(Advances in Parasitology 50) - Academic Press (2001)

SERIES EDITORS
D. ROLLINSON S. I. HAY
Department of Zoology, Spatial Epidemiology and Ecology Group
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London, UK University of Oxford, South Parks Road
d.rollinson@nhm.ac.uk Oxford, UK,
simon.hay@zoo.ox.ac.uk
EDITORIAL BOARD
M. G. BASÁÑEZ R. E. SINDEN
Reader in Parasite Epidemiology, Immunology and Infection Section,
Department of Infectious Disease Department of Biological Sciences,
Epidemiology, Faculty of Medicine Sir Alexander Fleming Building, Imperial
(St Mary’s campus), Imperial College College of Science, Technology and
London, London, UK Medicine, London, UK
S. BROOKER D. L. SMITH
Wellcome Trust Research Fellow and Johns Hopkins Malaria Research Insti-
Reader, London School of Hygiene and tute & Department of Epidemiology,
Tropical Medicine, Faculty of Infectious Johns Hopkins Bloomberg School of
and Tropical, Diseases, London , UK Public Health, Baltimore, MD,USA
R. B. GASSER R. C. A. THOMPSON
Department of Veterinary Science, Head, WHO Collaborating Centre for
The University of Melbourne, Parkville, the Molecular Epidemiology of Parasitic
Victoria, Australia Infections, Principal Investigator, Envi-
ronmental Biotechnology CRC (EBCRC),
N. HALL School of Veterinary and Biomedical
School of Biological Sciences, Bios- Sciences, Murdoch University, Murdoch,
ciences Building, University of Liverpool, WA, Australia
Liverpool, UK
R. C. OLIVEIRA X. N. ZHOU
Centro de Pesquisas Rene Rachou/ Professor, Director, National Institute
CPqRR - A FIOCRUZ em Minas Gerais, of Parasitic Diseases, Chinese Center
Rene Rachou Research Center/CPqRR for Disease Control and Prevention,
- The Oswaldo Cruz Foundation in the Shanghai , People’s Republic of China
State of Minas Gerais-Brazil, Brazil
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CONTRIBUTORS OF VOLUME 76
Daniel Adesse
Instituto de Biofı́sica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro; and Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de
Janeiro, Brazil
Daniele Andrade
Janeiro, CCS, Laboratório de Imunologia Molecular, Cidade Universitária
Rio de Janeiro, Rio de Janeiro, Brazil
Anthony W. Ashton
Department of Pathology, Albert Einstein College of Medicine, Bronx,
New York, USA; and Division of Perinatal Research, Kolling Institute for
Medical Research, University of Sydney, Sydney, New South Wales,
Australia
Barbara A. Burleigh
Department of Immunology and Infectious Diseases, Harvard School of
Public Health, Boston, Massachusetts, USA
Antonio Carlos Campos de Carvalho

Janeiro, Brazil; and Dominick P. Purpura Department of Neuroscience,
Albert Einstein College of Medicine, Bronx, New York, USA
Kacey L. Caradonna
Department of Immunology and Infectious Diseases, Harvard School of
Public Health, Boston, Massachusetts, USA
Streamson C. Chua
Departments of Medicine, and Neuroscience; and The Diabetes Research
Center, Albert Einstein College of Medicine, Bronx, New York, USA
Marina V. Chuenkova
Department of Pathology and Sackler School of Graduate Students, Tufts
University School of Medicine, Boston, Massachusetts, USA
xv
xvi Contributors of Volume 76
Edecio Cunha-Neto
Laboratório de Imunologia, Instituto do Coração, Hospital das Clı́nicas;
Disciplina de Imunologia Clı́nica e Alergia, Faculdade de Medicina,
Universidade de São Paulo; and Instituto de Investigação em Imunolo-
gia—INCT, São Paulo, SP, Brazil
Maria de Narareth Meirelles

Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
Mahalia S. Desruisseaux
Departments of Pathology and Medicine, Albert Einstein College of
Medicine, Bronx, New York, USA
Monisha Dhiman
Department of Microbiology and Immunology, University of Texas
Medical Branch, Galveston, Texas, USA
Lisia Esper
Department of Biochemistry and Immunology, Institute of Biological
Science, Federal University of Minas Gerais, Belo Horizonte, Brazil
Stephen M. Factor
Fabio S. Fortes
Colegiado de Ciencias Biologicas e da Saude (CCBS), Centro Universitario
Stadual da Zona Oeste (UEZO), Rio de Janeiro, Brazil
Nisha Jain Garg

Department of Microbiology and Immunology; Department of Pathology;
and Faculty of the Center for Tropical Diseases, Sealy Center for Vaccine
Development, and the Institute for Human Infections and Immunity,
University of Texas Medical Branch, Galveston, Texas, USA
Luciana Ribeiro Garzoni

Regina Coeli Goldenberg

Instituto de Biofı́sica Carlos Chagas Filho, Universidade Federal do Rio
de Janeiro, Brazil
Contributors of Volume 76 xvii
Shivali Gupta
Huan Huang
New York, USA
Dumitru A. Iacobas
Dominick P. Purpura Department of Neuroscience, Albert Einstein
College of Medicine, Bronx, New York, USA
Sanda Iacobas
Jasmin
Janeiro, Brazil; and Dominick P. Purpura Department of Neuroscience,
Albert Einstein College of Medicine, Bronx, New York, USA
Linda A. Jelicks
Department of Physiology & Biophysics, Albert Einstein College of
Jorge Kalil
Laboratório de Imunologia, Instituto do Coração, Hospital das Clı́nicas;
Disciplina de Imunologia Clı́nica e Alergia, Faculdade de Medicina,
Universidade de São Paulo; and Instituto de Investigação em
Imunologia—INCT, São Paulo, SP, Brazil
Fabiana S. Machado
Department of Biochemistry and Immunology, Institute of Biological
Science, Federal University of Minas Gerais, Belo Horizonte, Brazil
Shankar Mukherjee
New York, USA
Fnu Nagajyothi
New York, USA
xviii Contributors of Volume 76
Luciana Gabriel Nogueira

Laboratório de Imunologia, Instituto do Coração, Hospital das Clı́nicas,
Faculdade de Medicina, Universidade de São Paulo; and Instituto de
Investigação em Imunologia—INCT, São Paulo, SP, Brazil
Mercio PereiraPerrin
Department of Pathology and Sackler School of Graduate Students, Tufts
University School of Medicine, Boston, Massachusetts, USA
Cibele M. Prado
Department of Pathology, Laboratory of Cellular and Molecular Cardiology,
Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão
Preto, São Paulo, Brazil
Xiaohua Qi
Department of Microbiology and Immunology, Albert Einstein College of
Marcos A. Rossi
Department of Pathology, Laboratory of Cellular and Molecular Cardiology,
Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão
Preto, São Paulo, Brazil
Julio Scharfstein
Janeiro, CCS, Laboratório de Imunologia Molecular, Cidade Universitária
Rio de Janeiro, Rio de Janeiro, Brazil
Philipp E. Scherer
Touchstone Diabetes Center, Departments of Internal Medicine and Cell
Biology, University of Texas Southwestern Medical Center, Dallas, Texas,
USA
Milena B. Soares
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil
David C. Spray
André Talvani
Laboratório de doença de Chagas, Departamento de Ciências Biológicas
& NUPEB, Universidade Federal de Ouro Preto, Ouro Preto, Minas
Gerais, Brazil
Contributors of Volume 76 xix
Herbert B. Tanowitz
Departments of Pathology and Medicine; and The Diabetes Research
Center, Albert Einstein College of Medicine, Bronx, New York, USA
Mauro M. Teixeira
Laboratório de Imunofarmacologia, Departamento de Bioquı́mica e
Imunologia/ICB; and Faculdade de Medicina, Universidade Federal de
Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Priscila Camillo Teixeira

