By
From the Immunobiology Section, Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
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Abstract |
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Although interferon (IFN)--activated, mononuclear phagocytes are considered to be the major effectors of resistance to intracellular pathogens, it is unclear how they control the growth
of microorganisms that reside in nonhemopoietic cells. Pathogens within such cells may be
killed by metabolites secreted by activated macrophages or, alternatively, directly controlled by
cytokine-induced microbicidal mechanisms triggered within infected nonphagocytic cells. To
distinguish between these two basic mechanisms of cell-mediated immunity, reciprocal bone
marrow chimeras were constructed between wild-type and IFN-
receptor-deficient mice and
their survival assessed following infection with Toxoplasma gondii, a protozoan parasite that invades both hemopoietic and nonhemopoietic cell lineages. Resistance to acute and persistent
infection was displayed only by animals in which IFN-
receptors were expressed in both cellular compartments. Parallel chimera experiments performed with tumor necrosis factor (TNF)
receptor-deficient mice also indicated a codependence on hemopoietic and nonhemopoietic
lineages for optimal control of the parasite. In contrast, in mice chimeric for inducible nitric
oxide synthase (iNOS), an enzyme associated with IFN-
-induced macrophage microbicidal
activity, expression by cells of hemopoietic origin was sufficient for host resistance. Together,
these findings suggest that, in concert with bone marrow-derived effectors, nonhemopoietic cells can directly mediate, in the absence of endogenous iNOS, IFN-
- and TNF-
-dependent host resistance to intracellular infection.
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Introduction |
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Cell-mediated immune responses are critical for immune defense against a variety of intracellular infections as well as tumors. During the induction of cell-mediated immunity (CMI),1 T lymphocytes expressing IFN-
and TNF-
are generated within secondary lymphoid organs in an IL-12-dependent manner (1). Their extravasation into sites of infection results in the recruitment of
macrophages, which become activated by these T cell-
derived lymphokines. Nevertheless, the mechanisms by
which the multicellular interactions involved in CMI lead
to control of intracellular pathogens and protection of the
invaded tissue have only been partially elucidated.
Most of our understanding of CMI is based on studies of microbial agents such as Listeria, Mycobacteria, and Leishmania, which have an absolute or near absolute tropism for macrophages (4). Nonetheless, many intracellular pathogens infect not only cells of the mononuclear phagocyte lineage but also those of epithelial, endothelial, mesodermal, and neuronal derivation. The list of organisms belonging to this category include Salmonella, Shigella, Chlamydia, Rickettsiae, Trypanosoma, and Toxoplasma. Although the growth of such intracellular pathogens is generally thought to be controlled by toxic reactive nitrogen or reactive oxygen intermediates, it is unclear in many cases whether the latter effector molecules can be generated and/or function in the nonhemopoietic cell types that they invade. For example, although NO is potentially produced by a wide variety of host cells, its documented antimicrobial functions have been largely restricted to monocytes/macrophages (7). Thus, the question arises as to how T cell-derived lymphokines can act to restrict the growth of the above pathogens within cells of nonhemopoietic origin. Although many possible scenarios have been proposed, these can be distilled into two conceptually distinct "cis" and "trans" models (see Fig. 1 for schematization) that describe how intracellular infections might be controlled within nonmacrophage (i.e., nonhemopoietic) cells.
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The trans model argues that the main site for lymphokine-induced activation is at the level of a professional effector cell, such as the macrophage (8, 9). Upon activation,
the macrophage releases toxic metabolites, most notably
NO generated enzymatically by type 2 nitric oxide synthase
(iNOS), which diffuses to neighboring somatic cells, thereby
restricting the growth of the intracellular pathogen. In direct contrast, the cis model proposes that lymphokine receptor ligation on nonprofessional effector cells leads to metabolic changes resulting in inhibition of microbial growth
within the same cells (10). One prediction of the cis
model is that receptors for IFN- and TNF-
need to be
expressed and functional on somatic (in addition to bone
marrow [BM]-derived) cellular compartments for complete
resistance. In contrast, the trans model requires that these
receptors be expressed only on hemopoietic effector cells
and postulates that infected nonhemopoietic cells play no active role in host resistance.
