Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages

186

101

The development of Toxoplasma gondii was studied in mice fed bradyzoites. At one hour after oral inoculation (HAI), bradyzoites were found in cells of the surface epithelium and the lamina propria of the small intestine, primarily the ileum. Division into two tachyzoites was first observed at 18 HA1 in the intestine. At 24 HAI, organisms were also seen in mesenteric lymph nodes. Organisms were first detected in the brain at six days after oral inoculation with bradyzoites (DAI) but not consistently until 10 DAI. Immunohistochemical staining with bradyzoite specific (BAG-5 antigen) anti-serum showed that bradyzoites retained their BAG-5 reactivity even after the first division into two tachyzoites in the intestine at 18 HAL BAG-5 positive organisms were not seen 2-5 DAI. BAG-5 antigens reappeared in T. gondii at 6 DAI. Whole mice and individual tissues of mice fed bradyzoites were bioassayed in cats and mice for the presence of bradyzoites. Feces of cats fed murine tissues were examined for oocyst shedding for short prepatent periods. Bradyzoites were present in the intestines of mice up to 12 HA1 but not at 18 HAI, and tachyzoites and not bradyzoites disseminated to other tissues from the intestine. Bradyzoites were again detected 6 DAI. Using the mouse bioassay, T. gondii was first detected in peripheral blood at 24 HA1 and more consistently at 48 HAL Using a pepsin-digestion procedure and mouse bioassay, organisms were demonstrated in many tissues of mice 15 and 49 DAI.Supplementary key words. Cat feeding studies, immunohistochemical staining, oral infection, stage specific antibodies.NFECTIONS by Toxoplasma gondii are widely prevalent in hu-

Section: Discussionmentioning

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Bradyzoite‐Induced Murine Toxoplasmosis: Stage Conversion, Pathogenesis, and Tissue Cyst Formation in Mice Fed Bradvzoites of ‐ Different Strains of Toxoplasma gondii

Dubey

1997

186

101

“…Western blot studies have demonstrated that tachyzoites and bradyzoites are antigenically distinct [5,11,15,. Monoclonal antibodies specific to bradyzoites [3,15,20, 221 and tachyzoites [5] have been described. The development in vitro of bradyzoites and the conversion of bradyzoites isolated from mouse brains into tachyzoites has been demonstrated recently employing bradyzoite specific monoclonal antibodies [3, 191. In the present study, utilizing our previously developed bradyzoitespecific monoclonal antibodies, we describe a continuous cell culture system that allows for the in vitro study of bradyzoite development of T. gondii.…”

mentioning

confidence: 99%

A Cell Culture System for Study of the Development of Toxoplasma gondii Bradyzoites

Weiss

Laplace

Takvorian

et al. 1995

156

151

Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS-induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite-specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno-electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii. Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst-like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free-floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of gamma-interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL-6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.

“…Cysts from brains of mice that were mfected three months before were used as a controls for mature bradyzoites.In order to evaluate whether chemotherapeutic agents can also induce stage differentiation, murine bone marrowderived macrophages were infected with tachyzoites of T. gondii strain NTE, and were incubated in the presence of pyimethamine at different concentrations. Since previous experiments have shown that maximal expression of bradyzoite-specific antigens occurs on day 4 after infection, all cultures were evaluated on this day by determining the relative number of parasitophorous vacuoles expressing the MAb 4FS-reactive bradyzoite-specific antigen in an immunofluorescence assay [3].To determine whether the promoter of the bradyzoite-specific gene BAG1 [l] is stage-specifically regulated, the 5' promoter region of this gene was cloned upstream of the reporter gene encoding chloramphenicol acetyltransferase (CAT). Since the 3' flanking sequence has been shown to be not important for regulation of the B,4GI promoter, the 3' flanking region of SAGl was cloned downstream of the reporter gene.…”

mentioning

confidence: 99%

“…In order to evaluate whether chemotherapeutic agents can also induce stage differentiation, murine bone marrowderived macrophages were infected with tachyzoites of T. gondii strain NTE, and were incubated in the presence of pyimethamine at different concentrations. Since previous experiments have shown that maximal expression of bradyzoite-specific antigens occurs on day 4 after infection, all cultures were evaluated on this day by determining the relative number of parasitophorous vacuoles expressing the MAb 4FS-reactive bradyzoite-specific antigen in an immunofluorescence assay [3].…”

mentioning

confidence: 99%

Regulation of Developmental Differentiation in the Protozoan Parasite Toxoplasma gondii

Groß¹,

Bohne²,

Lüder³

et al. 1996

Toxoplasma gondii is an important pathogen of newborn infants and immunocompromised patients, such as those receiving organ transplants or suffering from the acquired immunodeficiency syndrome (AIDS). Whereas disease in newborns depends on congenital transmission of the parasite during primary infection of the mother, toxoplasmic encephalitis (TE) in AIDS patients seems to reflect reactivation of a latent infection [12]. It is still unclear whether conversion between bradqzoites and tachyzoites or formation and rupture of the cyst wall is important for pathogenesis of acute or reactivated toxoplasmosis [8].Recent progress in the generation of stage-specific monoclonal antibodies and genetic manipulation has made it possible to further dissect the mechanisms that are involved during tachyzoitebradyzoite interconversion [3, 151, In-vitro studies performed by independent investigators demonstrated that differentiation from the tachyzoite stage to the bradyzoite stage is inducable by external stress factors, such as pH modification of the cell culture medium or immunomodulation [4, 141. This study tnes to give new insights into the kinetics of tachyzoite to bradyzoite differentiation and what is known about the regulation of developmental differentiation of T. gondii.Mice were intraperitoneally infected with tachyzoites of T. gondii strain NTE. One day later, tachyzoites were harvested from the peritoneal cavity and were used to infect human foreslun fibroblasts. Differentiation into the bradyzoite stage was induced by shifting the pH of the cell culture medium above pH 8.0 [13]. The kinetics of the expression of stagespecific antigens, such as SAGl (tachyzoite) or BAGl (bradyzoite) and P115 (cyst wall) was determined by an immunofluorescence assay and by immunoelectron microscopy utilizing corresponding monoclonal antibodies (MAbs). Cysts from brains of mice that were mfected three months before were used as a controls for mature bradyzoites.In order to evaluate whether chemotherapeutic agents can also induce stage differentiation, murine bone marrowderived macrophages were infected with tachyzoites of T. gondii strain NTE, and were incubated in the presence of pyimethamine at different concentrations. Since previous experiments have shown that maximal expression of bradyzoite-specific antigens occurs on day 4 after infection, all cultures were evaluated on this day by determining the relative number of parasitophorous vacuoles expressing the MAb 4FS-reactive bradyzoite-specific antigen in an immunofluorescence assay [3].To determine whether the promoter of the bradyzoite-specific gene BAG1 [l] is stage-specifically regulated, the 5' promoter region of this gene was cloned upstream of the reporter gene encoding chloramphenicol acetyltransferase (CAT). Since the 3' flanking sequence has been shown to be not important for regulation of the B,4GI promoter, the 3' flanking region of SAGl was cloned downstream of the reporter gene. This construct was used to transiently transfect tachyzoites of the NTE strain followed b...