Compound 566C80, 2-[trans-4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone, was studied for its in vitro and in vivo activities against Toxoplasma gondii. Replication within human foreskin fibroblasts of tachyzoites of seven different strains, five of them isolated from AIDS patients, was inhibited by concentrations as low as 4.8 x 10-9 M. In vivo, a dose of 100 mg/kg of body weight per day, administered by gavage for 10 days, protected 100% of mice against death due to infection with five different strains of T. gondii, including the highly virulent RH strain. A dose of 50 mg/kg/day protected at least 80% 'of mice infected with the same inoculum, and a dose as low as 9.3 mg/kg/day protected 40 to 60% of mice. Treatment with 50 mg/kg/day for 30 days completely eradicated parasites from mice infected with four of five strains of T. gondii. 566C80 was active in vitro against the cyst stage of T. gondii at concentrations of 50 to 100 ,ug/ml. In vivo activity against this form of T. gondii was examined in mice infected for 6 weeks with strain ME49 and then treated orally with 100 mg of 566C80 per kg per day for 8 weeks. Treated mice sacrfficed at 2-week intervals revealed a steady decline in the numbers of cysts in their brains compared with untreated controls. In addition, mortality as well as clinical signs of brain infection was absent from treated mice, whereas control mice had a high mortality rate and showed clinical signs of central nervous system infection. These results reveal remarkable in vitro and in vivo activities of 566C80 against T. gondii.

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The immunoglobulin G (IgG) subclass response to Toxoplasma gondii antigens during the acute and chronic stages of T. gondii infection were studied by using immunoblots with reduced antigen (RA) and nonreduced antigen (NRA) preparations. Serum samples were from individuals with acute or chronic T. gondii infection, and sequential samples were from women who seroconverted during gestation and were treated with spiramycin. IgGl antibodies were predominant in sera from each of the groups and recognized a large number of RA and NRA. In the latter group of patients, IgGl and IgG3 were the first antibodies to appear in response to the infection. In all groups, an antigen with a molecular weight (MW) of 30,000 was the most intensely stained and frequently recognized by IgGl antibodies in NRA preparations. In RA preparations, antigens of MW 35,000 and 30,000 were the most intensely stained and frequently recognized by IgG1 antibodies. An antigen with an MW of 22,000 was intensely stained in the IgGl immunoblots of the NRA preparation and to a lesser extent in the RA preparation. In contrast to immunoblots with IgGl antibodies, very few antigens in the RA and NRA preparations were detected by IgG2 and IgG3 antibodies; IgG4 antibodies rarely detected any antigens. Of interest was that IgG2 antibodies detected antigens distributed over the entire MW range, whereas those detected by IgG3 antibodies were located mostly below the 35,000 MW marker. Enzyme-linked immunosorbent assay results paralleled those of the immunoblots in that IgGl antibodies were predominant.

The immunoglobulin A (IgA) antibody response to antigens of Toxoplasma gondii during the acute and chronic stages of T. gondii infection was studied by immunoblotting with a nonreduced antigen (NRA) preparation. Serum samples were from individuals with acute or chronic T. gondii infection, and sequential samples were from women who seroconverted during gestation or congenitally infected infants. IgA antibodies to a variety of antigens were present in sera from each of the groups, irrespective of the titer in an IgA enzyme-linked immunosorbent assay. The predominant NRA recognized by IgA antibodies in sera from each of the four groups had an approximate molecular weight of 30,000. Other prominent NRAs had approximate molecular weights of 35,000 and 5,000; however, the latter NRA was not present in sera of congenitally infected infants.

Toxoplasma antigens were detected in sera and urine of mice acutely infected with Toxoplasma gondii. The concentrations of antigens in the urine samples measured by enzyme-linked immunosorbent assay were similar to those detected in the sera of the corresponding mice. The major antigens were not dialyzable and were largely destroyed by treatment with trichloroacetic acid and heat (100°C for 1 h). Toxoplasma antigens were demonstrable on Western blots (immunoblots) of the urine samples.The alarming incidence of toxoplasmic encephalitis in patients with acquired immunodeficiency syndrome and the difficulty encountered in establishing the diagnosis without brain biopsy has served as an impetus for development of new, noninvasive methods for diagnosis of this entity (7). The earlier demonstration of Toxoplasma antigens in serum samples from acutely infected animals and humans (1,3,4,6,8,10,11) suggested to us that if antigens also appear in the urine, their detection might prove to be of diagnostic value. The experiments reported here were performed to determine whether Toxoplasma antigens can be detected in urine from a murine model of acute toxoplasmosis.Female Swiss Webster mice weighing 18 to 20 g (Simonson Laboratories, Gilroy, Calif.) were placed in metabolic cages, two mice per cage, 1 day prior to infection. A total of five experiments were performed. The first two were pilot experiments for determining the dose of Toxoplasma gondii to be used and to ascertain the optimal times for collection and the optimal storage conditions for urine and serum. Eight mice were used for each experiment.Urine was collected for 24 h and used as normal urine. Thereafter, the mice were infected intraperitoneally with 105 trophozoites of the RH strain of T. gondii obtained from the peritoneal fluid of mice infected 2 days previously (12). Urine was collected on days 4 through 7 after infection (all infected mice died by day 8). Urine samples collected on a given day from the 8 mice were pooled and centrifuged for 10 min at 1,300 x g, and the pellet was discarded. The supernatant was used undialyzed or was dialyzed overnight against 0.01 M phosphate-buffered saline, pH 7.2 (PBS), by using dialysis membrane (Spectrum Medical Industries Inc., Los Angeles, Calif.) with a 12,000to 14,000-molecularweight cutoff. The undialyzed and dialyzed specimens were stored at -20°C. Preliminary experiments revealed that results with urine placed at 4°C and studied within 10 days did not differ from those obtained with urine samples stored at -20°C for up to 30 days.Serum was collected on days 4, 5, and 6 of infection from mice infected at the same times and with the same inocula as those from which urine was collected. Sera obtained from uninfected mice were used as controls. The sera were stored at -20°C and run in parallel with the urine samples.Urine samples and sera were tested for the presence of Toxoplasma antigen in a modification (J. S. Remington, Y. * Corresponding author. Suzuki, and P. Stepick-Biek et al., manuscript in preparation) of...