We have designed an experimental approach for distinguishing between these two models of antimicrobial effector function that involves infection of BM chimeric mice
with the intracellular pathogen Toxoplasma gondii. This apicomplexan protozoan parasite invades a wide variety of cells,
including epithelial, mesodermal, and neuronal as well as
hemopoietic cells (15). Immunity to T. gondii is strictly dependent on IFN- production by CD8+ T lymphocytes as
well as NK and CD4+ T cells (15). Although the requirement for CD8 lymphocytes is not completely understood, one possibility is that class I-restricted recognition
of infected nonhemopoietic cells leads to the inhibition of
parasite growth through localized IFN-
secretion (18, 19). In addition to IFN-
, TNF-
has also been shown to play
a major role in host resistance to T. gondii. However, whereas
IFN-
is necessary for both acute and chronic resistance to
parasite infection (20), the requirement for TNF-
manifests primarily in the chronic phase, when the parasites reside in the central nervous system (CNS; 21, 23, 24). Similarly, although iNOS-dependent production of NO is also
critical for control of the parasite, its role becomes apparent
only during the chronic phase of T. gondii infection (25, 26).
In this study, we have used BM chimeric mice to evaluate the requirement for IFN-R, TNFR, and iNOS expression on hemopoietic versus nonhemopoietic cells in host resistance to acute and chronic T. gondii infection. Our findings
suggest that in both stages of infection, nonhemopoietic cells
function as iNOS-independent effectors of IFN-
/TNF-
-
dependent immunity. These observations directly support
the cis model of CMI outlined above.
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Materials and Methods |
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Mice.
IFN-Bone Marrow Transplantation.
Recipient mice were given lethal total body irradiation (950-1,000 rads) and reconstituted intravenously with 10-20 million bone marrow cells within 24 h. Marrow cell suspensions were prepared from donor tibial and femoral bones by flushing with RPMI 1640 (GIBCO BRL) supplemented with antibiotics (penicillin [100 U/ml] and streptomycin [100 U/ml]) using a 25-gauge needle syringe. Irradiated and reconstituted mice were given Bactrim (sulfamethoxazole [150 mg/ml] and N-trimethoprim [30 mg/ml]; Teva Pharmaceuticals) in their drinking water for 5 wk. Thereafter, they were switched to sterile drinking water, thus ensuring that the antibiotic treatment would not affect the ensuing experimental infection with T. gondii. Unless otherwise stated, mice were used for experimental infection or for analysis of chimerism 8-9 wk after BM cell transfer. For each infection experiment, groups of nonirradiated WT and KO animals were included as positive and negative controls for host resistance, respectively. In every instance except one, sham chimeric KOAnalysis of Chimerism.
The extent of hemopoietic cell replacement by donor phenotype cells upon reconstitution was analyzed 8 wk after transfer of BM cells using mice chimeric for iNOS gene deficiency. Spleen cells and d5 thioglycollate-elicited peritoneal cells were harvested from each of three mice per group. Cells were plated in 96-well plates at a concentration of 2 × 106 cells/ml and stimulated with 100 U/ml of IFN-Assessment of Cytokine Production in IFN-R KO Mice.
Experimental Infection with T. gondii and Listeria monocytogenes.