Laboratório de Imunologia, Instituto do Coração, Hospital das Clı́nicas,
Faculdade de Medicina, Universidade de São Paulo; and Instituto de
Investigação em Imunologia—INCT, São Paulo, SP, Brazil
Louis M. Weiss
Jian-jun Wen
PREFACE
Chagas disease or American trypanosomiasis is caused by the hemofla-

gellate Trypanosoma cruzi which was first described by Carlos Chagas
shortly after the turn of the twentieth century. Chagas disease has been
designated a neglected tropical disease designated by the World Health
Organization and the National Institutes of Health. Interestingly, palaeo-
parasitological studies have shown that T. cruzi was present in tissues
obtained from mummies in northern Chile and southern Peru from the
period 4000 BC to AD 1400, obviously long before it was discovered by
Carlos Chagas in 1909 (Aufderheide et al., 2004).
Carlos Justiniano Ribeiro das Chagas (Fig. 1A and B) was born in the
state of Minas Gerais, Brazil, on 9 July 1879, and after his basic education
and a brief brush with engineering in the city of Ouro Preto, he began his
studies of medicine in Rio de Janeiro where he trained under the guidance
of Dr. Oswaldo Cruz. After graduation from medical school, while work-
ing in 1909 as a malaria control officer in Lassance, Minas Gerias, Chagas
observed microscopic flagellated organisms in the blood of a febrile child
named Berenice. When the fever abated, there were no longer any organ-
isms in her blood. Chagas named the organism T. cruzi in honour of his
mentor. In a period of several months, working for the most part by
himself, he described the pathogen, its vector and the clinical features of
the disease that bears his name an accomplishment unique in the history
of medicine (Chagas, 1909; Lewinsohn, 2003). Although his achievements
ultimately gained widespread recognition, he was never awarded a
Nobel Prize for this important work. Carlos Chagas died in 1935. In the
1960s, Berenice was located and was found to be seropositive for Chagas
disease reflecting the typical lifelong infection with this parasite. She was,
however, free of clinical manifestations of her chronic infection and died
due to unrelated ‘‘natural causes’’ in 1973. In 2009 the scientific world
celebrated the centenary of Chagas achievements in numerous symposia
and review articles on the clinical and research aspects of T. cruzi and of
Chagas disease (Apt, 2010; Biolo et al., 2010; Buckner and Navabi, 2010;
Casadei, 2010; Epting et al., 2010; Junqueira et al., 2010; Rassi et al., 2010;
Villalta et al., 2009).
Since Chagas’s original observations were published, an enormous
amount of information has accumulated on the biology, pathology, path-
ogenesis, epidemiology and clinical manifestations of the disease. The
completion of the genome of the CL Brener strain and the anticipated
xxi
xxii Preface
completion of the genomes of other strains will continue to add to our

