Heat-shock protein 70 (HSP70) is highly expressed in Toxoplasma gondii-infected cells. However, the role of this protein is not well understood, especially during apoptosis. This study addresses the mechanism behind the antiapoptotic chaperone activity of HSP70 in Toxoplasma-infected host cells using a human macrophage cell line, THP-1 by Western blot, DNA fragmentation assay, immunoprecipitation, and a caspase-3/7 activity assay based on cleavage of the colorimetric substrate DEVD-pNA. Apoptosis induced by arsenic trioxide (As(2)O(3)) was inhibited in T. gondii-infected THP-1 cells, but not in uninfected cells. Without As(2)O(3) induction of apoptosis, T. gondii infection caused increased expression of Bcl-2 and HSP70, but not caspase-3. However, active form caspase-3 levels were lower in As(2)O(3)-treated infected cells as compared with As(2)O(3)-treated uninfected cells. Bcl-2 expression in As(2)O(3)-treated infected cells was similar to that in cells infected with T. gondii. Translocation of apoptosis-inducing factor (AIF) and release of cytochrome c from mitochondria were inhibited in As(2)O(3)-treated infected cells as compared with As(2)O(3)-treated uninfected cells. Increased parasite loads in Toxoplasma-infected macrophages caused higher HSP70 and Bcl-2 expression in whole-cell extracts and fractionated components, respectively. However, expression of AIF and cytochrome c was unaffected. Toxoplasma dose-dependently inhibited caspase-3 activation, thus revealing an anti-apoptotic parasite activity on cytochrome c-mediated caspase activation in subcellular components. In addition, immunoprecipitation analysis suggested that HSP70 is capable of binding to the pro-apoptotic factors AIF and Apaf-1, but not to cytochrome c or procaspase-9. Taken together, these data demonstrate that T. gondii infection inhibits mitochondrial apoptosis through overproduction of anti-apoptotic Bcl-2 as well as HSP70, which are increased parasite loads dependently.

ABSTRACT.Purpose: Toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, can lead to severe visual impairment. T. gondii inhibits or delays programmed cell death caused by various apoptotic triggers; however, the mechanisms involved in the T. gondii-induced suppression of apoptosis in retinal cells have not been analysed in detail. Methods:We investigated the role of T. gondii infection in H 2 O 2 -induced apoptosis in human retinal pigment epithelial cells (ARPE-19) by monitoring the activities of apoptosis-regulating molecules and mitogen-activated protein kinases (MAPKs), including p38 MAPK. We also examined the gene downstream from p38 MAPK.Results: T. gondii infection significantly inhibited the cellular toxicity of H 2 O 2 (500 lm) and increased cell viability in a multiplicity of infection (MOI)-dependent manner by reducing DNA fragmentation and reactive oxygen species (ROS) generation in ARPE-19 cells. Western blot analysis also showed that T. gondii infection prevented the host cell expression of proapoptotic factors, such as Bad and Bax, and the activation of caspase-3. Infection with T. gondii increased the expression of the anti-apoptotic factor Bcl-2 in ARPE-19 cells under oxidative stress. In accordance with these findings, Toxoplasma infection was protective enough to suppress the phosphorylation of p38 MAPK following H 2 O 2 treatment. Exposure to H 2 O 2 increased the expression of heme oxygenase-1 (HO-1) in ARPE-19 cells, and its expression was significantly inhibited in H 2 O 2 -treated infected cells. Conclusion:The protective function of T. gondii infection against ROS-induced apoptosis results from changes in the expression of apoptotic molecules and the downregulation of stressinduced intracellular signalling.

