epilepsy.

Animals↗

PubMed2001

DNA replication and daughter cell budding are not tightly linked in the protozoan parasite Toxoplasma gondii.

In the protozoan parasite Toxoplasma gondii, cell division occurs by an unusual internal budding process whereby two daughter cells develop within and eventually subsume the mother cell. We have examined this process using inhibitors targeted at specific events in the cell cycle. By adding inhibitors to newly established parasites we were able to examine the effects of the inhibitors on parasites treated at the start of intracellular development and many hours prior to the onset of daughter cell budding. As with other eukaryotes, inhibitors of nuclear DNA synthesis blocked parasite DNA synthesis and prevented cell division. Examination of parasites treated with the nuclear DNA synthesis inhibitor aphidicolin showed that the formation of daughter apical complexes and the initiation of budding occurred as normal and only the inability of the nucleus to become incorporated into the daughter cells prevented successful cell division. Moreover, these inhibitory effects of aphidicolin were not reversible. The initiation of nuclear DNA synthesis and cell division in newly invaded Toxoplasma required both gene transcription and protein synthesis, although inhibitors of mitochondrial DNA synthesis, transcription and protein synthesis did not block parasite division. Thus, unlike most eukaryotes, Toxoplasma tachyzoites have separated nuclear DNA replication and mitosis from the events associated with cell division (daughter cell budding). This implies that Toxoplasma tachyzoites may have dispensed with specific cell cycle checkpoints present in other eukaryotes with, in particular, a DNA-replication checkpoint control either missing, or downregulated in this stage of the parasite life cycle.

Animals↗

PubMed1990

Disruption of the choroidoretinal interface by toxoplasma.

Toxoplasmosis is a common zoonotic infection of the retina caused by Toxoplasma gondii. The disease causes severe damage that affects the retina and subjacent choroid. Invasion of the retinal cells by the parasite leads to the major damage seen in the retina. Subsequent reactions to the invasion by toxoplasma leads to the influx of inflammatory cells. Although hypersensitivity reactions have been described to both toxoplasma and to retinal autoantigens, the major disruption of the choroidoretinal interface is probably secondary to the tissue invasion by the parasite. Patients with AIDS may show extensive necrosis of the retina in the absence of inflammatory cells. Healing of the active lesion leads to scar formation. Toxoplasma retinochoroiditis may be pigmented, nonpigmented or punched-out. Vascular anastomoses between the retina and choroid, retinal and subretinal neovascularization may occur in the aftermath of choroidoretinal interface disruption.

Animals↗

PubMed1997

Detection of a novel 40,000 MW excretory Toxoplasma gondii antigen by murine Th1 clone which induces toxoplasmacidal activity when exposed to infected macrophages.

To analyse target molecules of the CD4+ T-cell response to toxoplasma infection, a panel of Toxoplasma gondii-specific murine CD4+ T-cell clones has been established. Clone 3Tx15, belonging to the T helper 1 (Th1) subtype, abolished intracellular parasite growth when co-cultured with macrophages and live toxoplasma at a ratio of 2:2:1. This effect results from macrophage toxoplasmicidal activity induced upon parasite-dependent cellular interaction, an irrelevant Th1 clone failed in this three-party system. Clone 3Tx15 detects its corresponding antigen in the supernatant of infected cells and also reacts with a host cell-free preparation of T. gondii-excreted/secreted antigens. T-cell blot analysis of two-dimensionally separated toxoplasma lysate revealed a molecular weight of about 40,000 for the fractions stimulating clone 3Tx15. As checked in parallel enzyme-linked immunosorbent assay, the 40,000 MW T-cell antigen co-migrates with the excretory protein GRA4, the sole 40,000 MW T. gondii antigen hitherto known to be recognized by T lymphocytes. Nevertheless, neither recombinant GRA4 nor immunoaffinity-purified natural GRA4 was stimulatory for clone 3Tx15. Our findings thus demonstrate that Th1 clone 3Tx15 which induces toxoplasmicidal activity during antigenic interaction with infected macrophages defines a new 40,000 MW excretory T. gondii antigen.

