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*
Department of Medicine, Division of Infectious Disease, and
Department of Medicine, Division of Medical Oncology, University of Colorado Health Sciences Center, Denver, CO 80220
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has long been appreciated that viruses subvert normal host cell functions to replicate or establish latency, and infection with certain intracellular pathogens, including viruses and bacteria, may induce host cell apoptosis (13, 14, 15, 16, 17, 18, 19, 20). Despite the medical importance of protozoans such as T. gondii, little is known regarding how these intracellular parasites subvert host cell processes to their own advantage (21). In this regard, T. gondii infects all nucleated cells, yet causes no obvious disturbance until the dividing parasites rupture the host cell (22). This is surprising in that the stress of infection could induce apoptotic cell death of the host cell (23). We wondered whether infected cells appeared unperturbed before rupture due to inhibition of apoptosis as has been reported for certain viral infections (24, 25, 26, 27, 28, 29). The following studies were performed to investigate this hypothesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tachyzoites of T. gondii were maintained in human foreskin fibroblasts (CRL 1634, American Type Culture Collection, Rockville, MD) as previously described (22). The T. gondii strain RH (from Lloyd Pfeffercorn) is a well-characterized laboratory isolate that has been used by us previously (22, 30).
Other cell lines
Murine MLR T lymphoblasts were derived by culturing 5 x 106 spleen cells from C57Bl/6 (B6) or B6smn.C3H-gld (B6-gld) mice (The Jackson Laboratory, Bar Harbor, ME; both H-2b) with 1 x 106 irradiated spleen cells from BALB/c mice (H-2d) in individual wells of a 24-well plate in medium R10, which consists of RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT), 4 mM glutamine (Sigma Chemical Co., St. Louis, MO), MEM nonessential amino acids (Sigma), MEM vitamins (Sigma), 1 mM pyruvate (Sigma), 50 nM 2-ME (Sigma), and penicillin/streptomycin (Life Technologies). After 5 days of culture, fresh medium containing 10 U/ml recombinant human IL-2 (Hoffmann La Roche, Basel, Switzerland) was added. The resulting lymphoblasts were isolated by Lympholyte M density gradient (Cedarlane, Hornby, Canada) on day 7 and used within 3 days of separation.
The H-2d-specific CD8-positive CTL line L3 was derived by Glasebrook and Fitch (31) from C57Bl/6 mouse spleen cells stimulated with DBA/2 spleen cells. L3 was propagated by periodic stimulation with irradiated BALB/c spleen cells. L1210-Fas, a lymphocytic cell derived from a DBA/2 mouse, which expresses H-2d, was obtained from Pierre Golstein (Centre d’Immunologie INSERM-CNRS, Marseille, France). The Fas (CD95) gene has been transfected into this cell line and is constitutively expressed (32). A20 (American Type Culture Collection, TIB 208) is a B cell lymphoma cell line derived from a BALB/c mouse that expresses H-2d. WEHI 231 (American Type Culture Collection, CRL 1702) is a pre-B leukemia cell that can be induced to undergo apoptosis by gamma irradiation (33). CTLL-2 (American Type Culture Collection, TIB 214) is a T cell line that requires IL-2 for survival and undergoes apoptosis when IL-2 is removed (34). The mastocytoma cell line P815 (American Type Culture Collection, TIB-64) is a DBA/2 (H-2d)-derived tumor obtained from the American Type Culture Collection. Cells were all maintained in R10 at 37°C and 5% CO2 in a humidified environment. Recombinant human IL-2 (10 U/ml) was added to cultures of normal T cells, L3, and CTLL-2 cells for routine growth.
T. gondii infection
Tachyzoites were recovered from infected fibroblasts by forced passage through a 27-gauge needle. Recovered tachyzoites and cells were then incubated together in medium R10 overnight. The multiplicity of infection (moi)3 ranged from 1 to 10, as indicated.
The number of infected cells was determined by UV microscopy of samples stained with acridine orange. Ten microliters of cell suspension was placed on a slide, and a coverslip was carefully placed on top. After the preparation had dried, 100 cells were examined for the presence of Toxoplasma.
