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Cerebral involvement during malaria is a complication that leads to seizure, coma, and death. The effect of new neuroprotective therapies has not yet been investigated, although cerebral malaria shares some features with neurological stroke. Erythropoietin (EPO) is one of the more promising drugs in this area. We measured the effect of EPO on the survival of mice infected with Plasmodium berghei ANKA and demonstrated that inoculations of recombinant human EPO at the beginning of the clinical manifestations of cerebral malaria protect >90% of mice from death. This drug has no effect on the course of parasitemia. The effect of EPO was not related to either the inhibition of apoptosis in the brain or the regulation of the increase and decrease of nitric oxide production in the brain and blood, respectively. Tumor necrosis factor-alpha and interferon-gamma mRNA overexpression was inhibited by EPO, and treated mice had fewer brain hemorrhages. EPO has been used in patients with chronic diseases for years, and more recently it has been used to treat acute ischemic stroke. The data presented here provide the first evidence indicating that this cytokine could be useful for the symptomatic prevention of mortality during the acute stage of cerebral malaria.

Present serological methods differentiate poorly between acute and chronic toxoplasmosis in pregnant women, particularly when immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to Toxoplasma gondii are present simultaneously. In the present study, a simple test for discriminating between high-avidity antibodies, which are usually present in chronic infections, and low-avidity antibodies, typical of acute infection, was evaluated. Sera were evaluated for Toxoplasma gondii antibodies using a commercial enzyme immunoassay, but a duplicate well was washed in 6M urea to disrupt low-avidity complexes. Results are expressed as the percentage of antibodies resisting elution by urea. Equivocal sera (n = 493) containing both IgG and IgM Toxoplasma gondii antibodies from 309 pregnant women whose status as chronically or acutely infected had been independently determined using standard methods were evaluated for antibody avidity. A value of > 35% elution-resistant antibodies was always associated with chronic infection and could absolutely exclude a recent (< 3 months) infectious incident. Values of < 35% require repeat testing four weeks later to confirm the patient's status, since a proportion of individuals with chronic toxoplasmosis maintain low-avidity antibodies over long periods. This inexpensive, simple method can provide reassurance to clearly chronically infected individuals and avoids the need for repeated testing in these cases.

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The parasite Toxoplasma gondii can infect most mammals and birds, sometimes causing severe pathology. Primary infection during pregnancy can result in abortion or fetal defects. Host immunity, particularly cellular immunity towards antigenic peptides, can control infection, but an efficient vaccine is not yet available. We have evaluated T-cell responses to a crude soluble toxoplasma antigen (ST-Ag) and to five recombinant peptide antigens of cells in whole-blood cultures from 22 pregnant women with preexisting infections and from 7 pregnant negative controls. Cells from all infected patients but from none of the controls responded specifically to ST-Ag by expressing surface CD25 on culture. Responses to the recombinant antigens showed considerable variation between individuals. rGRA1 elicited a response in 16 of the 22 samples (73%), rSAG1 in 13, rGRA7 in 9, rGRA6-CT in 4, and rGRA6-NT in only 1. Most responding cells were CD4؉ . Cells from infected subjects cultured with ST-Ag all released high levels of gamma interferon (IFN-␥) into the culture supernatant (4,343 ؎ 2,536 pg/ml). Cells from 12 patients released IFN-␥ after culture with rGRA1 (130 ؎ 98 pg/ml), those from 10 patients released it after culture with rSAG1 (183 ؎ 128 pg/ml), and those from 4 patients released it after culture with rGRA7 (324 ؎ 374 pg/ml). Intensity of IFN-␥ production in response to the latter two recombinant antigens correlated with responses to ST-Ag (r ‫؍‬ 0.61 and 0.53, respectively; P < 0.01). Interleukin-4 was always absent from supernatants of cells stimulated with toxoplasma antigens. The heterogeneity of human responses to individual recombinant toxoplasma antigens should be considered in the design of potential vaccines.The protozoan parasite Toxoplasma gondii infests a wide range of mammalian hosts and some birds, usually with few pathological consequences. Human infestation is widespread and originates mainly, but not only, with cats. Incidence varies widely between geographical sites, but disease is rare, except in immunodepressed individuals and, especially, as a result of congenital infection. Cellular immunity provides an essential component of protection against T. gondii, and J. K. Frenkel (reviewed by Darcy and Santoro [2]) first demonstrated the key role played by T lymphocytes. Infection during pregnancy, especially during the third trimester, carries a high risk of mother-to-fetus transmission with severe consequences for the child. Immunocompetent mothers infected before pregnancy do not pass the parasite to their offspring in utero, even if reexposure to the parasite occurs during the critical period. The crucial T-cell responses to T. gondii may be measured (10), and we have recently determined that cytofluorimetric detection of CD25 expression after stimulation of whole-blood cultures with T. gondii antigen for 7 days is a sensitive and specific test, comparable to the 3 H-thymidine incorporation assay (5). The assay, which uses a crude soluble T. gondii antigen preparation (ST-Ag), was found to be useful ...