knowledge of this complex human parasite and provide fertile ground for
researchers of this neglected tropical disease. Moreover, use of the mod-
ern tools of molecular biology, biochemistry, cell biology and immunol-
ogy has greatly expanded our knowledge of the complex biology of this
organism and the host responses to this infection. The advances made
through the application of the methods of these disciplines have raised
hopes for the development of sorely needed new therapeutic and
prophylactic agents for the management of T. cruzi infection.
This volume in Advances in Parasitology is not meant to be a compre-
hensive ‘‘novel’’ on Chagas disease but rather a collection of ‘‘short
stories’’ in which experts in the field have highlighted historical perspec-
tives and detailed descriptions of innovative experimental work based on
cutting-edge methodologies applied to the challenges of Chagas disease.
Our hope is that by bringing together in one place reviews of some of the
best current work in Chagas disease research, readers will be informed
and perhaps even stimulated to become involved in combating the illness
Preface xxiii
FIGURE 1 (A) Carlos Chagas on the 10,000 Cruzados Banknote from Brazil.
(B) A photograph of Carlos Chagas (white arrow) and Albert Einstein (black arrow)
taken during Einstein’s visit to the Oswaldo Cruz Institute in 1925 (Lewinsohn, 2003).
With permission of Casa de Oswaldo Cruz-Fiocruz, Arquivo e Documentação, Rio de
Janeiro, Brazil.
that, despite over 100 years of research, still is the most important para-
sitic disease in the Americas.
Chagas disease is present in the countries of Latin America with the
exception of the Caribbean. Vector-borne transmission of the T. cruzi
parasite usually occurs in individuals living in primitive houses in areas
where the sylvatic cycle is active. The parasite has a complex life cycle
which is detailed in the epidemiology chapter. One of the important
recent changes in the epidemiology of Chagas diseases has been the
increased immigration of infected, usually asymptomatic, individuals
from endemic areas to non-endemic areas such as North America, Eur-
ope, Japan and Australia. Thus, Chagas disease is being recognized with
increasing frequency worldwide. This immigration into non-endemic
areas of potentially chronically infected individuals has led to screening
of blood donors to identify people who are asymptomatic but have the
potential to transmit the infection via blood transfusion and organ trans-
plantation. Interestingly, as a result of the immigration of populations into
non-endemic areas, congenital Chagas disease has now been diagnosed in
Europe among immigrants from Latin America. The exacerbation of
xxiv Preface
Chagas disease in the setting of immune suppression has been documen-

ted among individuals with HIV/AIDS and among those who receive
immunosuppressive therapy in the setting of treatment for other immune
disorders or organ transplantation. Acute Chagas disease and congenital
Chagas disease are well described in the chapters in this volume. These
are areas that have not received intensive investigation in recent years.
The description of chronic Chagas disease has been dealt with in several
excellent recent reviews, and the reader is referred to these for discussion
of these topics (Carod-Artal et al., 2011; Lima-Costa et al., 2010; Rassi et al.,
2010; Tanowitz et al., 2009).
Diagnostic testing is not covered in a separate chapter, as this is in a
state of flux; however, there have been a number of reviews on this topic
(Britto, 2009; Shah et al., 2010). At the present time, the diagnosis of acute
T. cruzi infection is usually made by the detection of parasites in wet
mounts of blood or cerebrospinal fluid and in Giemsa-stained slides.
Testing for anti-T. cruzi IgM antibodies is not useful. Polymerase chain
reaction (PCR) tests can detect the parasite and are useful in diagnosis. In
acute or congenital Chagas disease, PCR is thought to be the most sensi-
tive method for detecting infection; however, it is not widely available.
The diagnosis of chronic Chagas disease is usually based on detecting
specific antibodies. Several serologic assays are employed such as the
indirect immunofluorescence (IFA) and enzyme-linked immunosorbent
assay (ELISA). Serologic assays are used widely for clinical diagnosis and
for screening of donated blood, as well as in epidemiologic studies. A
radio immunoprecipitation assay (RIPA) based on iodinated T. cruzi
proteins is specific and sensitive and is being used as the confirmatory
assay to test all donor samples that are positive in the screening test (Shah
et al., 2010). The utility of various diagnostic modalities have been pub-
lished and has generated controversy, and the reader is referred to the
following for a discussion of these issues (Otani et al., 2009; 2010; Shah
et al., 2010). A critical unmet need is for tests that deal with the issue of
parasitological cure; currently, no test can accurately predict that cure has
been achieved following drug treatment.
Other chapters in this Advances in Parasitology volume (Parts A and B)
deal with advances in the therapy of Chagas disease such as cell-based
therapy for chronic Chagas cardiomyopathy which could obviate the
need for heart transplantation. There are important chapters which
focus on advances in chemotherapy as well as the current state of vaccine
development. One of the intriguing questions of this disease is what role
human genetic variability contributes to susceptibility to infection and the
final clinical outcome of infection with T. cruzi. Chapters on molecular
biology, cell biology, host cell invasion, stage differentiation and parasite
signalling follow. For example, there is a comprehensive review of the
current state of knowledge of the unique organelle, the acidocalcisome, an
Preface xxv
organelle which the author of this chapter, was instrumental in describing

