Aspirin Treatment of Mice Infected with Trypanosoma
cruzi and Implications for the Pathogenesis of Chagas
Shankar Mukherjee1, Fabiana S. Machado2, Huang Huang1, Helieh S. Oz3, Linda A. Jelicks4, Cibele M.
Prado1,5,6, Wade Koba4, Eugene J. Fine4, Dazhi Zhao1,5, Stephen M. Factor1, J. Elias Collado1,5, Louis M.
Weiss1,5, Herbert B. Tanowitz1,5*., Anthony W. Ashton1,7.
1Division of Parasitology, Department of Pathology, Albert Einstein College of Medicine, New York City, New York, United States of America, 2Department of
Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil, 3Center for Oral Health Research, University of
Kentucky Medical Center, Lexington, Kentucky, United States of America, 4Department of Nuclear Medicine and the M. Donald Blaufox Laboratory for Molecular Imaging,
Physiology and Biophysics, Albert Einstein College of Medicine, New York City, New York, United States of America, 5Division of Infectious Disease, Department of
Medicine, Albert Einstein College of Medicine, New York City, New York, United States of America, 6Department of Pathology, University of Sa ˜o Paulo, Ribeira ˜o Preto,
Brazil, 7Division of Perinatal Research, Kolling Institute for Medical Research, University of Sydney, Sydney, Australia
Chagas disease, caused by infection with Trypanosoma cruzi, is an important cause of cardiovascular disease. It is increasingly
clear that parasite-derived prostaglandins potently modulate host response and disease progression. Here, we report that
treatment of experimental T. cruzi infection (Brazil strain) beginning 5 days post infection (dpi) with aspirin (ASA) increased
mortality (2-fold) and parasitemia (12-fold). However, there were no differences regarding histopathology or cardiac structure
or function. Delayed treatment with ASA (20 mg/kg) beginning 60 dpi did not increase parasitemia or mortality but improved
ejection fraction. ASA treatment diminished the profile of parasite- and host-derived circulating prostaglandins in infected
mice. To distinguish the effects of ASA on the parasite and host bio-synthetic pathways we infected cyclooxygenase-1 (COX-1)
null mice with the Brazil-strain of T. cruzi. Infected COX-1 null mice displayed a reduction in circulating levels of thromboxane
(TX)A2and prostaglandin (PG)F2a. Parasitemia was increased in COX-1 null mice compared with parasitemia and mortality in
ASA-treatedinfectedmiceindicatingthe effectsofASAonmortality potentiallyhadlittletodowith inhibitionofprostaglandin
metabolism. Expression of SOCS-2 was enhanced, and TRAF6 and TNFa reduced, in the spleens of infected ASA-treated mice.
Ablation of the initial innate response to infection may cause the increased mortality in ASA-treated mice as the host likely
succumbs more quickly without the initiation of the ‘‘cytokine storm’’ during acute infection. We conclude that ASA, through
both COX inhibition and other ‘‘off-target’’ effects, modulates the progression of acute and chronic Chagas disease. Thus,
eicosanoids present during acute infection may act as immunomodulators aiding the transition to and maintenance of the
chronic phase of the disease. A deeper understanding of the mechanism of ASA action may provide clues to the differences
between host response in the acute and chronic T. cruzi infection.
Citation: Mukherjee S, Machado FS, Huang H, Oz HS, Jelicks LA, et al. (2011) Aspirin Treatment of Mice Infected with Trypanosoma cruzi and Implications for the
Pathogenesis of Chagas Disease. PLoS ONE 6(2): e16959. doi:10.1371/journal.pone.0016959
Editor: Photini Sinnis, New York University School of Medicine, United States of America
Received November 14, 2010; Accepted January 13, 2011; Published February 15, 2011
Copyright: ? 2011 Mukherjee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the United States National Institutes of Health (HBT [AI-76248]) and the National Health and Medical Research
Council of Australia (AWA ). The work was also supported by a Career Development Award from the American Heart Association (SM [0735252N]), the
National Health and Medical Research Council of Australia (AWA ), the Conselho Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico (CNPq) (FSM
[576200/2008-5, 473670/2008-9]), and the Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado de Minas Gerais (FAPEMIG) (FSM ). CMP was supported by a Fogarty
International Training Grant (HBT [D43TW007129]). The funding bodies indicated had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work
In Latin America millions of people are at risk of infection with
the parasite Trypanosoma cruzi, the cause of Chagas disease. The
cardiac manifestations are the most prominent symptoms of
disease. Acute myocarditis is accompanied by an intense
inflammatory response including upregulation of inflammatory
mediators such as cytokines, chemokines, nitric oxide and
endothelin-1 [1–6]. As the acute infection wanes individuals may
remain asymptomatic; however, 10 to 30% of infected individuals
ultimately develop chronic cardiomyopathy . Manifestations
during this stage of the disease include congestive heart failure,
conduction abnormalities and thrombo-embolic events [7,8]. The
etiology of the chronic cardiomyopathy is primarily the result of
parasite persistence but may also result from microvascular spasm
with focal ischemia and autoimmune mechanisms [4,5,9–11]. Our
group has investigated the etiology of vascular spasm in the setting
of T. cruzi infection. In this regard, we suggested as early as 1990
that the eicosanoid, thromboxane (TX)A2contributed to T. cruzi-
associated vasospasm and platelet aggregation .