Tachyzoites of the RH strain of T. gondii were cultivated in human foreskin fibroblasts maintained in DMEM (GIBCO BRL) supplemented with 1% FCS and antibiotics. The ME49 strain of T. gondii was passaged as cysts in C57BL/6 mice. Experimental animals were infected with 20 ME49 cysts by intraperitoneal injection. An additional set of IFN- ![]() |
Results and Discussion |
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Measurement of the extent of replacement of tissue macrophages by donor-derived cells is required for proper interpretation of the results obtained with
BM chimeric mice. We performed this assessment in iNOS
BM chimeric mice, in which IFN-- and STAg-induced NO production can be used as a readout of macrophage
function. 8 wk after reconstitution, the ability of spleen
cells to produce NO in response to IFN-
and parasite antigen was clearly dictated by the genotype of the BM donor
(Fig. 2 A). For instance, spleen cells from iNOS KO mice
reconstituted with WT marrow exhibited robust NO production. Importantly, there was no residual NO-producing capacity detectable in WT mice reconstituted with iNOS
KO BM. Similarly, the capacity of thioglycollate-elicited
peritoneal cells to produce NO in response to IFN-
and
STAg stimulation was dictated by the BM donor genotype
(Fig. 2 B). Identical results were obtained using chimeric
IFN-
R KO mice (data not shown). Thus, in these chimeras, most if not all of the macrophages in a lymphoid organ
(spleen) or at a site of cellular recruitment (peritoneal cavity) appear to consist of donor-derived cells.
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Having demonstrated successful and complete reconstitution of macrophages by donor BM-derived
cells, we proceeded to construct chimeric mice using WT and
IFN-R KO on the same 129/SvEv genetic background.
IFN-
R KO mice fail to respond to IFN-
and exhibit increased susceptibility to a variety of intracellular pathogens (27, 31). Both the cis and trans models predict that a KO
WT chimera should exhibit susceptibility to infection because the BM-derived elements, including macrophages and
neutrophils, will not be able to control infection. More importantly, however, different outcomes are predicted by the
two models for the WT BM
KO chimera. The trans
model predicts resistance for this chimera, because the WT
marrow-derived cells would be armed and protect somatic cells, whereas the cis model predicts susceptibility for this chimera, because the somatic cells would be unable to
control infection due to a lack of IFN-
responsiveness. An
assumption made for the KO
WT chimera is that the
KO BM-derived cells remain competent for IFN-
production in the absence of responsiveness to IFN-
. Taking
into account previous reports of IFN-
amplification of the
IL-12 response (32, 33), a concern raised by these observations is that type 1 responses may not develop normally in
the absence of IFN-
responsiveness in APC and T cell
populations. To address these concerns, spleen cells from
uninfected IFN-
R KO and WT mice were stimulated
with tachyzoite extract (STAg), and their capacity to produce IL-12 p40 as well as IFN-
was measured by ELISA.
As shown in Fig. 3, A and B, production of both cytokines in response to STAg stimulation was not compromised by
IFN-
R deficiency. To assess possible defects in type 1 cell
development, IFN-
R KO mice were immunized with irradiated tachyzoites and their spleen cells restimulated with
STAg or mitogen in vitro. Robust, antigen-specific IFN-
production was observed in cultures from both WT and
KO mice (Fig. 3 C). On the basis of these control experiments, we predicted that in WT mice reconstituted with
IFN-
-unresponsive BM cells, IL-12-dependent NK as
well as T cell IFN-
production should not be impaired.
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As expected, sham (KO KO) chimeric mice completely
deficient in IFN-
R were acutely susceptible to T. gondii
infection, whereas WT
WT sham chimeric mice survived
infection for at least 60 d (Fig. 4 A). As predicted by both
trans and cis models, chimeric WT mice reconstituted with
IFN-
R-deficient BM were susceptible to acute T. gondii
infection. Importantly, however, in WT
KO chimeras,
IFN-
R deficiency in the recipient compartment, despite a WT BM donor genotype, resulted in acute mortality.
This observation indicates that IFN-
activation of WT
BM-derived cells is not sufficient to confer protection and
that nonhemopoietic cells responsive to this lymphokine
are required, an interpretation consistent with the cis model.
A possible caveat is that at week eight, WT
KO BM reconstitution may be incomplete in tissue sites other than the
spleen and the peritoneum, where chimerism was initially
assessed (Fig. 2). The latter explanation is unlikely, however, because even after an additional 2 mo of reconstitution, these animals remained fully susceptible to acute infection (data not shown).
