Cell Therapy in Chagas Disease.

Abstract

Chagas disease which is caused by the parasite Trypanosoma cruzi is an important cause of cardiomyopathy in Latin America. In later stages chagasic cardiomyopathy is associated with congestive heart failure which is often refractory to medical therapy. In these individuals heart transplantation has been attempted. However, this procedure is fraught with many problems attributable to the surgery and the postsurgical administration of immunosuppressive drugs. Studies in mice suggest that the transplantation of bone-marrow-derived cells ameliorates the inflammation and fibrosis in the heart associated with this infection. Cardiac magnetic resonance imaging reveals that bone marrow transplantation ameliorates the infection induced right ventricular enlargement. On the basis of these animal studies the safety of autologous bone marrow transplantation has been assessed in patients with chagasic end-stage heart disease. The initial results are encouraging and more studies need to be performed.

Full text

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Hindawi Publishing Corporation
Interdisciplinary Perspectives on Infectious Diseases
Volume 2009, Article ID 484358, 6 pages
doi:10.1155/2009/484358
Review Article
CellTherapy inChagas Disease
AntonioC.CamposdeCarvalho,1,2,3Regina C.S.Goldenberg,3LindaA.Jelicks,4
MilenaB.P.Soares,5RicardoRibeirodosSantos,5DavidC.Spray,2andHerbertB.Tanowitz6
1Instituto Nacional de Cardiologia, 22240-006 Rio de Janeiro, RJ, Brazil
2Dominick P. Purpura, Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA
3Instituto de Biof´ ısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil
4Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
5Centro de Pesquisas Gonc ¸alo Moniz, Fundac ¸˜ ao Oswaldo Cruz, 40296-70 Salvador, BA, Brazil
6Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
Correspondence should be addressed to Herbert B. Tanowitz, tanowitz@aecom.yu.edu
Received 5 April 2009; Accepted 11 May 2009
Recommended by Louis M. Weiss
Chagas disease which is caused by the parasite Trypanosoma cruzi is an important cause of cardiomyopathy in Latin America. In
later stages chagasic cardiomyopathy is associated with congestive heart failure which is often refractory to medical therapy. In
these individuals heart transplantation has been attempted. However, this procedure is fraught with many problems attributable
tothesurgeryandthepostsurgicaladministrationofimmunosuppressivedrugs.Studiesinmicesuggestthatthetransplantationof
bone-marrow-derived cells ameliorates the inflammation and fibrosis in the heart associated with this infection. Cardiac magnetic
resonance imaging reveals that bone marrow transplantation ameliorates the infection induced right ventricular enlargement. On
the basis of these animal studies the safety of autologous bone marrow transplantation has been assessed in patients with chagasic
end-stage heart disease. The initial results are encouraging and more studies need to be performed.
Copyright © 2009 Antonio C. Campos de Carvalho et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
1.Introduction
This year we celebrate the 100th anniversary of the discovery
by the Brazilian physician-scientist Carlos Chagas of the
disease that bears his name (Chagas disease). This represents
a rare instance in the history of medicine where a researcher
described the disease, identified the transmission method
and isolated the causal agent [1, 2]. Since 1909, many studies
have been performed to unravel disease mechanisms and
to find a cure for Chagas disease. Unfortunately, although
much progress has been achieved, until now there is no
consensus on the exact mechanisms that lead to the different
manifestations of the disease, nor is there an effective
treatment for this infection.
Chagas disease is caused by the hemoflagellate parasite,
Trypanosoma cruzi. The parasite has a complex life cycle
consisting of different life forms that are distinguished by
morphological and biochemical criteria. Blood form trypo-
mastigotes are found in infected mammalian hosts. After
the insect vector, from the Reduvidae family, ingests blood
containing blood form trypomastigotes they transform into
dividing epimastigotes in the midgut of the insect vector.
After3–4weeksepimastigotesbecomeinfective,nondividing
metacyclic trypomastigotes. These forms are present in the
hindgut of the vector and are deposited with the feces during
blood meals. Transmission to a new host takes place when
the parasite-laden feces contaminate oral or nasal mucous
membranes, the conjunctivas, or other vulnerable surfaces
such as the skin. In the mammalian host the metacyclic
trypomastigotes invade the cells of the host and once inside
the parasites transform into intracellular amastigotes that
multiply by binary fission. As the amastigotes accumulate
inside the host cells, signaling mechanisms that have not
been fully identified lead to their transformation into blood
form trypomastigotes. They are then released as the host
cell ruptures and disseminate through the lymphatics and
the bloodstream to invade new cells or, while in circulating
blood, may be ingested in meals taken by the insect vectors.