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Eicosanoids are a family of lipid mediators that participate in a
wide range of biological activities including vascular tone,
inflammation, ischemia and tissue homeostasis . In mammals,
the biosynthetic pathways for these important biological mediators
are well described. Arachidonic acid (AA), derived from
membrane phospholipids on the inner leaflet of the plasma
membrane by phospholipase A2, is hydrolyzed by the prostaglan-
din (PG)H synthase/cyclooxygenase (COX) enzymes to PGH2
. PGH2 is the central substrate for subsequent eicosanoid
synthesis which is mediated by species-specific synthases to
generate PGs and TXA2. Enzymes in the COX family are
structurally and enzymatically similar but have different patho-
physiological roles. COX-1 is constitutive and mediates gastric
mucus production, platelet activation and vascular tone while
COX-2 is inducible and functions in inflammation, cancer and
tissue damage [13,14]. The relevance of these enzymes, and the
bioactive lipids they produce, are not well understood in parasitic
Phospholipase A1 (PLA1) the enzyme that initiates the AA
metabolic pathway by cleaving the Sn-1 acyl chain was reported in
T. brucei [16,17]. PGF2a synthases have been identified in
Leishmania, T. cruzi (Old Yellow Enzyme) and T. brucei [18,19].
PGF2a is the predominant eicosanoid species produced in
Leishmania and T. brucei, along with smaller quantities of PGE2
and PGD2 [16,18,20]. Importantly, T. cruzi preferentially
synthesizes TXA2 . Eicosanoids released by T. cruzi may
contribute to parasite differentiation, phagocytosis  and host
survival  by acting as immunomodulators to aid transition and
maintenance of the chronic phase of the disease. Moreover, recent
studies have demonstrated that trypanosomes are capable of AA
metabolism complicating the interpretation of the potential
significance and source of these bioactive lipids [18–20].
Our recent data  indicated that host- and parasite-derived
prostaglandins potentially contribute to the pathogenesis of
Chagas disease. Given the increasing importance of eicosanoids
in T. cruzi infection, it is not unexpected that there should be
interest in non-steroidal anti-inflammatory agents (NSAIDS) in
the pathogenesis and clinical management of this infection.
However, administration of NSAIDS may enhance mortality in
patients [23,24] and in experimental T. cruzi infection .
Moreover, characterization of COX inhibition on disease
exacerbation in T. cruzi has not been fully addressed. We sought
to determine what effect NSAID use would have on the
development of acute and chronic Chagas disease. To examine
the consequences of COX inhibition we administered aspirin
(ASA) to T. cruzi infected mice either early in the course of
disease, 5 days post infection (dpi) or late in infection (60 dpi).
ASA exhibits irreversible inhibition of COX isoforms and is
widely used to treat the symptoms of Chagas disease making it the
most clinically applicable choice for these studies. COX
inhibition early in the disease increased parasitemia and
mortality. Administration of ASA during the chronic phase had
no effect on mortality or parasitemia but improved ejection
fraction. ASA ablated the increased release of PGF2aand TXA2
in response to T. cruzi infection; however, infection of COX-1
null mice only mimicked the effects of ASA on parasitemia,
primarily through decreased TXA2 release. The enhanced
mortality in response to ASA was likely due to ‘‘off-target’’
effects of ASA. ASA treatment of T. cruzi infected mice
suppressed TNF-a release through increased expression of
suppressor of cytokine signalling-2 (SOCS-2) and reduced
TNF-a receptor-associated factor (TRAF6) expression in the
spleen. Thus, the effects of ASA in T. cruzi infection may be via
dual mechanisms that operate during different phases of disease.
Global inhibition of eicosanoid production early in the
course of T. cruzi infection results in increased
parasitemia and mortality
Infected CD-1 mice were treated with either 20 or 50 mg/kg
ASA from 5 dpi to address the involvement of COX-derived
mediators during acute infection. Over the subsequent 50 days of
infection 40% of untreated mice died (Figure 1A). ASA treatment
increased mortality during acute infection in a dose dependent
manner with 60% and 80% mortality (50 dpi) in the groups
treated with 20 and 50 mg/kg ASA, respectively (Figure 1A).
Similarly, ASA treatment increased the parasitemia during acute
infection by 2.7 and 5.6 fold in the 20 and 50 mg/kg ASA treated
groups, respectively (Figure 1B). Conversely, treatment of mice
with ASA (20 mg/kg) during the chronic phase (60 dpi) produced
no exacerbation of disease (Figure 1C). Delayed administration of
ASA did not increase peripheral parasitemia nor did it augment
mortality (Figure 1C). Thus, eicosanoid production during acute
infection appears to modulate host response and disease evolution
in favor of progression to the chronic state.