Determination of apoptotic morphology
Cell nuclei were examined for apoptotic morphology following the method of Duke and Cohen (35). Briefly, cells were suspended in a saline solution containing 20 µg/ml of the nucleic acid-binding dyes acridine orange (Sigma) and ethidium bromide (Sigma) and examined by fluorescence microscopy. The differential uptake of these dyes by cells allows the identification of viable and nonviable cells (36). Acridine orange enters into live and dead cells and stains the chromatin green. Ethidium bromide, in contrast, enters only into cells that have lost membrane integrity, staining the chromatin red. Apoptotic vs necrotic cell death may also be quantified using this combination of dyes. In normal cells or cells that have died by necrosis, the euchromatin and heterochromatin are visualized as heterogeneous nuclear structures. In vivid contrast, apoptotic nuclei show condensed chromatin that appears as brightly staining, featureless regions, often resulting in distinct beads. One hundred host cell nuclei per condition were counted, and the percentage of nuclei with apoptotic morphology was calculated.
DNA ladder gel electrophoresis
DNA in apoptotic cells is no longer intact and appears as a ladder of DNA bands at an interval of about 200 bp on electrophoretic gels. Uninfected A20 cells, A20 cells infected with T. gondii at an m.o.i. of 10, and freshly obtained T. gondii tachyzoites were incubated overnight at 37°C. Half of each culture was then treated with 20 µM beauvericin (Sigma) for 1 h. The cells were harvested by centrifugation and resuspended in 15 µl of a solution containing 2 vol of sample buffer (glycerol containing 0.01 M Tris-HCl, pH 8.0, and 7.5% bromophenol blue) and 1 vol of 10 mg/ml RNase A. The samples were loaded onto a 2% agarose gel from which the section above the wells had been replaced with 1% agarose, 2% SDS, and 53 µg/ml proteinase K. After electrophoresis, the gel was stained with ethidium bromide and visualized with UV light.
JAM assay
As first described by Matzinger, the JAM assay is a quantitative measure of DNA fragmentation (37). In brief, cells were labeled overnight with tritiated thymidine (Amersham, Arlington Heights, IL). The next day the cells were washed to remove the unincorporated radiolabel and were infected with T. gondii. Experiments were performed following overnight infection. After experimental treatment and incubations were complete, the cells were harvested onto a unifilter plate using a 96-well harvester (Packard, Meridian, CT), and the resulting counts were determined on a beta counter (Packard). When DNA was fragmented, the small pieces passed through the filter, resulting in lower counts. The percent DNA fragmentation was calculated using the formula ((spontaneous counts - experimental counts)/spontaneous counts) x 100.
Lysis assay
Lysis of target cells by CTL was measured using a method similar to the JAM assay, as proposed by Eric Martz (38). A20 target cells were prepared in the same manner as that described for the JAM assay by labeling overnight with tritiated thymidine at between 0.5 and 2 µCi/ml in tissue culture medium. After overnight culture, the cells were washed once. Further washing was not necessary because unincorporated thymidine was not retained on the harvesting filter for any of the conditions tested. The target cells were diluted to 5 x 104 cells/ml in tissue culture medium. H-2d allospecific CTL L3 effector cells were washed and diluted to 2.5 x 105 cells/ml. One hundred microliters of target cells and 100 µl of effector cells were combined in 96-well microtiter plates with 50 µl of 125 µg/ml DNase I (Sigma). Target cells with medium alone were used as negative controls. The plate was centrifuged for 5 min at 500 rpm to bring the cells into contact and then incubated for 4 h at 37°C. After the incubation period, the plate was harvested on unifilter plates in the same manner as that described for the JAM assay. As lysis occurred, the cell membranes became permeable to the DNase. Loss of the radiolabel after digestion occurred as in the JAM assay. The results were calculated in the same manner as described for the JAM assay.
The DNase accessibility assay was used in this study instead of the chromium release assay so that identical targets cells could be used, eliminating variations in results due to culture conditions.