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To further confirm that full functional reconstitution was
achieved after 8 wk, similarly constructed sets of chimeric
mice were infected with the macrophage-trophic intracellular bacterium L. monocytogenes and survival of chimeric
mice assessed after challenge with a sublethal dose. In the
case of this pathogen, IFN-R expression on the BM but
not in the somatic cell compartment was required for resistance (Fig. 4 B). The latter finding confirms that after only
2 mo, WT BM reconstitution of IFN-
R KO recipients is
sufficiently complete to confer potential resistance to intracellular infections. The above experiments, by demonstrating
a differential requirement for IFN-
R expression on somatic cells for protection against Toxoplasma versus Listeria,
underscore the critical importance of host cellular niches in
determining the effector cell types and mechanisms required for control of these pathogens.
Resistance to T. gondii is exquisitely dependent on IFN-, not
only during acute infection but also during the chronic
phase of infection (20, 21). For instance, administration of
neutralizing antibodies to IFN-
30 d after inoculation allows uncontrolled cyst reactivation and tachyzoite replication in the brain parenchyma and invariably causes death of
the host within 10 d. To further explore the role of nonhemopoietic cells versus BM-derived inflammatory cells as
effectors in the chronic phase of the infection using the
IFN-
R KO chimeric mice, partial chemotherapy (commencing 3 d after parasite inoculation) was employed to
allow host survival through the acute phase and establishment of persistent infection (as evidenced by cyst formation
in brain tissue). Once infection was established (at day 20),
further drug treatment was terminated and the survival of
the chimeric mice monitored.
As shown in Fig. 5, even with oral drug treatment, a majority of sham IFN-R KO chimeric mice succumbed to
infection by week three postinfection. Chimeric mice with
IFN-
R deficiency in either the hemopoietic or nonhemopoietic compartments also died within 5-11 d following
cessation of chemotherapy. The kinetics of mortality in these
two sets of chimeric mice were essentially identical. As expected, WT sham chimeric mice survived the infection
even after drug treatment was withheld. Thus, as observed
for resistance to acute infection, IFN-
R expression on nonhemopoietic as well as BM-derived cells is essential for host
survival in established infection, as assessed in this drug
treatment model.
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Although IFN--mediated signals are
clearly important and required on both somatic and hemopoietic cells during the acute and chronic phases of infection,
IFN-
alone does not suffice, at least during the chronic
phase. At this stage, TNF-
is also crucially required for host
resistance. Thus, administration of anti-TNF Ab (without
additional neutralization of IFN-
) is sufficient to reactivate
infection and induce lethal encephalitis in chronically exposed C57BL/6 mice (21). Additionally, T. gondii infections are lethal in mice lacking either the TNF p55 receptor or
both the p55 and p75 receptors (23, 24). In the case of this
as opposed to IFN-
R deficiency, lethality occurs only after
infection establishes in the CNS (~3 wk after infection).
Because TNFRs are expressed not only on macrophages
and other hemopoietic cells but also on nonhemopoietic
cells, including astrocytes and neuronal cells, we evaluated
the requirement for TNFR signaling on these cellular compartments for resistance to chronic infection. Reciprocal
chimeras were constructed between WT (B6 × 129/J F1)
and TNFR p55/p75 KO mice on the same hybrid background. As shown in Fig. 6, TNFR KO mice reconstituted
with KO BM cells succumbed to infection within 20 d, as
previously reported for unmanipulated TNFR KO mice.
WT controls and WT WT sham chimeric mice survived
T. gondii infection for at least 60 d. However, TNFR KO
BM cells in the context of a WT recipient did not confer
the same susceptibility phenotype observed in completely deficient mice. Similarly, WT BM
KO chimeras also exhibited only partial resistance to chronic infection. Thus,
both compartments must be receptor deficient or WT to
exhibit a completely susceptible or resistant phenotype, respectively. In contrast to these findings, resistance to Listeria
monocytogenes has been shown to require TNFR expression
only on BM-derived cells (34). These divergent requirements for TNFR expression in Toxoplasma and Listeria systems further highlight the importance of the parasitized cellular niche in determining which effector cell populations
are required for host resistance.