ASA treatment during the chronic disease improves
Declining cardiac function is a significant source of mortality in
both experimental models and in patients suffering from Chagas
disease. The effects of early (beginning at 5 dpi) or delayed
(beginning at 60 dpi) treatment with ASA (20 mg/kg) on cardiac
structure and function were determined using magnetic resonance
imaging (MRI). Significant dilatation of the right ventricle was
observed in mice in response to infection (Figure 2A). Neither early
nor delayed treatment with ASA attenuated the right ventricular
dilation (Figure 2A and B) with a 2 fold increase in internal
diameter in all infected groups. Moreover, a 10% decrease in left
ventricular diastolic diameter was also noted across all groups
(Figure 2B). Despite this, delayed treatment significantly reversed
the reduction in ejection fraction observed in infected mice as
determined by echocardiography. The 38% decrease in ejection
fraction in infected mice was attenuated to 15% upon delayed
treatment with ASA. The improved ejection fraction in the
delayed treatment group may be due to reduced inflammation
during the chronic stage of the disease. Using microPET imaging
we previously observed increased glucose uptake in the hearts of
infected mice  which correlated with increased inflammation.
Thus, our microPET data demonstrating reduced left ventricular
glucose uptake (LVSUV; Figure 2D) in the delayed treatment
group suggest reduced inflammation in that group compared with
untreated infected mice. Unlike the mortality data, treatment at
the early timepoints did not restore cardiac function indicating
that the therapeutic window for the preservation of cardiac
function and host survival are not the same.
The effective preservation of myocardial function by ASA
prompted us to examine cardiac tissue from infected mice for
hallmarks of disease (Figure 3). Compared to hearts from
uninfected mice (Figure 3A and B) infected mice displayed
increased inflammation and pseudocysts along with increased
cellularity, mostly resulting from influx of inflammatory cells
(Figure 3C and D). No significant differences in myocardial
histopathology were observed between infected mice with or
without ASA treatment with parasite pseudocysts present in both
groups (arrows, Figure 3C through F). Thus, the action of ASA did
not prevent the pathological changes to the heart induced by
experimental T. cruzi infection.
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ASA treatment ablates eicosanoid production by both
the parasite and host
The improvements to cardiac function with ASA without
alteration to cardiac pathology or structure indicated a humoral
mediator in the suppression of cardiac function. TXA2is an ASA
sensitive mediator with robust links to cardiac damage post-
infarction and during failure [27,28]. To determine the extent of
COX suppression by ASA treatment we measured TXA2levels (as
the stable hydrolytic product TXB2) in the plasma of infected mice
by ELISA. TXB2levels in infected CD-1 mice increased linearly
from 10 dpi and peaked at 27 ng/ml plasma at 45 dpi (Figure 4A).
In ASA-treated, infected mice TXA2levels remained at or below
those observed in uninfected mice (constant at 8 ng/ml plasma
over 45 dpi). ASA treatment ablated TXA2release in uninfected
mice (Figure 4A) and the augmentation of TXA2release in ASA
treated mice due to T. cruzi infection was also blunted when
compared to vehicle treated controls. Thus, both host- and
parasite-derived eicosanoid synthesis in infected mice appear to be
sensitive to COX-inhibition by ASA.
Previously we demonstrated that in T. cruzi-infected TXA2
synthase null mice the majority of TXB2in the plasma is derived
from the parasite, and not the host . TXA2release in infected
TXA2synthase null mice (Figure 4B) was also ablated by treatment
with ASA (20 mg/kg). The reduction in total TXA2levels indicated
that either the TXA2 biosynthetic pathway in the parasite is
host-cell derived PGH2. Treatment of cultured epimastigotes, the
extracellular life-stage of T. cruzi, withASAhad no effect on parasite
proliferation in vitro (data not shown). These results were consistent
with previous data suggesting that the biosynthetic pathways of the
parasite are resistant to the effects of ASA [18,20,29]. Thus, it
appears that the scavenging of prostanoid precursors by the parasite
from the host was the most likely hypothesis for the observed effects.
Infection of COX-1 and TXA2Synthase null mice mimics
only some of the changes in ASA treated mice
To confirm the mechanismofASAaction in vivowe examined the
pathogenesis in genetically modified mice with attenuated biosyn-
thetic capacity. ASA has a 66 fold preference for COX-1 over
COX-2[30,31]; therefore,COX-1null mice were chosento test the
hypothesis. TXA2synthase null mice (normal COX-1 activity by
ablated TXA2synthesis) were used for comparison. Infection of
COX-1 and TXA2synthase null mice with T. cruzi yielded small
alterations in the mortality curves but no significant change in the
overall survival compared to wild-type (WT) littermates (Figure 5A).