CTL assay
Apoptosis induced by CTL was examined using target cells labeled with tritiated thymidine for JAM assay as described above. Effector cells were MLR lymphoblasts obtained as described above. Five thousand target cells per well were plated with effector cells to give E:T cell ratios of 50:1 or 20:1. The percent specific DNA fragmentation was determined after 4-h incubation at 37°C.
For the Fas-dependent killing assay, effector cells were incubated for 1 h in plates that had been coated overnight with 25 µl of anti-CD3 (145-2C11, American Type Culture Collection) at 20 µg/ml. EGTA-Mg2+ (5 mM; Sigma) was added to inhibit granule exocytosis at the end of the 1-h preincubation, before the addition of target cells.
Gamma irradiation
WEHI-231 cells were infected overnight at an moi of 10. The cells were then irradiated with 1000 rad using a cobalt source. Apoptosis was determined by nuclear morphology.
UV irradiation
The mastocytoma cell line P815 was irradiated with UV light by placing a flat-bottom 96-well plate directly on top of a UV transilluminator. Cells were allowed to settle to the bottom of the plate for 1 to 2 h before irradiation, to provide even exposure. Control cells were protected by aluminum foil.
IL-2 withdrawal
CTLL-2 were washed three times to remove IL-2 and resuspended in fresh R10 without IL-2. T. gondii was added at an moi of 1, 5, or 10 at the end of the three washes. The cells were then incubated overnight, and apoptosis was determined by nuclear morphology the next day.
Beauvericin treatment
Beauvericin stock was prepared by dissolving beauvericin in absolute ethanol at 2 mM. Cells were treated by adding cell suspensions to beauvericin solution at 10 or 20 µM (39). The cells were then incubated for 1 h and assayed for apoptosis by nuclear morphology.
Pyrimethamine treatment
Pyrimethamine stock was made by dissolving 5 mg/ml of pyrimethamine (Sigma) in DMSO and was used at a 5 µg/ml final concentration. Overnight treatment with 5 µg/ml pyrimethamine kills T. gondii under these conditions (our unpublished observation).
Cycloheximide and actinomycin D treatment
Cycloheximide (Sigma) stock was prepared by dissolving 250 µg/ml in ethanol. A final concentration of 2.5 µg/ml was used in the assays. Actinomycin D (Sigma) stock was made by dissolving 500 µg/ml in water. A final concentration of 500 ng/ml was used.
Statistical analysis
Comparisons were performed using unpaired Student’s t test, assuming unequal variances. The p values reported are for the greatest differences observed in the dose-response analyses, considering a two-tailed value of p < 0.05 significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTL are major effector cells for killing cells infected with
viruses and other intracellular pathogens. CTL induce apoptosis in
their targets by two distinct mechanisms (32). The first mechanism uses
the pore-forming protein perforin and a group of proteases collectively
referred to as granzymes. The second mechanism is mediated by Fas
ligand expressed on the CTL and Fas on the target. To investigate
whether T. gondii infection might inhibit CTL-mediated
apoptosis, we used established experimental conditions in which both or
only one mechanism of killing could function (32, 40, 41). In the
experiment depicted in Figure 1
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alloreactive (H-2b anti-H-2d) MLR
lymphoblasts were used as effector cells, and H-2d-bearing
L1210-Fas tumor cells were used as target cells. To examine both
mechanisms simultaneously, MLR lymphoblasts derived from B6 mice were
employed (Fig. 1a). These lymphoblasts rapidly
induced high levels of apoptosis in A20 tumor cells, which were
significantly inhibited by T. gondii infection of the target
cells (p < 0.001). However, it could not be
determined whether Fas-dependent or independent cytotoxic pathways were
inhibited using this experimental protocol.
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b). T cells from
these mice kill only by perforin/granzyme-mediated mechanisms; the
Fas-mediated mechanism is not used (41). Apoptosis was induced in
uninfected A20 cells by these effector cells, but was significantly
inhibited by infection with T. gondii
(p < 0.001).
