Article Figures & Data
Figures
- Fig. 1.
QC-PCR of DNA from cells treated with DNase. FreeT. gondii organisms were untreated (lanes 1 and 2) or treated with anti-T. gondii serum and complement (lanes 3 and 4), or both untreated and treated T. gondii organisms were mixed at 1:1 (lanes 5 and 6). Cells were untreated (lanes 1, 3, and 5) or treated (lanes 2, 4, and 6) with DNase. DNA was extracted and amplified in the presence of competitor DNA with SAG-1 primers. Each lane represents QC-PCR of the DNA template corresponding to 100 tachyzoites. M, molecular size marker (φX174 HaeIII digest); sizes (in bases) are shown at left. Experiments were performed in triplicate. Similar results were obtained in four independent experiments.
- Fig. 2.
Kinetics of the number of viable tachyzoites after host cells were lysed by CTL. Ten thousand M12-neo-1 cells were infected with RH tachyzoites (infection rate, 45%) and cultured in the presence (hatched bars) or absence (open bars) of CTL at an effector/target cell ratio of 10:1. The reaction was terminated at the times indicated, and the number of viable tachyzoites was determined by QC-PCR. Results are shown as the mean parasite number per 300 M12-neo-1 cells initially placed in the culture ± 1 SD (four samples/group). In the same experiment, a portion of the infected M12-neo-1 cells was51Cr labeled, and CTL activity was assessed by the51Cr release assay. The percentages of specific51Cr release by the target cells during 4 and 6 h of culturing were 76 and 84%, respectively. Similar results were obtained in two independent experiments.
- Fig. 3.
Resistance of the intracellular T. gondii to DNase treatment after CTL-mediated lysis of the host cells. M12-neo-1 cells (104) infected with tachyzoites for 15 h (infection rate, 40%) were cultured in the presence of anH-2d-specific CTL line at an effector/target cell ratio of 10:1 for 4 h. The samples were disrupted by repeated passage through a 30-gauge needle. One of the disrupted samples was treated with rat anti-T. gondii serum (Ab) and complement (C). Finally, the samples were treated or not treated with DNase, and DNA was extracted. QC-PCR of each DNA sample was performed, and the SAG-1 gene copy number was estimated as described in Materials and Methods. Mean data from one of two similar independent duplicate experiments are shown.
Tables
- Table 1.
Number of SAG-1 gene copies in live and dead T. gondii after DNase treatmenta
DNase treatment Results obtained with the following treatment of T. gondii prior to DNase treatment: Not treated (P = 0.268)b Anti-T. gondiiantibody + complement (P < 0.001) Mixture of not treated plus anti-T. gondii antibody + complement (P = 0.01) − 135 ± 8 149 ± 14 163 ± 16 + 128 ± 5 <7 88 ± 2 ↵a Tachyzoites were not treated or were treated with anti-T. gondii serum and complement. Their viability was nearly 100% (not treated) or 0% (treated), as determined by the trypan blue dye exclusion test. These tachyzoites or their 1:1 mixture was treated (+) or not treated (−) with DNase. DNA was extracted and subjected to QC-PCR of the SAG-1 gene in triplicate. The results are expressed as the mean SAG-1 copy number per template DNA, corresponding to approximately 100 input tachyzoites ± 1 SD. The detection limit was seven copies.
↵b Data are for Student’s t test between DNase-treated and untreated groups.
- Table 2.
Number of Neo gene copies of M12-neo-1 cells after lysis by CTL and DNase treatmenta
Time (h) Neo gene copy no. with the following CTL/DNase treatment: % Specific51Cr release −/− −/+ +/− +/+ 0 195 ± 30 180 ± 17 ND ND ND 1 ND ND 199 ± 24 64 ± 3 48 2 ND ND 210 ± 36 26 ± 12 67 4 ND ND 188 ± 35 4 ± 2 78 ↵a 51Cr-labeled M12-neo-1 cells (104) were incubated or not incubated with alloreactive CTL cells (105) for 1 to 4 h, and treated or not treated with DNase. The number of Neo gene copies was determined by QC-PCR and is expressed as the mean copy number per template DNA, corresponding to 300 input M12-neo-1 cells ± 1 SD. The percent specific 51Cr release was determined by a standard method in the same experiment. Similar results were obtained in three independent experiments. ND, not done.
- Table 3.
Effect of T. gondii-specific CTL on the viability of intracellular T. gondiia
CTL Infection of target cells with T. gondii No. of copies of the following gene with the indicated DNase treatment: Neo SAG-1 − + − + − − 136 ± 40 118 ± 30 ND ND − + 148 ± 32 144 ± 20 110 ± 14 140 ± 52 + − 124 ± 7 122 ± 25 ND ND + + 137 ± 27 60 ± 19 102 ± 8 110 ± 9 ↵a M12-neo-1 cells (104) which were infected (+) or not infected (−) with T. gondii were incubated or not incubated with CTL specific for T. gondiifor 4 h at an effector/target cell ratio of 50:1 prior to DNase treatment. The numbers of Neo and SAG-1 gene copies were determined by QC-PCR as described in Materials and Methods and are expressed as the mean copy number for triplicate experiments ± 1 SD. Fifty-one percent of the target cells were infected with T. gondii, as determined by light microscopy. ND, not done.