Conversely, peripheral parasitemia in infected COX-1 null mice
was increased 9.2 fold compared to WT littermates with peak
parasitemia increased 5.7 fold and prolongation of parasite levels
from 33 dpi to 42 dpi. Similarly, parasitemia in T. cruzi infected
TXA2synthase null mice was increased 7.1fold over the 50 dpi and
accompanied by a 7- fold increase in peak parasitemia and
prolongation of parasite persistence until 48 dpi. The kinetics in
both genetically modified mouse strains were almost identical
suggesting that the majority of these changes were accounted for by
a lack of TXA2production by the host in COX-1 null mice.
mice was significantly different from their WT controls. Plasma of
infected COX-1 null mice exhibited a 2.1 fold decreased rate of
Figure 1. Early administration of ASA increases mortality and parasitemia in response to T. cruzi infection. A and B. CD-1 mice were
infected with the Brazil strain of T. cruzi and mortality (A) and parasitemia (B) assessed in vehicle (N) and ASA treated mice (%, 20 mg/kg; &, 50 mg/
kg) over 55 days post infection (dpi). Treatment with ASA or vehicle started at 5 dpi. C. Table showing the effects of delayed ASA treatment on
chronic experimental T. cruzi infection. ASA treatment (20 mg/kg) was initiated 60 dpi until 120 dpi. Survival and parasitemia were assessed 120 and
75 dpi respectively. Data are represented as mean 6 SD are representative of at least 20 mice per group. * and # indicates significance (P#0.05) from
uninfected and infected mice, respectively. ND=not detected.
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synthesis and an overall 70% decrease in PGF2a production
(Figure 5C) compared to infected WT mice. Unlike WT mice, the
plasma of COX-1 null mice did not contain significant levels of
TXA2(Figure 5D). As stated above, our previous data suggested
that most of the circulating TXA2in experimental infection is
parasite derived . Thus, our current data suggest that
scavenging of metabolic intermediates from the host is likely
hypothesis the release of PGF2afrom infected TXA2synthase null
mice, which haveunaffected generation of precursor molecules, was
normal (Figure 5C). Collectively, these data indicate that decreased
eicosanoid biosynthesis by ASA only accounts for the control of
parasite proliferation in experimental infection while the enhanced
mortality observed with early administration of ASA may be a
response to alterations in other unrelated pathways.
Effect of ASA on cytokines in T. cruzi infected mice
The enhanced mortality when ASA treatment was initiated
early resulted in the hypothesis that ASA treatment might inhibit
priming of the innate immune system in order to decrease host
response to the parasite. Plasma of mice treated with ASA (20 mg/
kg) during acute T. cruzi infection displayed a reduction in TNF-a
and IFN-c levels 15 dpi (data not shown). Immunoblotting of
lysates from the spleen of infected mice confirmed that TNF-a
expression was significantly reduced by ASA treatment (Figure 6).
These findings were not replicated in cardiac tissue (no change in
TNFa production compared to uninfected mice) indicating that
the spleen was the likely source of the cytokines present in the
plasma and the target organ of ASA treatment. ASA has many off-
target effects that influence the inflammatory response including
inhibition of NFkB , augmentation of SOCS-2 and decrease
of TRAF6 expression [33,34]. Treatment with ASA early in
infection not only ablated TNF-a release from spleen but also
increased SOCS-2 and reduced TRAF6 expression (Figure 6).
Changes were observed as early as 15 dpi and were even more
pronounced at 30 dpi indicating they were present throughout the
acute phase of disease. Thus, the enhanced mortality observed in
infected mice treated with ASA early in infection may be due to
suppression of the innate immune response as a result of
suppression of cytokine synthesis/release and receptor signaling
associated with the initial ‘‘cytokine storm’’ during acute infection.
To examine the above hypothesis more directly, we treated
infected WT and COX-1 null mice with ASA (20 mg/kg) and
examined the effects on survival (Figure 7). Infection of COX-1
null mice with T. cruzi had little effect on host survival with 23.3%
mortality over the 50 days of infection. However, infected COX-1
null mice treated with ASA beginning 5 dpi significantly
exacerbated mortality with 65% of mice dying in response to
Figure 2. Effects of early and delayed administration of ASA on cardiac structure and function. A. Short axis MRI images showing cardiac
remodeling during T. cruzi infection of CD-1 mice with and without ASA treatment (20 mg/kg). Arrows indicate the wall of the right ventricle of the
heart. B. Cardiac dimensions were assessed in uninfected and infected with or without ASA at 5 (Early) or 60 (delayed) dpi. Parameters quantified
included left ventricular internal diastolic diameter (B, while squares), left ventricular wall diameter (B, grey squares), right ventricular internal
diameter (B, black squares). C. Ejection fraction measured using echocardiography. D. Left ventricular glucose SUV measured by microPET. Number of
mice in each group is indicted. * and # indicates significance (P#0.05) from uninfected and infected mice, respectively.
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T. cruzi infection over the subsequent 50 days and with a linear
rate of loss (1.995 mice/day) from day 26 onwards. As an
important target of ASA was removed in these mice we can only
speculate that the ‘‘off target’’ effects of ASA we have observed are
those that mediate the mortality in response to infection.