in several protozoa. The last several chapters are diverse and deal with
topics in pathogenesis including those on eicosanoids, oxidative stress,
vascular pathophysiology, and myocardial inflammation. One of the
chapters deals with the question of autoimmunity. This has intrigued
investigators for decades and the controversy continues as to what role,
if any, does autoimmunity play in the pathogenesis of Chagas disease.
The work on parasite-derived neurotropic factors is reviewed, and there
is a review on myocardial inflammation and Chagas disease. As there
have been excellent recent reviews on immunology in Chagas disease, the
editors decided to concentrate on myocardial inflammation rather than
the entire gamut of immunology which could be a topic of an entire
volume. In recent years, there has been an increase in obesity and diabetes
in Chagas endemic areas which has alarmed many and has resulted in an
increase in the investigation as to the role of adipose tissue and diabetes in
infections caused by parasites. To this end, we have included a review
which explores this relationship demonstrating the complex interaction
that is resulting from the intersection of the obesity epidemic with this
endemic tropical disease.
The editors wish to thank their families, friends, laboratory members
and colleagues who have made it possible to achieve all that we have
done. In addition, they acknowledge the efforts of the entire Chagas
disease research community who have contributed their talents to
unravel the complex interactions of T. cruzi and humans.
Only a life lived for others is a life worthwhile.
We can’t solve problems by using the same kind of thinking we used
when we created them
Albert Einstein
LOUIS M. WEISS
HERBERT B. TANOWITZ
April 2011
REFERENCES
Apt, W., 2010. Current and developing therapeutic agents in the treatment of Chagas disease.
Drug Des. Devel. Ther. 4, 243–253.
Aufderheide, A.C., Salo, W., Madden, M., Streitz, J., Buikstra, J., Guhl, F., et al., 2004. A 9,000-
year record of Chagas’ disease. Proc. Natl. Acad. Sci. USA. 101, 2034–2039.
Biolo, A., Ribeiro, A.L., Clausell, N., 2010. Chagas cardiomyopathy—where do we stand after
a hundred years? Prog. Cardiovasc. Dis. 52, 300–316.
Britto, C.C., 2009. Usefulness of PCR-based assays to assess drug efficacy in Chagas disease
chemotherapy: value and limitations. Mem Inst Oswaldo Cruz 104 (Suppl. 1), 122–135.
xxvi Preface
Buckner, F.S., Navabi, N., 2010. Advances in Chagas disease drug development: 2009–2010.
Curr. Opin. Infect. Dis. 23, 609–616.
Carod-Artal, F.J., Vargas, A.P., Falcao, T., 2011. Stroke in asymptomatic Trypanosoma
cruzi-infected patients. Cerebrovasc. Dis. 31, 24–28.
Casadei, D., 2010. Chagas’ disease and solid organ transplantation. Transplant. Proc. 42,
3354–3359.
Chagas, C., 1909. Nova tripanozomiase humana: Estudos sobre a morfolojia e o ciclo evolu-
tivo do schizotrypanum cruzi n. Gen., n. Sp., ajente etiolojico de nova entidade morbida
do homem. (New human trypanosomiasis. Studies about the morphology and life-cycle
of Schizotripanum cruzi, etiological agent of a new morbid entity of man). Mem Inst
Oswaldo Cruz 1, 159–218.
Epting, C.L., Coates, B.M., Engman, D.M., 2010. Molecular mechanisms of host cell invasion
by Trypanosoma cruzi. Exp. Parasitol. 126, 283–291.
Junqueira, C., Caetano, B., Bartholomeu, D.C., Melo, M.B., Ropert, C., Rodrigues, M.M., et al.,
2010. The endless race between Trypanosoma cruzi and host immunity: lessons for and
beyond Chagas disease. Expert Rev. Mol. Med. 12, e29.
Lewinsohn, R., 2003. Prophet in his own country: Carlos Chagas and the Nobel Prize.
Perspect. Biol. Med. 46, 532–549.
Lima-Costa, M.F., Matos, D.L., Ribeiro, A.L., 2010. Chagas disease predicts 10-year stroke
mortality in community-dwelling elderly: the Bambui cohort study of aging. Stroke 41,
2477–2482.
Otani, M.M., Vinelli, E., Kirchhoff, L.V., del Pozo, A., Sands, A., Vercauteren, G., et al., 2009.
WHO comparative evaluation of serologic assays for Chagas disease. Transfusion 49,
1076–1082.
Rassi, A., Jr., Rassi, A., Marin-Neto, J.A., 2010. Chagas disease. Lancet 375, 1388–1402.
Shah, D.O., Chang, C.D., Cheng, K.Y., Salbilla, V.A., Adya, N., Marchlewicz, B.A., et al., 2010.
Comparison of the analytic sensitivities of a recombinant immunoblot assay and the
radioimmune precipitation assay for the detection of antibodies to Trypanosoma cruzi in
patients with Chagas disease. Diagn. Microbiol. Infect. Dis. 67, 402–405.
Tanowitz, H.B., Machado, F.S., Jelicks, L.A., Shirani, J., de Carvalho, A.C., Spray, D.C., et al.,
2009. Perspectives on Trypanosoma cruzi-induced heart disease (Chagas disease).
Prog. Cardiovasc. Dis. 51, 524–539.
Villalta, F., Scharfstein, J., Ashton, A.W., Tyler, K.M., Guan, F., Mukherjee, S., et al., 2009.
Perspectives on the Trypanosoma cruzi-host cell receptor interactions. Parasitol. Res. 104,
1251–1260.
CHAPTER 1
Bioactive Lipids in Trypanosoma
cruzi Infection
Fabiana S. Machado,* Shankar Mukherjee,†
Louis M. Weiss,†,‡ Herbert B. Tanowitz,†,‡ and
Anthony W. Ashton†,§
Contents 1.1. Introduction 3