Animals↗

PubMed1996

[Cryopreservation of isolates of Toxoplasma gondii in cell culture].

For a better preservation and identification of Toxoplasma gondii isolates, we propose a new method of freezing of toxoplasma growth in THP-1 cell culture. A cystogenic strain isolated from foetal blood has been grown in these cells and frozen in liquid nitrogen. After thawing, toxoplasma recover the same growth rate and morphology in vitro and the same capacity to form brain cysts into mice compared to the initial strains. The freezing of the cell suspension provides a simple and appropriate method for preservation of Toxoplasma gondii within "bank" isolates.

Animals↗

PubMed2000

Anti-Toxoplasma activity of vegetal extracts used in West African traditional medicine.

Both Toxoplasma gondii and Plasmodium are Apicomplexan protozoa that share common metabolic pathways and potential drug targets. The objective of this study was to examine the anti-Toxoplasma activity of nine West African plants with known activity against P. falciparum. The extracts were obtained from parts of plant commonly used, by most traditional healers, in the form of infusion or as water decoction. The in vitro activity of plant extracts on T. gondii was assessed on MRC5 tissue cultures and was quantified by enzyme-linked immunoassay. Aqueous extracts from Vernonia colorata were found to be inhibitory for Toxoplasma growth at concentrations > 10 mg/L, with an IC50 of 16.3 mg/L. A ten-fold gain in activity was obtained when organic solvents such as dichloromethane, acetone or ethanol were used to extract V. colorata's active principles. These extracts were inhibitory at concentrations as low as 1 mg/L, with IC50 of 1.7, 2.6 and 2.9 mg/L for dichloromethane, acetone and ethanol extracts respectively. These results indicate a promising source of new anti-Toxoplasma drugs from V. colorata and African medicinal plants.

Animals↗

PubMed1999

A Toxoplasma lectin-like activity specific for sulfated polysaccharides is involved in host cell infection.

Toxoplasma gondii is one of the most widespread parasites of humans and animals. The parasite has a remarkable ability to invade a broad range of cells within its mammalian hosts by mechanisms that are poorly understood at the molecular level. This broad host cell specificity suggests that adhesion should involve the recognition of ubiquitous surface-exposed host molecules or, alternatively, the presence of various parasite attachment molecules able to recognize different host cell receptors. We have discovered a sugar-binding activity (lectin) in tachyzoites of T. gondii that plays a role in vitro in erythrocyte agglutination and infection of human fibroblasts and epithelial cells. The ability to agglutinate erythrocytes can be reversed by a variety of soluble glycoconjugates, of which heparin, fucoidan, and dextran sulfate were the most effective. Interestingly, infectivity of tachyzoites for human foreskin fibroblasts, cells that are commonly used to grow T. gondii in vitro, was increased by low concentrations of the sulfated glycoconjugates that inhibited hemagglutination activity (i.e. dextran sulfate and fucoidan) whereas high concentrations inhibited parasite infection. Furthermore, inhibition of glycosaminoglycan biosynthesis and sulfation on the host cells reduced Toxoplasma infectivity. Finally, Toxoplasma tachyzoites showed a reduced ability to infect epithelial cell mutants deficient in the biosynthesis of surface proteoglycans. The probable identity of the hemagglutinin(s) was investigated by 1) direct binding of red blood cells to filter blots of Toxoplasma proteins separated by polyacrylamide gel electrophoresis, and 2) binding of metabolically labeled parasite proteins to fixed mammalian cells. Three parasite bands were thus identified as candidate adhesins. These results suggest that attachment of T. gondii to its target cell is mediated by parasite lectins and that sulfated sugars on the surface of host cells may function as a key parasite receptor.

Animals↗

PubMed2005

Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii.

Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.

Animals↗

PubMed2001

A parafusin-related Toxoplasma protein in Ca2+-regulated secretory organelles.