The ability of T. gondii to inhibit Fas-dependent apoptosis
mediated by CTL was examined using a modification of a protocol first
described by Rouvier and colleagues (32). In brief, B6 MLR lymphoblasts
were preincubated for 1 h with anti-CD3 Ab to induce Fas
ligand expression, and EGTA was then added before incubation with
target cells to prevent granule exocytosis (40). As in the case of
perforin/granzyme-mediated cytotoxicity, Fas-dependent induction of DNA
fragmentation by CTL was inhibited by T. gondii infection of
target cells (Fig. 1c; p < 0.0007).
CTL-mediated lysis in the presence of T. gondiiinfection
The abilities of T. gondii to inhibit DNA fragmentation
and lysis were also compared (Table I).
The allospecific cytotoxic T cell clone L3, which is able to use both
granules and Fas ligand, was used to test for cytotoxicity against A20
tumor cells. Significant inhibition of CTL-induced DNA fragmentation by
T. gondii infection was seen, but with no significant
concomitant inhibition of target cell lysis.
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Inhibition of gamma irradiation-induced apoptosis
To assess protection against radiation-induced apoptosis, we
irradiated WEHI 231 cells, which are sensitive to gamma irradiation and
undergo apoptosis when exposed to it (33). At 24 and 48 h
following irradiation, the percentage of apoptotic cells in uninfected
WEHI-231 was always higher than that in the infected population (Table II).
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We also tested the effect of T. gondii infection on UV
irradiation-induced apoptosis. In this assay, P815 mastocytoma cells
infected overnight with T. gondii were protected from DNA
fragmentation as measured by JAM assay. The fragmentation resulting
from UV irradiation was inhibited in a dose-dependent manner by
T. gondii infection (Fig. 2).
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We next tested the effect of T. gondii infection on
apoptosis resulting from IL-2 withdrawal using the IL-2-dependent cell
line CTLL-2 (34). At 24 h after removal of IL-2, uninfected CTLL-2
were mostly apoptotic (Fig. 3). Infection
with T. gondii protected against apoptosis, and as observed
with CTL-mediated apoptosis, greater inhibition was observed at
higher moi.
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Finally, we examined the effect of T. gondii infection
on induction of apoptosis by beauvericin (39). Beauvericin is a calcium
ionophore that activates the apoptotic process in many cell types in as
little as 5 min after addition, with nearly 100% of cells having
apoptotic nuclei by 1 h (our unpublished data). Following
overnight infection with T. gondii, cells were incubated for
1 h with beauvericin. Two doses of beauvericin and three moi were
tested (Fig. 4). Increasing proportions
of cells were protected at higher moi for both doses of beauvericin
(p < 0.03). Similar results were obtained
using A20 or WEHI-231 cells (data not shown).
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) and by JAM assay
(Table III and data not shown).
Beauvericin did not induce detectable DNA fragmentation in T.
gondii tachyzoites (Fig. 5).
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We wondered whether T. gondii would continue to mediate
protection against apoptosis if it were killed following cell entry. To
address this question, A20 cells were infected, and pyrimethamine,
which kills intracellular tachyzoites (22), was added at various times
after infection, but before addition of beauvericin. Pyrimethamine
treatment abolished most of the apoptosis inhibitory effect in a
time-dependent manner (Table III), suggesting that protection against
apoptosis required the continued presence of live organisms.
Ciprofloxacin, which also kills intracellular T. gondii,
yielded similar results (Table III).
Effects of cycloheximide and actinomycin D on protection
To further substantiate the need for continual production of an
inhibitory factor, the induction of apoptosis in target cells in which
RNA or protein synthesis was blocked was tested. Cells were treated
with cycloheximide to block protein synthesis or with actinomycin D to
block RNA synthesis for 5 h before beauvericin treatment (Table IV). By visual examination with acridine
orange, 33 of 100 of these target cells were infected, resulting in
33% inhibition of apoptosis. Treatment with either cycloheximide or
actinomycin D significantly decreased the inhibition of apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A variety of viruses have been shown to encode proteins capable of inhibiting apoptosis. The majority of these proteins prevent apoptosis triggered by specific inducers working in a limited range of induction pathways. Only two viruses are known to be broadly active in inhibiting apoptosis: the cowpox virus, which encodes the crmA gene product, and baculovirus, which encodes the p35 protein. Both the crmA gene product and the baculovirus p35 protein act by blocking protease activity. Cells expressing crmA are rendered resistant to Fas or TNF receptor ligation (28, 43, 44, 45, 46). Baculovirus p35 protein blocks Fas and TNF receptor activities, nerve growth factor withdrawal, and expression of the Drosophila reaper gene (24, 47, 48, 49, 50).