Toxoplasmosis is usually diagnosed using serological tests with increases in specific IgG and IgM antibodies. However, these tests are not reliable in immunocompromised patients, nor is it possible to differentiate clearly between the acute and latent forms of toxoplasmosis [1,2]. Because a relatively high percentage of individuals have antibodies without a recognized parasite infection, the early detection of Toxoplasma gondii antigen in the serum or other body fluids of patients would help to diagnose correctly and prevent late complications. For detecting Toxoplasma antigen, several laboratory procedures are available. Direct detection procedures, such as microscopic examination, immune histology, or cell culture are reliable, but they are either insensitive or time-consuming [1,2]. PCR is highly sensitive and specific, although heme, heparin, and other poorly characterized substances have been reported to decrease the efficiency of PCR [3]. ELISA is considered to be a highly sensitive, practical method for detecting the parasite antigen [2]. Many reports have discussed titrating serum antibodies in hosts after Toxoplasma infection, however, little information is available on the correlations among parasitemia, circulating antigens, and antibody titers in T. gondii-infected hosts.To assess the sequential changes in parasite antigen and antibody responses in blood, 5 New Zealand white rabbits weighing 2-3 kg were infected with 1,000 tachyzoites of the RH strain of T. gondii subcutaneously. Then, blood samples were drawn from an ear vein of each rabbit every other day for 20 days. To check parasitemia in the rabbits, 0.5 ml of heparinized blood from each rabbit was injected intraperitoneally into 4 mice, and their survival was monitored for 20 days after infection.The ELISA for detecting circulating antigens was performed in microtitration trays [4,5]. To obtain mouse anti-Toxoplasma antisera, mice were infected with 20 brain cysts of avirulent Me49 strain of T. gondii orally. The mice were then sacrificed at 6 months after infection, and the sera were precipitated with saturated ammonium sulfate solution, resuspended in 0.01 M phosphate buffered saline. Mouse anti-Toxoplasma antisera were diluted with 0.1 M carbonate-bicarbonate buffer (pH 9.6, 10 mg/ml). Then, 100 ml were pipetted into 96-well microtiter plates (Nunc, Roskilde, Denmark) and incubated at 4℃ overnight. The plates were washed with PBS containing 0.05% Tween 20 (PBS/Tween 20), to which 0.1 ml of rabbit serum diluted 1 : 50 with PBS/ Tween 20 containing 0.1% bovine serum albumin was added. Toxoplasma lysate antigen (TLA) was prepared as a control. The plates were incubated at room temperature (RT) for 2 hr, and then 0.1 ml sample serum from the infected rabbit was added. After washing, 150 ml of horseradish peroxidase (HRP)-conju- Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondiiKorean J Parasitol. Vol. 47, No. 4: 409-412, December 2009 DOI: 10.3347/kjp.2009 Abstract: In this experiment, the correla...

The genetic diversity of Schistosoma haematobium remains largely unstudied in comparison to that of Schistosoma mansoni. To characterize the extent of genetic diversity in S. haematobium among its definitive host (humans), we collected S. haematobium eggs from the urine of 73 infected schoolchildren at 5 primary schools in White Nile State, Sudan, and then performed a randomly amplified polymorphic DNA marker ITS2 by PCR-RFLP analysis. Among 73 S. haematobium egg-positive cases, 13 were selected based on the presence of the S. haematobium satellite markers A4 and B2 in their genomic DNA, and used for RFLP analysis. The 13 samples were subjected to an RFLP analysis of the S. haematobium ITS2 region; however, there was no variation in size among the fragments. Compared to the ITS2 sequences obtained for S. haematobium from Kenya, the nucleotide sequences of the ITS2 regions of S. haematobium from 4 areas in Sudan were consistent with those from Kenya (> 99%). In this study, we demonstrate for the first time that most of the S. haematobium population in Sudan consists of a pan-African S. haematobium genotype; however, we also report the discovery of Kenyan strain inflow into White Nile, Sudan.

Ascaris suum eggs are inactivated by composting conditions; however, it is difficult to find functional changes in heat-treated A. suum eggs. Here, unembryonated A. suum eggs were incubated at 20℃, 50℃, and 70℃ in vitro, and the gene expression levels related to viability, such as eukaryotic translation initiation factor 4E (IF4E), phosphofructokinase 1 (PFK1), and thioredoxin 1 (TRX1), and to apoptosis, such as apoptosis-inducing factor 1 (AIF1) and cell death protein 6 (CDP6), were evaluated by real-time quantitative RT-PCR. No prominent morphological alterations were noted in the eggs at 20℃ until day 10. In contrast, the eggs developed rapidly, and embryonated eggs and hatched larvae began to die, starting on day 2 at 50℃ and day 1 at 70℃. At 20℃, IF4E, PFK1, and TRX1 mRNA expression was significantly increased from days 2-4; however, AIF1 and CDP6 mRNA expression was not changed significantly. IF4E, PFK1, and TRX1 mRNA expression was markedly decreased from day 2 at 50℃ and 70℃, whereas AIF1 and CDP6 mRNA expression was significantly increased. The expressions of HSP70 and HSP90 were detected for 9-10 days at 20℃, for 3-5 days at 50℃, and for 2 days at 70℃. Taken together, incremental heat increases were associated with the rapid development of A. suum eggs, decreased expression of genes related to viability, and earlier expression of apoptosis-related genes, and finally these changes of viability- and apoptosis-related genes of A. suum eggs were associated with survival of the eggs under temperature stress.