We cloned a gene, PRPI, of Toxoplasma gondii encoding a 637-amino-acids protein having a calculated mass of 70 kDa. The sequence showed high homology to parafusin, a protein that in Paramecium tetraurelia participates in Ca2+-regulated exocytosis and is a paralog of phosphoglucomutase. We show that Toxoplasma gondii homogenate and an expressed recombinant PRP1 fusion protein cross-react with a specific peptide-derived antibody to parafusin in Western blots. Antibodies to the recombinant PRP1 showed cross-reaction with parafusin and recognized PRP1, as bands at M, 63 x 10(3) and 68 x 10(3), respectively. PRP1 is labeled when Toxoplasma gondii cells are incubated with inorganic 32P and appears as the major band on autoradiograms of SDS-PAGE gels. The localization of PRP1 was examined in secretory organelles of Toxoplasma gondii by deconvolution light microscopy followed by three dimensional reconstruction using pairwise combinations of specific antibodies. PRP1 localized to the apical third of the cell. It co-localized with micronemes, the only secretory organelle the secretion of which is Ca2+ dependent. Quantification of the co-localized stain suggests that only mature micronemes ready for exocytosis have PRP1. These findings suggest that PRP1, parafusin and other members of the phosphoglucomutase superfamily have a conserved role in Ca2+-regulated exocytic processes.

Amino Acid Sequence↗

PubMed1980

Chronic Toxoplasma infections and familiarity-novelty discrimination in the mouse.

Male mice were exposed to a black arm and the stem of a Y-maze; entrance to a white arm was blocked by a transparent door. Toxoplasma-infected mice were significantly less active and tended to produce fewer faecal boluses than uninfected controls. In a subsequent free-choice trial, in which both arms were black, the uninfected mice spent significantly more time in the novel (previously blocked) arm; the infected mice showed no preference for either choice arm. Possible explanations are discussed. In particular, it seems that Toxoplasma-infected mice may be less responsive to novel stimuli. This suggestion has important implications for our understanding of one of the major ways in which Toxoplasma passes from host to host. If Toxoplasma infections impair responsiveness to novel stimuli, then infected mice are more likely to be taken by predators.

Animals↗

PubMed1990

Ultrastructural immunocytochemistry of the intact tissue cyst of Toxoplasma in the brains of mice with congenital toxoplasmosis.

Electron immunocytochemistry, using rabbit polyclonal anti-Toxoplasma IgG as the primary layer in immunogold staining, was used to assess the distribution of Toxoplasma antigen within, and in relation to, intact tissue cysts in brain tissue from mice with congenital toxoplasmosis. Toxoplasma antigen was widely distributed in the matrix of the cyst, being most concentrated in the proximity of the inner surface of the parasitophorous vacuole membrane (PVM). Relatively little Toxoplasma antigen was detected directly associated with cystozoites. Small amounts of antigen were detected within the host cell components exterior to the PVM and in the host neuropil immediately adjacent to the tissue cyst.

Animals↗

PubMed1999

A retrospective study of seroconversion against Toxoplasma gondii during 3,000 pregnancies in Stockholm.

The incidence of Toxoplasma gondii infections in Swedish women during pregnancy is largely unknown. This retrospective pilot study was performed on consecutive sera sampled from 3,094 women at delivery during 1992-93. Specific IgG anti-toxoplasma antibodies were found in 14% (450/3,094). Serum drawn from the women during the first trimester and cord blood from their children were analysed for IgG and IgM anti-toxoplasma antibodies. A seroconversion during pregnancy was found in 4 women, whose children were followed up at 4 years of age. No signs of sequelae, either neurological or ophthalmological, were found in 3 of the children. The fourth child died at 1 year of age of a disease of different aetiology. An incidence of a primary toxoplasma infection of >1/1,000 pregnancies in this pilot study justifies a larger study.

Adolescent↗

PubMed2006

Reactivation of toxoplasma retinochoroiditis under atovaquone therapy in an immunocompetent patient.