Similar to cells overexpressing p35 or crmA, cells infected with T. gondii are resistant to multiple inducers of apoptosis. However, neither of these two viral gene products is known to inhibit apoptosis over as broad a range of inducers as that in the case of T. gondii we now report. The mechanism by which T. gondii infection protects cells from induction of apoptosis has not yet been elucidated. However, according to current models of apoptotic induction, T. gondii probably blocks either the activation or the function of the three proteases Cpp-32 (51, 52, 53), ICE-LAP3 (54, 55), and Mch-2 (56). Our observation that T. gondii infection blocks apoptosis induced by granzyme B (CTL), which probably activates all three of these proteases directly (57), supports this concept.
Although the mechanism by which T. gondii infection inhibits the induction of apoptosis is unknown, it is an active process, requiring the presence of live parasites, as confirmed by the time-dependent abrogation of apoptosis protection upon killing of intracellular parasites with antibiotics. Furthermore, inhibition of apoptosis was directly dependent on the moi of T. gondii in a dose-dependent fashion, and inhibition of protein synthesis or mRNA transcription also abrogated this protection.
Although T. gondii-infected cells were resistant to
induction of CTL-mediated apoptosis, target cell lysis induced by CTL
was only minimally reduced (Table I). We interpret the 59% DNase
accessibility results in the uninfected cells as due to a combination
of both host cell DNA fragmentation and lysis, but that in this case,
the additional contribution to loss of the radiolabel by direct lysis
is negligible, since target cells engaged by CTL will fragment DNA
anyway. This is evident from the observation that the level of DNase
accessibility and the level of DNA fragmentation are essentially the
same. On the other hand, in the infected cells, since fragmentation
(JAM assay) is inhibited to 11%, we interpret the 51% DNase
accessibility results to be due primarily to host cell lysis, with only
a small component due to DNA fragmentation.
The observations that T. gondii infection renders host cells resistant to CTL-mediated DNA fragmentation, while not protecting against host cell lysis are not contradictory. Perforin, which can form lytic lesions independent of apoptotic induction, is probably responsible for the lysis of infected cells. It is thus possible that Fas-mediated bystander activity is inhibited by T. gondii infection, reducing early destruction of infected hemopoietic cells, thereby enhancing the spread of the parasite throughout the host. Lysis, in the absence of apoptosis, mediated by direct killing may also not be as detrimental to the parasite as the normal apoptotic effects. Alternatively, T. gondii may not specifically evade immune destruction through its ability to inhibit apoptosis, but, rather, may prolong the life of the host cell, which might otherwise undergo apoptosis due to the stress of infection.
One other intracellular parasite, Leishmania, has been shown
to inhibit apoptosis in infected cells (21). The system used to examine
the effect of infection by Leishmania was the induction of
apoptosis in bone marrow macrophages deprived of TNF-
. When
macrophages were infected with Leishmania, the cells were
activated to produce their own supply of TNF-, thus protecting
themselves from apoptosis. Not only the infected cells but also the
bystander cells were protected in this manner. This demonstration of
protection contrasts with our description of T. gondii
protection in that the Leishmania system involved an
external source of rescue induced by the parasite, limiting the rescue
to a single system, whereas T. gondii infection rescued
cells from a variety of apoptotic inducers, implying inhibition of the
internal apoptotic signaling pathway.