1.2. Eicosanoid Synthesis in Vertebrates 4
1.2.1. AA metabolism 4
1.2.2. The LO pathway of AA metabolism 6
1.2.3. The cytochrome P-450 pathway of AA
metabolism 7
1.2.4. The COX pathway of AA metabolism 7
1.3. Lipid Metabolism and Eicosanoid Biosynthetic
Pathways in Trypanosoma cruzi 10
1.4. Endogenous Regulation of Eicosanoids During
Experimental Chagas Disease 12
1.4.1. Acute infection 13
1.4.2. Chronic infection 16
1.4.3. Insect vectors 16
1.5. Lessons from Pharmacological Manipulation and
from Null Mice 17
1.5.1. Pharmacological intervention 17
1.5.2. Phenotypes of transgenic/knockout mice 20
Acknowledgements 23
References 24
* Department of Biochemistry and Immunology, Institute of Biological Science, Federal University of Minas
Gerais, Belo Horizonte, Brazil
{
Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA
{
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA
}
Division of Perinatal Research, Kolling Institute for Medical Research, University of Sydney, Sydney,
New South Wales, Australia
Advances in Parasitology, Volume 76 # 2011 Elsevier Ltd.

ISSN 0065-308X, DOI: 10.1016/B978-0-12-385895-5.00001-3 All rights reserved.
1
2 Fabiana S. Machado et al.
Abstract Chagas disease is caused by Trypanosoma cruzi, a protozoan para-