PURPOSE: To report a case of toxoplasma retinochoroiditis reactivation in an immunocompetent patient under atovaquone therapy. METHODS: Case report. RESULTS: A healthy woman with a history of bilateral toxoplasma retinochoroiditis since childhood presented with a reactivation of toxoplasma retinochoroiditis. Because earlier treatment regimens had either produced intolerable side effects and/or were deemed ineffective for the prevention of reactivation, the patient was started on atovaquone suspension (750 mg three times a day). After initial regression of the lesion and still under atovaquone therapy, the patient presented again five weeks later with worsened best-corrected visual acuity. Examination showed that the lesion had expanded again and more cells were present in the vitreous. CONCLUSIONS: To our knowledge, this is the first report of a reactivation of toxoplasma retinochoroiditis in an immunocompetent patient under atovaquone therapy, possibly indicating tachyzoite resistance to atovaquone.

Adult↗

PubMed2006

Immunochemical identification and detection of a 36-kDa Toxoplasma gondii circulating antigen in sera of infected women for laboratory diagnosis of toxoplasmosis.

The detection of Toxoplasma gondii circulating antigens has been indicated to be a reliable diagnostic approach of active human toxoplasmosis. However, few reports have appeared in the literature regarding the diagnostic potential of T. gondii circulating antigens. Here, a specific antibody and western blot analyses were used to demonstrate the presence of a highly reactive antigen of 36-kDa, not only in the extract of T. gondii tachyzoites, but also in selected sera of women with confirmed laboratory and clinical signs of recent toxoplasmosis. The 36-kDa Toxoplasma antigen was purified from T. gondii tachyzoites and human serum using electroelution from preparative polyacrylamide gels. The purified polypeptides showed a single peak at 10.9 min when analyzed by capillary zone electrophoresis. Based on the above encouraging results, we have developed an ELISA format for the detection of target Toxoplasma antigen (TAg-ELISA) in human serum samples. The TAg-ELISA detected the target antigen in 88% sera of acutely infected women and showed high degree of specificity (91%) among sera from non-infected women. In conclusion, the detection of 36-kDa Toxoplasma circulating antigen in human sera appears to be a promising alternative approach for laboratory diagnosis of active T. gondii infection.

Adult↗

PubMed1974

Cell-mediated immunity against Besnoitia and toxoplasma in specifically and cross-immunized hamsters and in cultures.

The capacity of hamster peritoneal cell populations to control viability and growth of Besnoitia and Toxoplasma organisms was assessed in vivo and in vitro. Immunized hamsters reduced the homologous organisms 100- to 10,000-fold over a 5-day period, but the heterologous infection increased 100- to 1,000-fold in numbers, similar as in the nonimmune controls. Passively administered antibody was ineffective although lytic cofactors were supplied by hamsters. In cultures, peritoneal cells from Besnoitia-immune hamsters delayed the growth of homologous parasites to an average of 38.5 h per division; however, in Toxoplasma-immune and nonimmune cells, Besnoitia divided every 12.8 h. Specificity of immunity was pronounced against both infections. With cross-infections, Toxoplasma-immune cultures did not effectively delay Besnoitia growth; however, Besnoitia-immune cultures reduced Toxoplasma growth by one-half. Co-cultivation experiments demonstrated that specifically committed lymphocytes could instruct macrophages to reduce the homologous organism 10-fold, whereas heterologous organisms were reduced only 2-fold. Lymphocyte supernatants initiated hypersensitivity as indicated by macrophage activation and giant cell formation in culture. However, these supernatants did not transfer infection immunity. Lymphokines could account for the hypersensitivity phenomena, but cell-mediated infection immunity in this model required close lymphocyte-macrophage proximity. These studies indicate that a number of distinct processes including delayed hypersensitivity, macrophage activation, and specific cellular immunity are acting simultaneously during latent Besnoitia infection of hamsters. All three processes are mediated by lymphoid cells and appear to be specifically induced. Although activated macrophages develop some heightened nonspecific capabilities, these were several orders of magnitude below the specific effects.

Analysis of Variance↗

PubMed1991

Human platelet-mediated cytotoxicity against Toxoplasma gondii: role of thromboxane.