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2Interdisciplinary Perspectives on Infectious Diseases
Although any nucleated mammalian cell can be para-
sitized by T. cruzi, cells of the reticuloendothelial, nervous
and muscle systems, including the heart, appear to be
favored. Chagas disease is characterized by three phases,
acute, indeterminate and chronic. In the acute infection,
which usually lasts for approximately two months, there are
nonspecific signs and symptoms such as fever and myalgias
associated with tissue parasitism, inflammation and high
peripheral blood parasitemia. The indeterminate phase may
last for months to a lifetime during which individuals are
relatively asymptomatic. In the chronic phase parasitemia
is low or nonexistent but there is an intense inflammatory
process in the affected organs. The gastrointestinal tract and
heart are the main targets of the chronic stage of the disease
and in these organs dilatation is present, constituting the so-
called mega syndromes.
The disease is endemic in all Latin America countries
with the exception of the Caribbean nations. In these
countries it is estimated that 16–18 million individuals are
infected with the parasite, with many new cases reported
each year. In the past transmission of T. cruzi to humans
has essentially been vector-borne. Presently this situation has
changed drastically due to the successful implementation of
vector control programs in many of the endemic countries.
The Southern Cone Initiative (SCI), which began in 1991
in Argentina, Bolivia, Brazil, Chile, Paraguay, and Uruguay,
was instrumental for the success of the control program.
The transmission of T. cruzi by blood transfusion has been
essentially eliminated throughout much of the endemic
range by obligatory testing of donated blood for evidence
of T. cruzi infection. In recent decades the rate of emigra-
tion from Chagas-endemic countries to the United States,
Canada, and the European Union has increased markedly.
Currently an estimated 100000 immigrants from these areas
are chronically infected with T. cruzi. Although these regions
are free of the vector transmission, transmission by blood
transfusion and by organ transplantation have been reported
in Canada and the United States.
During the chronic phase, cardiomyopathy is the most
important clinical manifestation of Chagas disease. It is
estimated that 10%–30% of all infected individuals will
acquire chronic chagasic cardiomyopathy. This represents
anywhere between 1.6 to 5.4 million patients with chronic
chagasic cardiomyopathy in Latin America, making Chagas
disease one of the most important causes of heart disease in
this region. Additionally, the chronic cardiac manifestations
of Chagas disease have created an immense social and eco-
nomic burden in endemic areas because of unemployment
and increased health care costs. It has been estimated that
20000 deaths occur annually in endemic countries due to
complications of chronic chagasic cardiomyopathy [3].
Dilated congestive cardiomyopathy is an important
manifestation of chronic chagasic cardiomyopathy that
typically occurs years or even decades after a person first
becomes infected. Apical aneurysm of the left ventricle is
one of the hallmarks of chronic chagasic cardiomyopa-
thy. Chronic chagasic cardiomyopathy is characterized by
focal or disseminated inflammatory infiltrates, myocytolysis,
myonecrosis and progressive fibrosis [4, 5]. Remodeling of
the myocardium and vasculature is the result of damage
to the extracellular matrix and the replacement of cardiac
myocytes and/or vascular cells by fibrous tissue. This results
in thinning of the myocardium and hypertrophy of the
remaining cardiac myocytes and also leads to thromboem-
bolic events. In that regard chronic chagasic cardiomyopathy
is similar to other dilated cardiomyopathies that lead to
congestive heart failure. The clinical correlation between
intensity of the myocarditis varies considerably from mild
cardiac symptoms to intense chronic cardiomyopathy, lead-
ing to heart failure and death [6]. Patients with chronic
chagasic cardiomyopathy may have a variety of arrhythmias
causing heart malfunction. The ECG abnormalities include
right bundle-branch block, left anterior fascicular block,
ventricular premature beats and A-V block [7, 8].