It is now appreciated that the release of eicosanoids during
infection with T. cruzi regulates host responses and controls disease
progression . The role of these bioactive lipids in acute and
chronic Chagas disease is largely unexplored and potentially
further complicated by whether the host or the parasite is the
primary source of synthesis. In this study we found that ASA
treatment increased mortality and parasitemia in a dose-
dependent manner during acute infection with the Brazil-strain
of T. cruzi in mice. These changes were due, in part, to suppression
of eicosanoid production (primarily TXA2) which controls parasite
proliferation and may participate in cytokine release/signaling
during early disease. Importantly, our data strongly suggest that
control of fever and pain with ASA during acute Chagas disease
should be used with caution. Conversely, use of ASA in the
chronic phase of disease may improve cardiac function suggesting
the same COX-1 products that mediate host-survival during the
acute disease are likely to contribute to the progression of cardiac
damage and heart failure in the chronic phase. Importantly, we
established an essential host-parasite interdependence that dictates
the biosynthetic activity of the parasite. This interdependence is an
exploitable target for therapy to manage the chronic phase of
disease and potentially prevent disease progression.
Previous studies have attempted to document the role of
eicosanoids in early disease using pharmacological intervention with
mixed results [22,24,25,35,36]. Pharmacological antagonists selective
for either COX-1 (ASA), COX-2 (celecoxib) or both (indomethacin)
increase mortality and parasitemia (both peripheral blood counts and
cardiacparasite nests) regardlessofmouse orparasite strainused[23–
25,36]. Conversely, others have found inhibition of prostaglandin
synthesis/release ablates parasitemia and extends survival in mice
infected with T. cruzi [22,35,37]. Our data correlates well with the
former group of studies regarding the changes in parasitemia and
degree of mortality. Moreover, use of the COX-1 null mice in this
study confirms that COX-1 derived mediators from the host
contribute to the suppression of parasite proliferation but perhaps
not mortality in acute disease. None of the other studies have utilized
null mice to confirm the observed effects and therefore it is difficult to
know whether mortality and parasitemia are coordinately regulated
in other reports or the response to separate properties of the
pharmacological antagonists used (as suggested by our study).
The mechanism for the enhanced mortality with NSAID
treatment during acute disease may lie with more complete
inhibition of prostaglandin synthesis or ‘‘off-target’’ effects of these
agents. ASA is not mono-specific and will also inhibit COX-2 .
Figure 3. Cardiac pathology in ASA-treated mice is no different to vehicle treated controls. Representative histopathology of infected
CD-1 mice with and without ASA treatment (20 mg/kg) at 35 dpi compared with uninfected controls. Sections were stained with H&E. Parasite
pseudocysts are observed (arrows). Total magnification of either 1006(A, C, E) or 4006(B, D, F). Images are representative of al least five mice in
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Conversely, the COX-1 null mice have ‘‘normal’’ COX-2 levels
and synthesis of many of the most potent immunosuppressive
prostaglandins, e.g. PGE2and PGI2, are closely linked to COX-2
expression . Therefore, a significant reason for why ASA, but
not deletion of COX-1, might be lethal in mice is the presence of
COX-2-associated immunosuppressive prostaglandins in the
COX-1 null mice. Aside from the inhibition of prostaglandin
synthesis ASA induces the synthesis of aspirin triggered lipoxin
(ATL) which is COX-2-dependent with little contribution from
COX-1. ALT induces SOCS-2 expression and TRAF6 degrada-
tion. Importantly, Machado and colleagues  demonstrated
that ASA-treated SOCS-2 null mice given LPS by the intra-
peritoneal route could not inhibit neutrophil migration and TNFa
signaling. Thus, mortality may have more to do with modulation
of the impending ‘‘cytokine storm’’ during acute disease than
actual prostaglandin production.
result from the different combination of agents, mice and parasite
strains previously employed. The expression of both COX isoforms
remains unchanged during infection and there is no increase in
COX-2 levels in COX-1 null mice as detected by immunoblotting
(data not shown). While the role of COX-2 in T. cruzi infection is
largely undefined both COX-1 and -2 appear to play different roles
during acute infection. Inhibition of COX-2 (celecoxib), but not
COX-1(ASA), prevented the thrombocytopenia and leukopenia
associated with acute infection and increased reticulocyte counts in
response to infection . Inhibition of COX-1 and -2 reciprocally
regulates NO release from M1 and M2 macrophages which may
correlate with resistanceto disease. Consistent with this observation,
COX-2-derived prostaglandins mediate most of the immunosup-
pressive effectsduringtheinitialphase ofT.cruzi infection .This
may result from the observations that PGI2and PGE2are more
closely linked to COX-2 metabolism while COX-1 is aligned with
TXA2synthesis [39,40]. Thus, the selectivity of the NSAIDs used
may determine whether parasite or host production of PGs is the
primary target of the treatment regimen used.
Our data with COX-1 null mice and pharmacological antagonism
strongly indicate that host-derived PGH2is involved in PG synthesis
throughout infection. A key question is whether the host or parasite is
the primary source of the lipid mediators regulating the pathogenesis
of disease. Pharmacological inhibition does not distinguish between
these two sources of eicosanoids. The reduction in PGF2arelease in
COX-1 null, but not TXA2synthase null, mice indicates that COX
activity in the host provides precursor molecules required for the
biosynthetic pathways of this parasite.This ‘‘scavenging’’ hypothesis is
confirmed bythe inabilityofthe parasite (the primarysource ofTXA2
during infection) to sustain TXA2release in the COX-1 null mice. If
the parasite is scavenging precursors from the host then they would
biosynthetic pathways in trypanosomes are poorly defined and little
homology is reported between the mammalian enzymes and their
PGF2asynthase ‘‘old yellow enzyme’’ , have been identified.
