Fausto G. Araujo

Remarkable in vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against tachyzoites and tissue cysts of Toxoplasma gondii.

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 micrograms/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 sacrificed 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.

Effect of clarithromycin and azithromycin on production of cytokines by human monocytes

We examined the in vitro effect of clarithromycin and azithromycin on cytokine production by LPS and Pansorbin stimulated human monocytes. At concentrations that are physiologically achievable, both antibiotics affected in vitro production of IL-1alpha, IL-1beta, IL-6, IL-10, GM-CSF and TNF-alpha to varying degrees. Of those individuals in whom a significant increase or decrease in cytokine production was noted, clarithromycin treatment resulted in a significant suppression of production of each cytokine in 71% and a significant increase in 29% of the individuals. Similar results were noted with azithromycin. The results with IL-6 and TNF-alpha in the clarithromycin studies were most striking. A significant decrease was noted in 60% of individuals for IL-6 and 86% for TNF-alpha. For azithromycin, the most interesting results were for IL-1alpha (decrease in 100% of individuals) and for TNF-alpha (decrease in 100% of individuals). These results show that both clarithromycin and azithromycin alter cytokine production in human monocytes and thus possess immunomodulatory activity.

Primary and Reactivated Toxoplasma Infection in Patients with Cardiac Transplants: Clinical Spectrum and Problems in Diagnosis in a Defined Population

We have attempted to define the serologic criteria for diagnosis of toxoplasmosis in heart transplant recipients. Of 31 patients who were seronegative before transplantation, 4 received a heart from a seropositive donor, and 3 of these 4 had seroconversion and developed life-threatening toxoplasmosis; the remaining 27 did not have seroconversion or develop clinical toxoplasmosis. Of 19 patients who had antibodies to Toxoplasma before transplantation, 10 developed significant increases in test titers of the dye test or double-sandwich IgM enzyme-linked immunosorbent assay but did not develop a clinical illness that could be attributed to toxoplasma infection. Significant serologic changes occurred more often in patients who received azathioprine, corticosteroids, and antithymocyte globulin than in those who received cyclosporine, corticosteroids, and antithymocyte globulin (p less than 0.05). These data show the wide clinical spectrum and differences in kinetics of antibody response of patients who develop toxoplasma infection after transplantation, and suggest that clinical disease occurs in those who have seroconversion but is rare in patients with preexisting antibody who have serologic evidence of recrudescence.

Azithromycin, a macrolide antibiotic with potent activity against Toxoplasma gondii.

Doses of 200 mg of azithromycin per kg (body weight) administered by the oral route daily for 10 days completely protected mice against death caused by intraperitoneal infection with Toxoplasma gondii. The same treatment regimen also protected 80% of mice infected intracerebrally, which suggests that azithromycin attains active concentrations in the inflamed central nervous system.

Effect of Clindamycin on Acute and Chronic Toxoplasmosis in Mice

The effect of clindamycin on survival of mice during acute infection with the RH and C56 strains of Toxoplasma and the ability of this drug to prevent congenital transmission during the acute stage of the infection in the mother and to eradicate the parasite from tissues of mice chronically infected with the C56 strain were evaluated. The drug effectively prevented death due to the acute infection and, in the experimental model employed, eradicated the organism at least from the liver, spleen, and brain of approximately 30 to 50% of the acutely infected animals which survived. Clindamycin also effectively prevented congenital transmission during the acute infection in the mother. During short-term treatment (7 days), persistent parasitemia in the chronic infection was effectively diminished or eradicated. More prolonged treatment resulted in a significant clearing of the organisms from the spleens and livers, but not from the brains, of chronically infected mice.

SERODIAGNOSIS OF RECENTLY ACQUIRED TOXOPLASMA GONDII INFECTION USING AN ENZYME-LINKED IMMUNOSORBENT ASSAY WITH A COMBINATION OF RECOMBINANT ANTIGENS

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) using four recombinant antigens of Toxoplasma gondii (rP22, rP25, rP29, and rP35) was used in an attempt to differentiate pregnant women with toxoplasma serologic profiles (TSPs) indicative of recently acquired infections (acute profile) from those with TSPs indicative of infections acquired in the distant past (chronic profile). In general, immunoglobulin G antibodies in sera from women with the acute profile reacted more strongly with the recombinant antigens than did those in sera from women with the chronic profile. However, reactivities differed significantly between antigens that reacted with a single serum and between sera that reacted with a single antigen. Because of these variations, we employed a combination of the four antigens in an ELISA (Comb-ELISA) and evaluated its ability to distinguish pregnant women with the acute profile from those with the chronic profile. Eighteen of 20 (90%) sera from acute-profile women were positive in the Comb-ELISA, whereas 69 of 70 (98.6%) sera from the chronic-profile women were negative. Thus, the Comb-ELISA may be useful for diagnosis of toxoplasmosis in pregnant women and for differentiation between recently acquired infections and infections acquired in the more distant past.