The virtual absence of parasites, both circulating and
within the heart and the presence of a focal and widespread
inflammatory process in the myocardium have generated
multiple hypotheses to explain the etiology of chronic cha-
gasic cardiomyopathy. However, the general opinion is that
the etiology of chronic chagasic cardiomyopathy is multi-
factorial involving parasite persistence, vascular impairment,
destruction of ganglia of the autonomic nervous system and
autoimmunity[9].Withsuchcomplexdiseasemechanismsit
isnotsurprisingthatoncethecardiomyopathyisestablished,
the prognosis for the chagasic patient is rather bleak. In fact,
chronic chagasic cardiomyopathy has been reported to be
the main prognostic mortality factor among patients with
heart failure of various etiologies [10]. Therapies for chronic
chagasic cardiomyopathy are identical to those for conges-
tive heart failure and often include β-blockers, diuretics,
angiotensin-enzyme inhibitors angiotensin receptor blockers
and amiodarone. There is no consensus about the use of
anti-trypanosomal agents such as benznidazole. In fact, a
large trial designed to address the efficacy of benznidazole in
chronicchagasiccardiomyopathyisunderway;theBENEFIT
Multicenter Trial. As the disease progresses few therapeutic
options are left for the chronic chagasic cardiomyopathy
patient other than heart transplantation. Although survival
in chagasic heart transplant patients has been reported
to be longer than that of persons transplanted for heart
disease resulting from other etiologies [11], the limited
number of donors and the complications of immunosup-
pressive therapy, including parasite reactivation, make this
therapeutic option a very limited one for the majority of
chronic chagasic cardiomyopathy patients. In that scenario,
cell transplantation appears as an alternative to standard
therapies in the setting of chronic chagasic cardiomyopathy.
2. CellTherapy for ChagasicCardiomyopathy
The use of cell therapy for chagasic cardiomyopathy followed
closelythedevelopmentofresearchontheuseofthistherapy
in patients with myocardial infarction. The pioneering
work of Soonpaa et al. [12] demonstrated conclusively
that exogenous cells could be integrated into the host
myocardium. Although initially most of the studies in this
area of research focused on transplantation of fetal cardiac

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Interdisciplinary Perspectives on Infectious Diseases3
myocytes, embryonic stem cells, or skeletal myoblasts into
hearts that were damaged cryogenically or by myocardial
infarction, more recently bone marrow derived cells have
become an important cell source. A major development
in the use of cell therapies to improve cardiac function
was based on the observations that stromal bone marrow
cells could be induced to differentiate into cardiac myocytes
in vitro [13] and that when they were transplanted into
cryo-injured rat hearts, myocardial function improved and
angiogenesis was promoted [14]. Another significant devel-
opment was the report by Orlic et al. [15] that hematopoietic
stem cells from transgenic mice expressing enhanced green
fluorescent protein (EGFP) transplanted into myocardial
infarction-damaged hearts of syngeneic mice differentiated
into cardiac muscle and vascular cells. Importantly, they
demonstrated complete integration of the transplanted c-
Kit+bone marrow derived cells, including formation of
connexin 43 gap junctions between the newly formed
myocardium and the surviving tissue. Many others have
reported that hematopoietic and mesenchymal stem cells
derived from bone marrow improve myocardial function
in animal models of both cryo-injured and ischemic heart
lesions [16–19].
Cardiac regeneration by bone marrow-derived cells has
been questioned [20–22] and remains controversial [23].
However, even in cases where cardiac regeneration by bone
marrow-derived cells has not been demonstrated, func-
tional measurements have detected improvements in heart
function after cell transplantation [22]. More recently, the
beneficialeffectsofcelltherapiesusingbonemarrow-derived
cells in heart disease have been increasingly attributed to
paracrine effects [24, 25].
In most of the reported studies the damage to the heart
is circumscribed to a specific area since the lesions are
ischemicinnature.Accordingly,celldeliveryhasbeenmostly
intramyocardial, especially in small animals. Due to the
global nature of chronic chagasic cardiomyopathy, systemic
delivery of cells was chosen for studies in a mouse model
of Chagas disease. Our reasoning was that direct myocardial
injections would have to be performed in various areas of the
left and right ventricle, creating the possibility of myocardial
damage due to the multiple injections. Thus, to validate the
therapy it was necessary to demonstrate that cells injected
intravenously established themselves in the chagasic hearts.
In initial experiments bone marrow mononuclear cells were
preincubated with Hoechst 33258 stain prior to injection
into tail veins of normal and chagasic mice, and cell-treated
mice were sacrificed at various time points thereafter. In
chagasic mice Hoechst+cells were observed in the heart 1–
7 days after BM cell injection, but were not found in heart
sectionsofnormalmiceinjectedwithHoechst33258-stained
cells (see Figure 1). Hoechst+cells were also found in the
spleen and liver of chagasic and control bone marrow cell-
treated mice 1-2 days after transplant. Heart sections of
mononuclear cell-treated mice were also stained for stem cell
markers by immunofluorescence, and Sca-1+and c-Kit+cell
clusters were found in hearts of mononuclear cell-treated
mice after cell injection [26]. As a result of these and other
experiments in which bone marrow cells from EGFP positive
mice were used, it was concluded that bone marrow stem
cellshometothechagasicheart,validatingsystemicinjection
as a viable approach for cell therapy in this context.
