Ji-Young Kim, et al. cleosome-sized fragments of DNA, which produce ladders when analyzed with agarose gel electro- phoresis. Our results indicated that the induction of DNA fragmentation in AD treated spleen cells was inhibited by T. gondii infection (Fig. 1). Apoptotic cells can be detected by the binding of fluorescent labeled annexin V to the plasma membrane. As such, early and late apoptotic cells were measured by flow cytometric analysis after double staining with FITC-conjugated annexin V and PI.14 While flow cytometric analysis could not differentiate late apoptosis and necrosis of cells, it demonstrated that concomitant infection with T. gondii abrogated AD induced apoptosis. Specifi- cally, in the AD treated population, 80.3% of cells were apoptotic, as opposed to 30.1% of the cells treated with both AD and T. gondii after incuba- tion for 18 h (Fig. 2). Proteins of the Bcl-2 family with pro-apoptosis or anti-apoptotic functions reside in the outer mitochondrial membrane. Bax, a pro-apoptotic protein, triggers the release of cytochrome c from mitochondria.1 Conversely, Bcl-2 and Bcl-XL are capable of blocking the release of cytochrome c, thereby preventing the activation of caspase 9. The induction of apoptosis by T. vaginalis is associated with a reduction of expression of anti-apoptotic Bcl-XL protein, but not Bcl-2, and the activation of caspase 9 suggests that mitochondria play a role in T. vaginalis- induced RAW 264.7 programmed cell death.13 RT- PCR analysis demonstrated that T. gondii infection promotes the activation of anti-apoptotic Bcl-2 family members but not pro-apoptotic pathways.15 Our studies showed that the expression of Bax mRNA and protein was indistinct in AD/T. gondii-treated mouse spleen cells, compared to uninfected cells (Fig. 3). Likewise, expression of Bcl-2 and Bcl-XL mRNA and Bak protein were unchanged in mouse spleen cells treated with AD and infected with T. gondii (Figs. 3 & 4). These results suggested that the anti-apoptotic Bcl-2 protein was not affected in T. gondii infection and that the other anti-apoptotic protein, Bcl-XL may be involved. Generally T. gondii infection pro- motes the suppression of pro-apoptotic Bax, but not anti-apoptotic Bcl-2 proteins (Fig. 4). Caspase 3 is a critical downstream member of the caspase family, and is an essential effector of apoptosis. The resistance of T. gondii infected cells Yonsei Med J Vol. 47, No. 6, 2006 to apoptosis may be attributed to prevention of caspase 3 activity.1,7,16 In the present study, inacti- vation of caspase 3 at 120 min post infection and decreased PARP were present in cells treated AD and infected with T. gondii as determined by Western blotting (Fig. 4). Cleaved caspase 8, which was present in cells treated with AD and T. gondii infection, may have induced a weak apoptosis response. We propose that AD did not trigger apoptosis via the caspase 8 pathway and that infection by the parasite induced apoptosis slightly. Activation of the caspase cascade leads to cleavage of a variety of target proteins including PARP, nuclear lamins, and protein kinase C (PKC); both PARP protein levels and cleavage of nuclear target proteins are down regulated by T. gondii.16 Our results showed that T. gondii mediates inhibition of apoptosis by a blockade of Bax ex- pression and caspase 3 activation. Inhibition of apoptosis by T. gondii is regulated at the transcriptional level of NF-κB, as infection with T. gondii induces the activation of NF-κB and the transcription of anti-apoptotic genes.17-21 Phos- phorylation of IκB activates and promotes NF-κB translocation to the nucleus, where NF-κB asso- ciates with the transcription of proinflammatory mediators. Further, expression of NF-κB is correlated with anti- apoptotic protein expression after T. gondii infection. In this experiment, spleen cells treated with AD and infected with T. gondii showed increased NF-κB DNA binding activity by EMSA (Fig. 5). Condensation of chromatin, disruption of nuclear architecture, vacuolation of the cytoplasm, membrane blebbing, detachment from substrate, and cell shrinkage are some of the morphological changes associated with apoptosis. We observed some of these morphologic changes in cells treated with AD and/or T. gondii using TEM. Condensed chromatin and fragmented nuclei of cells were apparent 6 h after treatment of the cells with AD; however, there were no apoptotic changes in cells treated with AD and T. gondii (Fig. 6C & D). In contrast to the inhibition of apoptosis in T. gondii infected cells, parasite infection induced apoptosis has been reported in neighboring non-invaded cells.5,6,22 However, we were unable to identify such changes in uninfected mouse spleen cells on TEM (Fig. 6D & E). In conclusion, infection with T. gondii induces