An enzyme-linked immunosorbent assay (ELISA) using two recombinant antigens of Toxoplasma gondii (GRA1 and GRA6 Nt) was developed in order to differentiate between pregnant women with a serological profile of recently acquired infection and those with chronic infection. Both proteins were expressed in Escherichia coli as glutathione S-transferase fusion proteins. Thirty-two serum samples from subjects who presented seroconversion within 3 months before sampling (group 1; acute profile), 46 serum samples from women who had a positive serology at least 1 year before sampling (group 2; chronic profile), and 100 serum samples from pregnant women who were not infected by T. gondii (group 3) were examined for immunoglobulin G (IgG) reactivity. For both antigens, the specificity reached 98%. In both groups of infected patients, the overall sensitivity scored was 60% for GRA1 and 83% for GRA6 Nt. In group 1, 34% of sera reacted with GRA1 whereas 84% of sera reacted with GRA6 Nt; in group 2, however, sensitivities were 78.2 and 82.6%, respectively. Combination of the readings obtained with both antigens yielded a sensitivity of 91%. A serological follow-up of 10 women who seroconverted during pregnancy displayed three different serological patterns: (i) a GRA profile paralleling the IgG curve, as detected by the commercial kit, (ii) a GRA1 profile, or (iii) GRA1 and GRA6 Nt profiles remaining negative for at least 8 weeks after the reference test gave positive results. Taken together, these results suggest that neither GRA1 nor GRA6 Nt is sensitive enough to be used routinely to differentiate between acute and chronic toxoplasmic infections.Maternal infection by Toxoplasma gondii during pregnancy may result in a fetal infection with consequences ranging from severe neurological abnormalities and chorioretinitis to subclinical infection at birth, which is marked by a risk of late onset of ocular lesions (19). The risk of transmission to the fetus and the severity of congenital infection depend on when, during pregnancy, the woman becomes infected: the probability of fetal infection varies from 6% at the end of the first trimester to 72% at 36 weeks of gestation (8). The severity of infection is greater in fetuses infected early in pregnancy than in those contaminated later (8). Because only 5% of women who seroconvert present clinical signs, diagnosis of maternal infection relies on serological tests. For pregnant women, these should not only detect seroconversion but also estimate the date of maternal contamination in order to estimate the risk of mother-to-child transmission and the severity of fetal infection.

A polyclonal antibody was raised against a Toxoplasma gondii 14-3-3^gluthatione S-transferase fusion protein obtained by cloning a 14-3-3 cDNA sequence determined from the T. gondii database. This antibody specifically recognized T. gondii 14-3-3 without any crossreaction with mammalian proteins. Immunofluorescence microscopy studies of the tachyzoites or the T. gondii-infected cells suggested cytosolic and membranous localizations of 14-3-3 protein. Different subcellular fractions were prepared for electrophoresis analysis and immunodetection. 14-3-3 proteins were found in the cytosol, the membrane fraction and Triton X-100-resistant membranes. Two 14-3-3 isoforms were detected. The major one was mainly cytoplasmic and to a lesser extent membrane-associated, whereas the minor isoform was associated with the detergent-resistant lipid rafts. ß