Protection against lipopolysaccharide-induced death by fluoroquinolones.

ABSTRACT Because fluoroquinolones have an immunomodulatory effect on cytokine production by lipopolysaccharide (LPS)-treated human monocytes, we examined the effect of fluoroquinolones on the survival of mice injected with a lethal dose of LPS. Trovafloxacin (100 mg/kg), ciprofloxacin (250 mg/kg), and tosufloxacin (100 mg/kg) protected 75% (P = 0.0001), 25% (P = 0.002), and 50% (P = 0.002), respectively, of mice against death. The fluoroquinolones significantly reduced serum levels of interleukin-6 and tumor necrosis factor alpha in LPS-treated mice. The protective effects of fluoroquinolones in LPS-induced shock in mice may also occur in humans.

Chronic infection with Toxoplasma gondii does not prevent acute disease or colonization of the brain with tissue cysts following reinfection with different strains of the parasite.

Two strains of Toxoplasma gondii with different capacities to induce disease and brain lesions in mice were used to study the effects of reinfection with the parasite on a previously infected host. In spite of marked antibody and cell-mediated immune responses, chronically infected mice developed disease and died of acute toxoplasmosis when reinfected with a strain different from the one causing the primary infection. Moreover, the marked antibody and cell-mediated immune responses of the chronically infected mice did not prevent invasion of their brains and formation of tissue cysts by the reinfecting strain. Tissue cysts of the reinfecting strain were demonstrated in the brains of the chronically infected and subsequently reinfected mice. These results highlight the importance of strain differences in the pathogenesis of toxoplasmosis.

Cloning of cDNAs encoding a 28 kilodalton antigen of Toxoplasma gondii

By screening cDNA libraries in lambda gt11 with antibodies raised against the previously described protective F3G3 antigen of Toxoplasma gondii, and subsequently screening with nucleic acid probes, we have isolated cDNA clones that encode a 28 kDa antigen of T. gondii that is likely one of the two antigenic components of the F3G3 antigen. The gene apparently exists as a single copy in the tachyzoite haploid genome of the three strains of T. gondii examined. Northern blot analyses revealed that the cDNAs hybridize with a major T. gondii RNA species of 1.1 kb. Together the cDNAs encompass 1051 bp of cDNA sequence containing an open reading frame with the capacity to encode a 28 kDa protein. Antibodies that were affinity purified using recombinant fusion proteins produced by two of the clones reacted on protein blots of whole T. gondii lysate with a single antigen having an apparent molecular mass of 28 kDa. Both recombinant fusion proteins reacted with IgG antibodies in sera of mice and humans infected with T. gondii and therefore might be useful for the development of diagnostic assays for T. gondii infection.

A morphological study of chronic cerebral toxoplasmosis in mice: comparison of four different strains of Toxoplasma gondii

The pathological changes, host-parasite relationship and structure of the tissue cysts in the brains of mice chronically infected with four different strains ofToxoplasma gondii were examined by light and electron microscopy. In mice infected with the mouse-adapted ME49 strain for 4, 8, 12, 16 and 25 weeks, the pathological changes consisted of moderate to severe meningitis and cuffing of blood vessels by inflammatory cells. At 4 weeks post-infection (p.i.), lymphocytes were the major cell type, but at later time points, plasma cells predominated. Large numbers of cysts were observed at between 4 and 12 weeks p.i., with a decrease being seen at 16 weeks p.i. Microglial nodules, many containing tachyzoites or bradyzoites, were present at all time points. In contrast, the three strains isolated from patients with the acquired immune deficiency syndrome (AIDS) resulted in no meningitis in two cases (DEY, DAG) and in mild meningitis in one case (WIL), although all three showed some cuffing of blood vessels. In addition, only very low numbers of cysts and nodules were observed. Ultrastructurally, the cysts of all four strains were seen to be located within host cells. The cysts of the ME49 strain differed from those of the other strains in that a proportion contained immature and dividing bradyzoites at all time points, whereas those of the other strains contained only mature bradyzoites. From the observation of nodules with parasites and cysts with immature zoites, it would appear that the ME49 strain may result in an unstable chronic infection with a continuous turnover of cysts, a feature that should be taken into consideration when this strain is used as an experimental model of chronic toxoplasmosis.