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We have previously
proposed that TNFRs expressed on nonhemopoietic, somatic cells such as neurons and astrocytes may be important in activating them to control intracellular tachyzoite replication by an iNOS-independent mechanism (24). This hypothesis was based on the finding that although TNFR
(p55/p75) KO mice develop necrotizing encephalitis and
die at the same rate as iNOS-deficient mice, they nevertheless display significant iNOS induction in brain tissue. Because macrophages from the same TNFR KO mice can exhibit both NO production and microbicidal activity in vitro
and ex vivo, these observations suggested that the defect responsible for death of the infected TNFR-deficient animals
resides in nonhemopoietic effector cells. The intermediate
level of resistance displayed by KO WT and WT
KO
TNFR chimeric mice (Fig. 6) argues instead that control of
chronic infection may require TNFR expression on both
hemopoietic and nonhemopoietic compartments.
If the extreme susceptibility of the TNFR KO mice is
explainable solely by iNOS deficiency, then the resistance
patterns in iNOS chimeric mice should be identical to
those exhibited by TNFR chimeric animals. As shown in
Fig. 7, this is clearly not the case. In contrast to the parallel
TNFR-deficient chimera, iNOS WT BM chimeric mice
displayed susceptibility indistinguishable from either iNOS
iNOS (Fig. 7 A) or nonirradiated iNOS KO mice (Fig. 7 B).
Furthermore, the WT
KO reciprocal chimera exhibited a
survival pattern virtually indistinguishable from the control WT
WT chimera. For reasons that are not clear (see Materials and Methods), these sham chimeric mice succumbed
early, at ~30 d, in contrast to nonirradiated WT C57BL/6
mice, which, as previously reported (26), survived greater
than 60 d (Fig. 7 B). Nevertheless, the findings clearly indicate that, in the iNOS system, the BM genotype is the main
determinant of the resistance phenotype of the chimera. This
is in direct contrast to the situation in both IFN-
R and
TNFR chimeric animals, in which both hemopoietic and nonhemopoietic cells contribute to host resistance during
the chronic phase of infection.
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Taken together, these results indicate that the trans
mechanism, whereby IFN-- and TNF-
-dependent production of toxic metabolites such as NO by mononuclear
phagocytes is, by itself, not sufficient to account for immune control of a pathogen that infects both hemopoietic
and nonhemopoietic cells. Instead, our findings are more
consistent with the cis model of host resistance against intracellular pathogens, which proposes that nonhemopoietic
cells need to be directly activated by lymphokines and play
an active role as effectors of IFN-
/TNF-
-dependent CMI
to T. gondii. The latter requirement may explain why, although not essential for cell survival or tissue homeostasis
(24, 27), the expression of IFN-
R and TNFR has been
retained in all nucleated cell types during the course of
evolution (35, 36).
Nevertheless, it remains unclear whether or not the direct
activation of nonhemopoietic cells by IFN- and TNF-
is
sufficient to completely restrict parasite growth within
these cells. Although in vitro experiments have clearly documented that nonhemopoietic cells can do so in the absence of macrophages (11), our in vivo results do not exclude potential synergism between trans-acting diffusible
metabolites such as NO derived from hemopoietic cells and
the cis-acting IFN-
/TNF-
-dependent mechanism in limiting tachyzoite replication within nonhemopoietic cells.