Once the homing of the cells to the diseased myocardium
was established, Soares et al. [26] demonstrated that bone
marrow mononuclear cells from normal syngeneic donors
significantly reduced cardiac inflammation and fibrosis in
mice with chronic T. cruzi infections. Importantly, the
improvement was long lasting, being observed up to six
months after cell therapy. The reduction in inflammation
likely resulted from increased apoptosis of the infiltrating
inflammatory cells as determined by TUNEL staining. The
decrease in fibrosis may result from activation of met-
alloproteases, since MMP9 expression is increased in the
chagasic hearts after cell therapy. Cell dosing experiments
demonstrated that 105cells were necessary for a significant
reduction in the number of inflammatory cells and injection
of 106or 107cells induced similar effects [26].
The mechanisms of action of the mononuclear cells in
chagasic mouse hearts have not yet been fully elucidated.
Trans differentiation/fusion appears to occur at an extremely
low frequency and paracrine effects may be the major cause
of improvement in myocardial function. In clinical human
trials autologous bone marrow cells would be employed.
Therefore, bone marrow cells from chronically infected mice
were used to ameliorate the pathology of infected mice [26].
Theseexperimentshighlightthetranslationalaspectsofthese
animal studies.
Recently, using cardiac MRI, it was demonstrated that
tail vein injection of 107bone marrow mononuclear cells
prevented and reversed the right ventricular dilatation
induced by T. cruzi-infection [27] which correlates with
the pathologic improvement reported by Soares et al. [26].
Furthermore it was determined that repeated injections
of Granulocyte-colony stimulating factor (G-CSF), which
mobilizes stem cells from the bone marrow, decrease inflam-
mation and fibrosis in the hearts of chagasic mice Garcia
et al. personal communication. This finding is consistent
with observations of Harada et al. [28] that demonstrated
improvement in heart function in an ischemic mouse model.
The combination of mononuclear cells and G-CSF enhances
the effect of the cell therapy in the reduction of the
inflammatory infiltrate.
In a rat model of chagasic cardiomyopathy Guarita
et al. [29] reported that direct left ventricular injection
of cocultured skeletal myoblasts and mesenchymal bone
marrow derived cells improved heart function in chronically
infected rats as determined by echocardiography. Injection
of the cocultured cells increased ejection fraction and
decreased end-systolic and diastolic volumes. These findings
demonstrated that local injection of stem cells is also
effective and suggest that cells are able to diffuse from the
injection site to reach other regions of the heart. This is an
important observation, given the widespread involvement of
the myocardium in chagasic cardiomyopathy.
Based on the encouraging results in animal models,
investigators in Brazil initiated a clinical trial to examine
the feasibility and safety of autologous bone marrow cell
transplantation in patients with congestive heart failure due

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4Interdisciplinary Perspectives on Infectious Diseases
(a) (b)
(c) (d)
Figure 1: Heart sections of T. cruzi-infected mice. (a) BALB/c mouse during the acute phase of infection with Colombian strain T. cruzi,
showing a parasite nest (green), DAPI-stained nuclei (blue) and myofibers (red). The majority of the DAPI nuclei belong to inflammatory
cells that infiltrate the heart in areas infected with parasites. (b) Inflammation of chronic chagasic BALB/c mouse, showing inflammatory
cells adhered to myofibers causing myocytolysis. (c) and (d), Detection of bone marrow stem cells (BMC) in the myocardium of chronic
chagasic mice. BMC obtained from normal BALB/c mice were injected i.v. into chronic chagasic mice (18 months of infection). BMC were
incubated with the fluorescent DNA stain Hoechst 33258 prior to injection into chagasic mice. Sections of frozen heart fragments were
prepared 7 (c) and 15 (d) days after BMC injection and fixed with cold acetone. Sections were observed in an Olympus spectral confocal
microscope FV1000 observed by fluorescence microscopy. In chagasic mice Hoechst+cells could be observed 1–7 days after BMC injection,
some of which were already beginning cell division cycles (c). Hoechst+cells proliferated and formed clusters of cells bearing a dotted
nuclear fluorescent pattern that could be observed up to 30 days after BMC transplant (d).