site. Chagas disease remains a serious health problem in large parts
of Mexico and Central and South America, where it is a major cause
of morbidity and mortality. This disease is being increasingly recog-
nized in non-endemic regions due to immigration. Heart disease
develops in 10–30% of infected individuals. It is increasingly clear
that parasite- and host-derived bioactive lipids potently modulate
disease progression. Many of the changes that occur during acute
and chronic Chagas disease can be accounted for by the effects of
arachidonic acid (AA)-derived lipids such as leukotrienes, lipoxins,
H(P)ETEs, prostaglandins (PGs) and thromboxane. During the course
of infection with T. cruzi, changes in circulating levels of AA
metabolites are observed. Antagonism of PG synthesis with cyclo-
oxygenase (COX) inhibitors has both beneficial and adverse effects.
Treatment with COX inhibitors during acute infection may result in
increased parasite load and mortality. However, treatment insti-
tuted during chronic infection may be beneficial with no increase in
mortality and substantial improvement with cardiac function.
Recently, T. cruzi infection of mice deficient in AA biosynthetic
enzymes for various pathways has yielded more insightful data
than pharmacological inhibition and has highlighted the potential
deleterious effects of inhibitors due to ‘‘off-target’’ actions. Using
COX-1 null mice, it was observed that parasite biosynthesis is
dependent upon host metabolism, that the majority of TXA2
liberated during T. cruzi infection is derived from the parasite and
that this molecule may act as a quorum sensor to control parasite
growth/differentiation. Thus, eicosanoids present during acute
infection may act as immunomodulators aiding the transition to,
and maintenance of, the chronic stage of the disease. It is also likely
that the same mediators that initially function to ensure host
survival may later contribute to cardiovascular damage. Collec-
tively, the eicosanoids represent a new series of targets for therapy
in Chagas disease with defined potential therapeutic windows in
which to apply these agents for greatest effect. A deeper under-
standing of the mechanism of action of non-steroidal anti-inflam-
matory drugs may provide clues to the differences between host
responses in acute and chronic T. cruzi infection.
ABBREVIATIONS
AA arachidonic acid
AhR aryl hydrocarbon receptor
ALX lipoxin receptor
ARNT aryl hydrocarbon receptor nuclear translocator
ASA aspirin
Bioactive Lipids in Trypanosoma cruzi Infection 3
ATL aspirin-triggered lipoxin

BLT1/2 B leukotriene receptor 1 or 2
CD cell differentiation antigen
COX cyclooxygenase
Cyslt1/2 cysteinyl leukotriene receptor 1 or 2
cys-LTs cysteinyl leukotrienes
FLAP 5-LO activating protein
FPRL-1 fMLP phagocyte receptor with low affinity
H(P)ETE hydro(peroxy)eicosatetraenoic acid
IFN interferon
IL interleukin
LO lipoxygenase
LT leukotriene
LX lipoxin
lyso PA lysophospholipid
NDGA nordihydroguaiaretic acid
NO nitric oxide
NOS nitric oxide synthase
NSAID non-steroidal anti-inflammatory drug
PAF platelet-activating factor
PG prostaglandin
PKC protein kinase C
PL(A/ phospholipase A/C/D
C/D)
PMA phorbol myristal acetate
SOCS-2 suppressor of cytokine signalling 2
Tb T. brucei
TbPGFS T. brucei PGF2a synthase
Tc T. cruzi
TcOYE old yellow enzyme
TGF-b transforming growth factor b
TNF-a tumour necrosis factor-a
TP, DP, T-, D-, F-, E- and I-type prostanoid receptors
FP, EP, IP,
TRAF TNF receptor-associated factor
TXA2 thromboxane A2
1.1. INTRODUCTION
In Latin America, millions of people are at risk for infection with the
parasite Trypanosoma cruzi the causative agent of Chagas disease. Clini-
cally, T. cruzi (Tc) infection causes acute myocarditis followed by chronic
cardiomyopathy and vasculopathy in humans and experimental models.
There are three stages of infection: acute, intermediate and chronic. Acute
myocarditis is characterized by an intense inflammatory response typified
by upregulation of inflammatory mediators such as cytokines, chemokines,
nitric oxide (NO) and endothelin-1 (Huang et al., 1999a, b; Machado et al.,
2008; Machado and Aliberti, 2009; Petkova et al., 2000, 2001; Tanowitz et al.,
2005). As the acute infection wanes, individuals may remain asymptomatic,
but 10–30% of infected individuals may ultimately develop chronic
cardiomyopathy (Tanowitz et al., 2009). The manifestations of the chronic
disease include dilated cardiomyopathy with congestive heart failure, con-
duction abnormalities and thromboembolic events (Tanowitz et al., 1992,
2009). Parasite persistence is central to the aetiology of the cardiomyopathy
and is aggravated by microvascular spasm with focal ischaemia and
autoimmune mechanisms (Factor et al., 1985; Petkova et al., 2000, 2001;
Tanowitz et al., 1996). As early as the 1990s, it was suggested that eicosa-
noids may play a significant role in the vasospasm and platelet aggregation
that characterize Chagas disease (Tanowitz et al., 1990).
1.2. EICOSANOID SYNTHESIS IN VERTEBRATES