Human platelets, in the absence of antibody, are cytotoxic to tachyzoites of Toxoplasma gondii as determined by vital staining, transmission electron microscopy, and the failure of Toxoplasma to survive and replicate in mice after in vitro interaction of the organisms with platelets. Platelet to T. gondii ratios as low as 1:3 were toxic to the organisms with direct cell-cell contact essential for platelet-mediated cytotoxicity. Adherence of platelets to T. gondii and disruption of surface membranes and cytoplasmic contents of the organisms were observed ultrastructurally. Reactive oxygen species were not implicated in the platelet-mediated toxicity. The interaction of T. gondii with platelets resulted in a marked increase in thromboxane B2 (TXB2) production compared with that by unstimulated platelets. The cyclooxygenase inhibitors acetylsalicylic acid and indomethacin inhibited platelet-mediated cytolytic activity as did the selective TXA2 synthetase inhibitor dazmegrel, indicating a role for thromboxane in the platelet-induced cytotoxicity. Further, toxoplasmacidal activity was retained in the TXA2 synthetase-containing microsomal fractions of platelets disrupted by freezing and thawing; cytolytic activity was absent in microsome-depleted platelet supernatant fractions. Both the TXA2-generating platelet microsome system and a stable TXA2 analogue induced damage to the cellular membranes of the Toxoplasma as noted by transmission electron microscopy. These findings suggest that platelets may play a role in the host defense against Toxoplasma and that release of thromboxane may be important in this cytolytic process.

Animals↗

PubMed1993

Detection of Toxoplasma gondii by competitive DNA amplification of bronchoalveolar lavage samples.

The prevalence of pulmonary toxoplasmosis was assessed by a prospective analysis of 144 bronchoalveolar lavage (BAL) samples using competitive polymerase chain reaction (PCR) to avoid false-negative results due to PCR techniques. Six samples were excluded because they contained amplification reaction inhibitors. None of the samples from the 37 immunocompetent patients and only 1 sample (1.7%) from the 59 immunocompromised patients without human immunodeficiency virus infection were PCR-positive. In contrast, Toxoplasma gondii DNA was found in 6 (14%) of 42 samples from patients with AIDS. All 6 patients had < or = 40 CD4 cells/microL and anti-Toxoplasma antibodies, and 5 had other sites of Toxoplasma infection. Six other AIDS patients who had received treatment for cerebral toxoplasmosis were PCR-negative. Thus, pulmonary toxoplasmosis is frequent in AIDS patients who have other sites of Toxoplasma infection and low CD4 lymphocyte counts and who are not receiving prophylaxis.

Adolescent↗

PubMed1994

Polymerase chain reaction for Toxoplasma gondii DNA in the cerebrospinal fluid of AIDS patients with focal brain lesions.

OBJECTIVE: To study the accuracy of polymerase chain reaction (PCR) for Toxoplasma gondii DNA in the cerebrospinal fluid (CSF) of AIDS patients for the diagnosis of T. gondii encephalitis. PATIENTS: Eighty-two AIDS patients with brain lesions. At autopsy, 19 patients (group A) had toxoplasmic encephalitis and 33 (group B) primary brain lymphoma or other infections. Brain histology was not available for 30 patients; cerebral lesions improved after anti-Toxoplasma therapy in 16 (group C), but there was no improvement in 14 patients (group D). METHODS: T. gondii RH strain was serially diluted in microplate wells. After heat denaturation, nested PCR was performed on diluted tachyzoites and on 10 microliters CSF with primers flanking the B1 repetitive region of T. gondii genome. RESULTS: DNA from one to five tachyzoites was detected in each experiment. PCR was positive in eight (42.1%) out of 19 group A samples, none of the group B samples, 10 (62.5%) out of 16 group C samples and none of the group D samples. Among group A and C patients, PCR was positive in all 11, and in seven out of 24 (29.1%; P < 0.04) patients who had received anti-Toxoplasma therapy for less or more than 1 week at the time of rachicentesis, respectively. CONCLUSIONS: Nested PCR for T. gondii in CSF may improve early differential diagnosis of AIDS-associated focal brain lesions. Higher diagnostic accuracy was achieved when lumbar puncture was performed in the first week of anti-Toxoplasma therapy.

AIDS-Related Opportunistic Infections↗