Himeno and Hiseada recently described the role of heat shock protein 65
(hsp65) in protection against T. gondii infection (58). They
suggested that hsp65 expression protects T. gondii-infected
macrophages from apoptosis. Their description of the induction of
apoptosis and its inhibition are not yet published, so direct
comparison of their work and ours is not yet possible. However, it is
unlikely that we are observing an hsp65-mediated event, as hsp65
expression, according to Himeno and Hiseada, was not induced by the RH
strain of T. gondii, which is what we used, and hsp65
expression was dependent of the presence of 
T cells, which were
not present in our system.
Several important observations regarding our experimental system must be emphasized. First, our experimental model of CTL-mediated target cell lysis involved CTL not specific for T. gondii. Nonetheless, this model is valid for assessment of the consequences of CTL-mediated target cell lysis or DNA fragmentation, as these phenomena are end results of common CTL killing mechanisms and are Ag independent. In this regard, another group recently used a similar strategy to assess the fate of intracellular Mycobacteria following CTL-mediated host cell destruction by using influenza-specific CTL and influenza Ag-expressing, Mycobacteria-infected target cells (59). Further, if we had used T. gondii-specific CTL, we would not have been able to compare apoptosis inhibition in infected and uninfected cells, as the latter would not express the appropriate Ags and thus would not be recognized and lysed by T. gondii-specific CTL. We reason, however, that as mechanisms of CTL killing are Ag independent, cells infected with T. gondii would be relatively more resistant to CTL-mediated apoptosis than if they were not infected. How this inhibition of apoptosis influences the course of acute or chronic T. gondii infection will require other strategies for study.
Second, the level of apoptosis inhibition varied substantially between experiments, as did the degree of T. gondii infection. Nonetheless, the general observations we describe were highly reproducible. Furthermore, the variability in T. gondii infection between experiments was used to underscore the role of active T. gondii infection in mediating apoptosis protection. Thus, in the four experiments shown here in which the extent of T. gondii infection was quantitated, we observed that from 33 to 79% of cells were infected, yielding apoptosis inhibition of from 33 to 84%, with the level of apoptosis inhibition being directly proportional to the level of infection. In experiments in which different moi were used, the level of inhibition of apoptosis varied in direct relation to the number of parasites.
In summary, these experiments demonstrate that T. gondii infection inhibits apoptosis induced in numerous cell types by six different agents: CTL (Fas-dependent or independent), gamma irradiation, UV irradiation, growth factor deprivation, and a toxin. Inhibition of apoptosis at a common point in the apoptotic pathway is thus likely. Elucidation of the mechanism(s) and gene(s) involved probably will enhance our understanding of the apoptotic process. The ability to extend the life of infected host cells would be a substantial advantage to T. gondii or other obligate intracellular pathogens for enhancing their own survival and may be crucial for allowing T. gondii to survive long enough to produce cysts and provide for the spread of the organism. The consequences of apoptosis inhibition for the immunopathogenesis of T. gondii infection remain to be defined.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul Nash, Heska, 1825 Sharp Point Dr., Fort Collins, CO 80525. E-mail address: ; or Dr. Tyler Curiel, Campus Box B168, University of Colorado Health Sciences Center, Denver, CO 80262. E-mail address:
3 Abbreviations used in this paper: moi, multiplicity of infection; hsp, heat shock protein.
Received for publication January 17, 1997. Accepted for publication October 28, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced apoptosis in human neuronal cells: protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA. Mol. Cell. Biol. 15:2359.[Abstract]
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E. Aga, D. M. Katschinski, G. van Zandbergen, H. Laufs, B. Hansen, K. Muller, W. Solbach, and T. Laskay Inhibition of the Spontaneous Apoptosis of Neutrophil Granulocytes by the Intracellular Parasite Leishmania major J. Immunol., July 15, 2002; 169(2): 898 - 905. [Abstract] [Full Text] [PDF]
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J.-L. Perfettini, T. Darville, A. Dautry-Varsat, R. G. Rank, and D. M. Ojcius Inhibition of Apoptosis by Gamma Interferon in Cells and Mice Infected with Chlamydia muridarum (the Mouse Pneumonitis Strain of Chlamydia trachomatis) Infect. Immun., May 1, 2002; 70(5): 2559 - 2565. [Abstract] [Full Text] [PDF]
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S. Wei, F. Marches, J. Borvak, W. Zou, J. Channon, M. White, J. Radke, M.-F. Cesbron-Delauw, and T. J. Curiel Toxoplasma gondii-Infected Human Myeloid Dendritic Cells Induce T-Lymphocyte Dysfunction and Contact-Dependent Apoptosis Infect. Immun., April 1, 2002; 70(4): 1750 - 1760. [Abstract] [Full Text] [PDF]
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A. Orlofsky, L. M. Weiss, N. Kawachi, and M. B. Prystowsky Deficiency in the Anti-Apoptotic Protein A1-a Results in a Diminished Acute Inflammatory Response J. Immunol., February 15, 2002; 168(4): 1840 - 1846. [Abstract] [Full Text] [PDF]
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 K. M. EDWARDS, M. H. CYNAMON, R. K. R. VOLADRI, C. C. HAGER, M. S. DESTEFANO, K. T. THAM, D. L. LAKEY, M. R. BOCHAN, and D. S. KERNODLE Iron-cofactored Superoxide Dismutase Inhibits Host Responses to Mycobacterium tuberculosis Am. J. Respir. Crit. Care Med., December 15, 2001; 164(12): 2213 - 2219. [Abstract] [Full Text] [PDF]
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D. G. Mordue, F. Monroy, M. La Regina, C. A. Dinarello, and L. D. Sibley Acute Toxoplasmosis Leads to Lethal Overproduction of Th1 Cytokines J. Immunol., October 15, 2001; 167(8): 4574 - 4584. [Abstract] [Full Text] [PDF]
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S. Goebel, U. Gross, and C. G. K. Luder Inhibition of host cell apoptosis by Toxoplasma gondii is accompanied by reduced activation of the caspase cascade and alterations of poly(ADP-ribose) polymerase expression J. Cell Sci., January 10, 2001; 114(19): 3495 - 3505. [Abstract] [Full Text] [PDF]
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K. Yoshiie, H.-Y. Kim, J. Mott, and Y. Rikihisa Intracellular Infection by the Human Granulocytic Ehrlichiosis Agent Inhibits Human Neutrophil Apoptosis Infect. Immun., March 1, 2000; 68(3): 1125 - 1133. [Abstract] [Full Text] [PDF]
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D. F. McCole, L. Eckmann, F. Laurent, and M. F. Kagnoff Intestinal Epithelial Cell Apoptosis following Cryptosporidium parvum Infection Infect. Immun., March 1, 2000; 68(3): 1710 - 1713. [Abstract] [Full Text] [PDF]
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 V. T. Heussler, J. Machado Jr, P. C. Fernandez, C. Botteron, C.-G. Chen, M. J. Pearse, and D. A. E. Dobbelaere The intracellular parasite Theileria parva protects infected T cells from apoptosis PNAS, June 22, 1999; 96(13): 7312 - 7317. [Abstract] [Full Text] [PDF]
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M. S. Hu, J. D. Schwartzman, G. R. Yeaman, J. Collins, R. Seguin, I. A. Khan, and L. H. Kasper Fas-FasL Interaction Involved in Pathogenesis of Ocular Toxoplasmosis in Mice Infect. Immun., February 1, 1999; 67(2): 928 - 935. [Abstract] [Full Text] [PDF]
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C. G. Freire-de-Lima, M. P. Nunes, S. Corte-Real, M. P. Soares, J. O. Previato, L. Mendonca-Previato, and G. A. DosReis Proapoptotic Activity of a Trypanosoma cruzi Ceramide-Containing Glycolipid Turned on in Host Macrophages by IFN-{gamma} J. Immunol., November 1, 1998; 161(9): 4909 - 4916. [Abstract] [Full Text] [PDF]
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D. M. Ojcius, P. Souque, J.-L. Perfettini, and A. Dautry-Varsat Apoptosis of Epithelial Cells and Macrophages Due to Infection with the Obligate Intracellular Pathogen Chlamydia psittaci J. Immunol., October 15, 1998; 161(8): 4220 - 4226. [Abstract] [Full Text] [PDF]
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