However, reports have indicated that parasitic biosynthetic pathways
effect on parasite biology [18,20,29]. Conversely, the recent report of
anti-parasitic activity of indomethacin derivatives  indicates that
the active sites of parasite enzymes, if not their primary sequences, are
sufficiently homologous to their mammalian counterparts. Recent
structural characterization of the target enzyme (TcCYP51), which
participates in sterol biosynthesis of T. cruzi, has facilitated
understanding of the integral nature of this enzyme to T. cruzi and
has revealed much of the kinetics of the mechanism of action of
indomethacin amides . Interestingly, no enzyme other than COX
isoforms has been identified as sensitive to indomethacin. However, it
remains to be determined whether TcCYP51 is an integral
component of the eicosanoid biosynthetic pathway in T. cruzi. .
The identification of the PGH2 derivatives that are most
important for disease remains unsolved. Several species of
eicosanoids have been implicated in both acute and chronic
Chagas disease. Plasma from infected mice displayed increased
levels of PGF2a, PGI2, TXA2 and PGE2 compared to
uninfected mice from 10 dpi onwards. Previously, we determined
that the main prostaglandins derived from T. cruzi are TXA2and
PGF2a, indicating that host is the likely source of the elevated
PGI2 and PGE2. No specific role has been delineated for the
elevated PGI2and PGF2aobserved in plasma from experimental
T. cruzi infection. PGF2alevels in the TXA2synthase null and WT
mice were similar indicating this prostaglandin was likely not
involved with the augmentation of parasitemia observed in the
COX-1 null and ASA treated mice or in the regulation of
mortality. This leaves the potential role of PGF2a in Chagas
disease largely unexplored; however, the significant amounts of
PGF2aproduced by T. cruzi, and the fact that all members of the
trypanosomatids have an identifiable synthase for PGF2a, indicate
that it is of significant value to the parasite.
During acute infection, PGE2has been shown to modulate the
virulence of the T. cruzi strain. A non-lethal strain (K98) provoked
elevated circulating PGE2while lethal strains (RA or K98-2) did not
. Inhibition of COX activity (and therefore PGE2 release)
increased mortality in K98-strain infected mice but PGE2infusion
did not attenuate the virulence of the RA strain. Inhibition of PGE2
synthesis reduces both inflammatory infiltrates and cardiac fibrosis
during acute infection . Conversely, preventing host response to
parasite-derived TXA2 augmented death and parasitemia .
TXA2likely regulates vasospasm, thrombosis, vascular permeability
and endothelial cell dysfunction during acute disease. TXA2also
displays immunosuppressive properties as WT mice display
minimal pathology but TXA2 receptor null mice exhibited
pronounced myocardial inflammation with an almost 3-fold
Figure 4. ASA inhibits both host- and T. cruzi-derived prosta-
glandin production. Plasma TXA2 levels, measured as the stable
hydrolytic product TXB2by ELISA, in uninfected or infected CD1 (A) or TXA2
synthase null (B) mice. ASA treatment (20 mg/ml) was initiated on 5 dpi.
Circulating levels were assessed at 30 and 20 dpi, respectively. Data (mean
6 SD) are derived from at least 5 mice per group. * and # indicate
significance (P#0.05) from uninfected mice and infected mice, respectively.
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increased in parasite load in cardiac tissue. Thus, it appears that the
eicosanoids present during acute infection largely act as immuno-
modulators that aid in the transition to and maintenance of the
chronic phase of the disease . It is unclear whether T. cruzi
generates prostaglandins as a defense against host immune system
or whether it hijacks the hostprostaglandinmetabolicpathway in its
favor. To this end, further studies using null mice missing
biosynthetic enzymes or receptors are required to fully elucidate
the role of the identified prostaglandins in Chagas disease.
In contrast to acute infection, where plasma levels of multiple PGs
are elevated, only increased levels of TXA2are observed in chronic
disease (.180 dpi) . In chronic disease the effects of TXA2
largely promote tissue damage, especially in the heart where it may
exacerbate myocyte apoptosis and enhance progression to dilated
cardiomyopathy and heart failure, a major cause of death in patients
with this disease. Thus, disproving the adage that the things that
don’t kill you make you stronger. In addition to the maelstrom of
changes that TXA2mediates during acute infection, the secretion of
TXA2would prevent the initiation of an adaptive immune response
by the host , enabling progression to and maintenance of the
chronic phase of the disease. Finally, the role for TXA2in chronic
disease is made more complicated by its control of parasite
proliferation. While we have confirmed that TXA2 plays a
prominent role in Chagas disease the hypothesis that parasite-
derived TXA2is the primary quorum sensor for the parasite 
may need to be re-visited. Unlike in acute infection (,30 dpi)
parasite-derived TXA2 release does not function to suppress
peripheral parasitemia in the long term with overall parasite load
in the TXA2synthase null mice 7-fold higher than WT littermates.