The identical susceptibility observed in iNOS iNOS
and iNOS
WT chimeric mice underscores the critical
importance of NO production by cells derived from the
hemopoietic lineage in host resistance. This conclusion
agrees with previous reports of the iNOS dependence of
the antitoxoplasmic activity of lymphokine-activated macrophages and microglial cells (37, 38). In parallel, the comparable survival curves exhibited by either iNOS-deficient
or WT recipients reconstituted with WT BM suggests that
iNOS expression in the nonhemopoietic cell compartment
plays a limited if not minimal role relative to that in hemopoietic cells. Consistent with this conclusion is the recent observation that lymphokine-induced control of intracellular tachyzoite growth occurs effectively in astrocytes (putative nonhemopoietic effector cells in the brain) derived from iNOS-deficient mice (39). Nevertheless, because of
technical problems uniquely observed in the iNOS BM
chimera experiments, we cannot definitively conclude that
iNOS expression in the nonhemopoietic compartment is
absolutely without antimicrobial function. Thus, the premature mortality exhibited by sham chimeric WT mice
suggests that lethal irradiation may have damaged a subset
of nonhemopoietic cells required for long-term survival after infection and could have, in theory, masked any protective effect of iNOS in the nonhemopoietic compartment.
Notwithstanding, it is reasonable to conclude, based on the
marked differences in mortality observed between iNOS
WT and WT
iNOS animals, that the IFN-
- and TNF-
-
dependent resistance mechanism(s) operating within nonhemopoietic cells has a major iNOS-independent component.
The nature of the cis-acting effector mechanism(s) responsible for restricting the growth of T. gondii within nonhemopoietic cells is presently undefined. A primary candidate mechanism is the depletion of intracellular tryptophan
stores by the enzyme indoleamine dioxygenase (IDO; 12).
IFN- and TNF-
synergistically induce the transcription
and activation of this enzyme in many human cell types,
including fibroblasts, retinal pigmented epithelium, and
neurons as well as macrophages (12, 40). Nonetheless, the evidence for the importance and contribution of the
IDO mechanism to host resistance in murine cells is more
tenuous and controversial than in human cells (43). IFN-
reportedly fails to induce IDO and toxoplasmastatic activity
in mouse fibroblasts (44). Furthermore, in murine macrophages, NO induction by IFN-
results in cross-inhibition of IDO gene transcription and enzymatic activity (42,
45). The above observations suggest that other as yet unidentified iNOS- and IDO-independent mechanism(s) are
responsible for resistance to T. gondii infection, a conclusion also reached in a study involving IFN-
-induced control of the parasite by endothelial cells (46).
The existence of iNOS-independent, IFN--dependent
mechanisms of host resistance is not unique to T. gondii infection. A similar divergence in the resistance phenotypes
of IFN-
- and iNOS-deficient mice has been described in
Chlamydia infections (47). In the case of these two pathogens, in vitro transfection experiments have directly implicated the IDO pathway in the control of microbial growth
within nonhemopoietic cells (48, 49). An IFN-
/TNF-
- dependent but iNOS-independent mechanism of CD8 T
cell-mediated host resistance has also been described for
hepatitis B infection, based on adoptive transfer experiments
in a transgenic mouse model (50). Clearly, the identification
of these important and potentially novel effector pathways is
a highly relevant area for future investigation and represents a
major frontier for the field of microbial immunity.
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Footnotes |
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Address correspondence to George S. Yap, Immunobiology Section, Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, MD 20892. Phone: 301-496-4881; Fax: 301-402-0890; E-mail: gyap{at}atlas.niaid.nih.gov
Received for publication 22 September 1998 and in revised form 29 December 1998.
G. Yap was supported by a Visiting Fellowship from the Fogarty International Center, National Institutes of Health.We are grateful to Dr. K. Elkins (Center for Biologics Evaluation and Research, Food and Drug Administration) for generously providing the Listeria stocks and H. Adams, A. Foxworth, and D. Adams (Animal Care Branch, National Institutes of Allergy and Infectious Diseases) for maintaining the BM chimeric mice. We also thank Drs. K. Elkins, S. James, D. Jankovic, D. Sacks, and T. Wynn for critical reading of the manuscript, Dr. M. Schito for preparing the diagram in Fig. 1, and Dr. T. Scharton-Kersten for her encouragement during the initiation of this project.
Abbreviations used in this paper BM, bone marrow; CMI, cell-mediated immunity; CNS, central nervous system; IDO, indoleamine dioxygenase; iNOS, inducible nitric oxide synthase; KO, knockout; STAg, soluble tachyzoite antigen; WT, wild-type.
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