tochronicchagasiccardiomyopathy.Thesepatientsgenerally
have a poor prognosis, with mortality rates reaching 40%
within two years of onset [30]. At the most advanced stage
of congestive heart failure the only therapy possible is heart
transplantation, but this procedure is feasible in only a very
small number of patients. Due to uncertainties regarding
the mechanisms of action of the mononuclear cells, the
trial was designed for patients with end-stage congestive
heart failure whose only therapeutic option would be heart
transplantation. This was an open label, uncontrolled, single
center clinical trial that enrolled 30 patients. Inclusion
criteria required patients to be 18–70 years old, of either
gender, with congestive heart failure due to Chagas’ disease,
in New York Heart Association (NYHA) class III or IV, with
an ejection fraction of less than 40% while on optimized
pharmacologic therapy for at least 4 weeks before enrollment
[31]. Bone marrow cell aspiration was performed on the
day of the injection and the mononuclear fraction was
obtained through Ficoll density gradient centrifugation. The
cell suspension was diluted in 20mL of saline with 5%
autologous serum and injected in the coronary arteries
usinganangioplastycatheterwiththefollowingdistribution:
10mL in the left descending coronary artery, 5mL in the
circumflex and 5mL in the right coronary artery. Mean
number of injected cells was 2.7 × 108. At the 25th day
after cell injection patients received 5μg/kg of G-CSF for
5 days. Patients were followed for six months. Importantly
there was no detectable increase in arrhythmias after cell
therapy nor were troponin I levels increased during or after
the procedure. Results indicated that cell therapy induced a
small but significant increase in ejection fraction. Quality of
lifeimprovedasdeterminedbytheMinnesotaQuestionnaire
and by NYHA class. The six minute walking test also showed
significant improvement. These results were observed 1
month after therapy and persisted for the 6 month follow-
up period. However, since the trial was not designed to
test for efficacy the only conclusion possible is that bone
marrow mononuclear cell therapy by intracoronary delivery
is feasible and safe in chronic chagasic cardiomyopathy
patients.
In a patient with chagasic cardiomyopathy, bone marrow
mononuclear cells delivered by the intracoronary route were

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Interdisciplinary Perspectives on Infectious Diseases5
preferentially retained in diseased, hypoperfused areas of
the myocardium [32]. Further studies using labeled cells
confirmed these results (Barbosa, personal communication).
Given the promising results of the phase II trial, a larger,
multicenter, randomized, double-blind and placebo con-
trolled trial was designed to test for efficacy of the intracoro-
nary delivery of bone marrow-derived mononuclear cells in
chronicchagasiccardiomyopathy.Inclusioncriteriaincluded
diagnosisofheartfailurebytheFraminghamcriteria,regular
visits to a cardiology service with at least two independent
serological diagnoses of Chagas disease, ages between 18–
75 years, NYHA class III or IV, ejection fraction below 35%
by echocardiography according to Simpson’s rule, and opti-
mized pharmacologic therapy. Main exclusion criteria were
valvular diseases (except for functional mitral or tricuspid
regurgitation),coronaryangiographywithsignificantlesions
(more than 50% of obstruction), sustained ventricular
tachycardia,abusiveuseofdrugsoralcohol,serumcreatinine
>2.5mg/dl, neoplasia and other diseases that might impact
life expectancy within two years. Primary endpoint for the
trial is the difference in ejection fraction between the cell
therapy and the placebo group as determined by Simpson’s
rule in echocardiography. The trial was powered to detect an
absolute 5% difference as significant. Secondary endpoints
include difference in ejection fraction, life quality assessment
by Minnesota Quality of Life Questionnaire, six minute
walking distance, NYHA class and brain natriuretic peptide
levels at baseline and 6 and 12 months after therapy, among
others.Thetrialisstillrecruitingpatients,butinitial6month
follow-up results are expected to be published by July 2009.
Transplantation of bone marrow derived-cells may prove
to be an important therapeutic modality in the management
of end-stage chagasic heart disease. Undoubtedly, identifying
which cell type(s) is(are) responsible for the effects observed
in the animal and the preliminary human experiments will
be an important step toward improvement of this therapy.
Since the percentage of stem cells, either hematopoietic
or mesenchymal, is minimal in the mononuclear fraction,
use of purified stem cell populations has the potential to
significantly increase the therapeutic potential of cell therapy
in chagasic cardiomyopathy.
Acknowledgments
This work was supported in part by grants from the NIH
HL-73732 (ACC) CA123334 (LAJ) and AI076248 (HBT),
from FINEP (ACC) and from the Brazilian Ministry of
Health (ACC). Regina Goldenberg was supported by Fogarty
International Grant - D43TW007129 (HBT).
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