This produced a late increase in the mortality of TXA2synthase null
mice which was not significant but may point to a need for host-
derived TXA2for control of the severity of the chronic disease. In
fact, in the group with delayed ASA treatment there was an
improvement inthe infection-associated decreaseinejection fraction
which may have resulted from negating the detrimental effects of
TXA2on myocyte contractility, platelet function and vascular tone.
In conclusion, our results demonstrate for the first time, that
parasitemia and mortality in response to COX blockade during T.
cruzi infection may be due to different pathways related to
inhibition of prostaglandin synthesis and cytokine release respec-
tively. Our data also show, for the first time, an interdependence of
the parasite on host metabolism for prostanoid biosynthesis. These
findings advance our understanding of host–parasite relationships
and reveal a potential new avenue for pharmacological treatment
for a disease with few therapeutic options.
Materials and Methods
Male CD-1 mice were obtained from Charles River Labora-
tories (Wilmington, MA). C57Bl/6 and CH3/HeJ mice were
obtained from Jackson Laboratories (Bar Harbor, ME). COX-1
Figure 5. Deletion of COX-1 mimics the effects of ASA on parasitemia but not survival in T. cruzi infected mice. A and B. Survival curves
(A) and peripheral parasitemia (B) for WT (black circle), TXA2synthase null mice (grey circle) and COX1 null mice (#) mice after inoculation with 105
trypomastigotes of the Brazil strain. C and D. Measurement of plasma PGF2a(C) and TXB2(D) in infected WT (black square), TXA2synthase null mice
(grey square) and COX-1 null mice (white square) mice. Data are represented as mean 6 SD are representative of at least 20 mice per group. * and #
indicates significance (P#0.05) from uninfected and infected WT mice, respectively.
Aspirin in Experimental Chagas Disease
PLoS ONE | www.plosone.org7 February 2011 | Volume 6 | Issue 2 | e16959
null mice and their wild type counter parts (originally from
Taconic Farms [Hudson, NY]), TXA2synthase null (originally
from Dr. Kenneth Wu, University of Texas Health Science
Center, Houston, TX), were bred in our facility. This study was
carried out in strict accordance with the recommendations in the
Guide for the Care and Use of Laboratory Animals of the National
Institutes of Health. All experiments involving mice were approved
by the Albert Einstein College of Medicine Institutional Animal
Care and Use Committee (Approval Number: 20100204). All
efforts were made to minimize suffering during surgical proce-
Parasitology and pathology
The Brazil strain of T. cruzi was used in our experiments. The
Brazil strain was maintained in C3H/HeJ. Hearts were obtained
from infected and uninfected mice, fixed in 10% buffered formalin,
paraffin embedded and stained with H&E. Male mice (8–10 weeks)
were infected by an intra-peritoneal route with 56104trypomas-
tigotes Brazil strain of T. cruzi at the inoculums indicated. ASA
(Sigma-Aldrich, Saint Louis, MO) was administered daily via
intraperitoneal route at a dose of either 20 or 50 mg/kg of body
weight beginning 5 (early) or 60 (delayed) dpi and was continued for
60 days. Mortality was recorded and blood drawn for the
determination of parasitemia at the intervals stated. Parasitemia
was assessed by counting in a hemocytometer chamber.
Cardiac magnetic resonance imaging (MRI)
Cardiac MRI of mice infected with T. cruzi was first described
by our laboratory group . Briefly, the mice were anesthetized
with 1.5% of isoflurane. A set of standard, shielded, nonmagnetic
electrocardiographic leads ending in silver wires were attached to
the four limbs. The ECG signal was fed to a Gould ECG amplifier
Figure 6. Treatment of T. cruzi-infected mice with ASA modulates cytokine signaling in the spleen of infected mice. Splenic extracts
were prepared from uninfected and infected CD1 mice treated with ASA (20 mg/kg) from 5 dpi. Immunoblotting for TNF-a, SOCS-2 and TRAF-6 was
performed on 15 and 30 dpi. b-actin was used as a loading control. Immunoblots are representative from at least three separate experiments.
Aspirin in Experimental Chagas Disease
PLoS ONE | www.plosone.org8 February 2011 | Volume 6 | Issue 2 | e16959
(Gould Instrument Systems, Inc. Valley View, OH) associated
with the Ponemah Physiology data acquisition system (Gould
Instrument Systems, Inc. Valley View, OH) for monitoring the
ECG and the R wave triggered a 5 volt signal to gate the
spectrometer. Images were acquired with a GE/Omega 9.4 T
vertical wide-bore spectrometer operating at a 1H frequency of
400 MHz and equipped with 50-mm shielded gradients (General
Electric, Fremont, CA) and a 40-mm1H imaging coil (RF Sensors,
New York, NY). Temperature within the coils was maintained at
30uC using a water cooling unit (Neslab Instrument, Inc.,
Portsmouth, NH). This temperature prevented hypothermia in
the anesthetized mice. After attachment of the cardiac gating
leads, the mice were wrapped in a Teflon sheet and multi-slice spin
echo imaging was performed to obtain short axis images of the
heart. The gating delay was adjusted to collect data in systole or
diastole. The following parameters were used to obtain 8 short axis
slices: echo time, 18 msec; field of view, 51.2 mm; number of
averages, 4; slice thickness, 1 mm; repetition time, approximately
0.2 sec; matrix size, 1286256 (interpolated to 2566256).
Several sets of 8 slices were acquired to define the entire heart
and to obtain images in diastole and systole taking approximately
20–30 minutes per mouse. Data were transferred to a PC and
analyzed using MATLAB-based software. Left ventricle (LV) and
right ventricle (RV) dimensions in millimeters were determined
from the images representing end-diastole. The left ventricular
wall is the average of the anterior, posterior, lateral, and septal
walls. The right ventricular internal dimension is the widest point
of the right ventricular cavity.
Echocardiography of mice infected with T. cruzi has been
described previously by our laboratory . Briefly, the mice were
lightly anesthetized with 1.5% isoflurane in 100% O2; the chest
wall was shaved and a small gel standoff was placed between the
chest and a 30-MHz RMV-707 B scanhead interfaced with a
Vevo 770 High-Resolution Imaging System (VisualSonics, Tor-
onto, ON, Canada). High-resolution, two-dimensional electrocar-
diogram-based kilohertz visualization (EKV Mode) and B mode
images were acquired. Continuous, standard electrocardiogram
was recorded using electrodes placed on the animal’s extremities.
Diastolic measurements were performed at the point of greatest
cavity dimension, and systolic measurements were made at the
point of minimal cavity dimension, using the leading edge method
of the American Society of Echocardiography (http://www.
asefiles.org/ChamberQuantification.pdf). Ejection fraction was
calculated and used as a determinant of LV cardiac function.
Micro-positron emission tomography (microPET)
We were the first group to describe the utility of microPET in the
mouse model of Chagas disease . Briefly, the mice were imaged
after 3 hours of fasting. Mice were anesthetized with 1.5%
isoflurane-oxygen mixture, which continued throughout the
imaging portion of the procedure. Each mouse was placed on a
heating pad before and during scanning to maintain normal body
temperature.Mice wereadministered300–400 mCi(12–15 MBq)in
0.1 mL normal saline, [18F] fluoro-2-deoxyglucose (FDG), via tail
vein and imaging was started at 1 hour after injection as previously
described. Imaging was performed using a Concorde Microsystems
R4 microPET Scanner, with 24 detector modules, without septa,
providing 7.9 cm axial and 12 cm transaxial field of view.
Acquisitions were performed in three-dimensional (3D) list mode.
Image analysis was performed using ASIPRO VM (Concorde
Microsystems, LLC) dedicated software. Manual regions of interest
(ROI) were defined around areas of visually identified heart activity
in the LV. Successive scrolling through 2 dimensional slices (each
1.2 mm thick in the axial images) permitted identification of a pixel
of maximum measured decay-corrected uptake, termed the
standardized uptake value, or SUV max. The SUV max is the
maximum value of the percentage injected dose per gram of cardiac
tissue multiplied by the body weight of each animal.
Measurement of PGF2aand TXB2by ELISA
Blood was drawn from the retro orbital plexus in anti-
coagulated tubes with heparin (100 U/ml). The blood was
centrifuged at 1500xg to remove cellular components and the
platelet-poor plasma used for the assessment of prostaglandins.
Plasma TXA2and PGF2alevels were determined using sensitive
ELISA kits according to manufacturer’s instructions (Cayman
Chemical Company, Ann Arbor, MI). TXA2 levels were
determined by measuring the stable hydrolytic product TXB2.
Immunoblotting of Signaling Molecules
For immunoblotting, aliquots of whole cell lysates (30 mg) from
spleen and heart were separated by SDS-PAGE under reducing
conditions. Proteins were transferred onto nitrocellulose mem-
brane (Protan BA 85 Nitrocellulose from Whatman, Dassel,
Germany) and analyzed by immunoblotting using antibodies
against SOCS-2, TNFa, and TRAF6 (Santa Cruz Biotechnology,
Santa Cruz, CA). Antibodies against b-actin (Ana Spec Inc, San
Jose, CA) was used to control for loading.
Data were pooled and statistical analysis was performed using
the Mann-Whitney U-test using Sigma Stat Version 2.0. Statistical
differences (p#0.05) are indicated on each figure using * or # to
denote significance from control and infected groups, respectively.
Conceived and designed the experiments: LMW HBT AWA. Performed
the experiments: SM FSM HH DZ HSO. Analyzed the data: LAJ SMF
LMW HBT AWA. Contributed reagents/materials/analysis tools: CMP
WK EJF JEC. Wrote the paper: LAJ LMW HBT AWA.
Figure 7. Treatment of T. cruzi infected COX-1 null mice with
ASA increases mortality. Survival curves for COX-1 null mice treated
with vehicle (N; n=10) or ASA (20 mg/kg) (#; n=14) beginning on
5 dpi. Mice were inoculated with 104trypomastigotes of the Brazil
strain and observed over 50 dpi.
Aspirin in Experimental Chagas Disease
PLoS ONE | www.plosone.org9 February 2011 | Volume 6 | Issue 2 | e16959
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