Eicosanoids are a family of lipid mediators that participate in a wide
range of biological activities including vascular tone, inflammation,
ischaemia and tissue homeostasis (Haeggstrom et al., 2010). In mammals,
the biosynthetic pathways for these important biological mediators are
dependent upon liberation of arachidonic acid (AA) from the inner leaflet
of the plasma membrane. The biosynthetic pathways for eicosanoid bio-
synthesis are well described in vertebrates and are outlined below.
1.2.1. AA metabolism
AA is a 20-carbon polyunsaturated fatty acid derived from linoleic acid.
Once synthesized, AA is stored as a part of glycerophospholipids that
compose the lipid bilayer of the plasma membrane and can be released
via the action of phospholipases A2, C and D (PLA2, PLC and PLD,
respectively; Fig. 1.1). AA can be reincorporated into cellular lipids via
reacylation and recombination with lysophospholipid. AA is metabolized
predominantly by the following three independent metabolic pathways:
1. The cyclooxygenase (COX) pathway: producing prostaglandins (PGs) and
thromboxane A2 (TXA2).
2. The lipoxygenase (LO) pathway: producing leukotrienes (LTs), lipoxins
(LXs), hydroxyeicosatetraenoic acids (HETE) and hydroperoxyeicosa-
tetraenoic acids (HPETE).
3. Cytochrome P-450 monooxygenase pathway: producing epoxides and
hydroxyeicosatetraenoic acids.
Phospholipids (PL) Phosphatidylinositides
Phosphatidic
acid (PA) PLC
Lyso PL
DG kinase
Lyso PA Diglyceride
(DG)
PLA2
DG lipase
COOH
Free arachidonic
acid
Cytochrome P-450
Cyclooxygenase Lipoxygenase
Monooxygenase
Epoxides PGs H(P)ETEs

TXs LTs
LXs
Inflammation, vascular tone, haemostasis, broncheconstriction
FIGURE 1.1 Production and use of free arachidonic acid from various intracellular
sources.
In addition to its role in eicosanoid synthesis, AA itself is capable of

regulating cellular responses. AA controls the activity of PLA2 and PLC
via a negative feedback mechanism (Sumida et al., 1993), triggers mobili-
zation of intracellular calcium stores in a manner similar to that of inositol
1,4,5-phosphate (Chow and Jondal, 1990) and activates the classical iso-
forms of protein kinase C (PKC) (Naor, 1991). Activation of PKC by fatty
acids may form a positive feedback loop to enhance fatty acid liberation
through amplification of PLA2 activity (Sumida et al., 1993). AA also
suppresses tumour necrosis factor (TNF)-a, interleukin (IL)-1a and lipo-
polysaccharide (LPS)-induced activation of endothelial cells (Stuhlmeier
et al., 1996), indicating that AA may negatively regulate endothelial cell
activation.
1.2.2. The LO pathway of AA metabolism

There are four LO enzymes, 5-, 8-, 12- and 15-LO, that metabolize AA by
oxygenation of a single carbon resulting in the formation of a variety of
compounds with diverse biological activities (Sumida et al., 1993). The
nomenclature of the LO enzymes is derived from the position of the
hydroperoxy group in the product of that enzyme (Chiang et al., 2006).
For example, 5-LO converts AA into a hydroperoxide by insertion of
molecular oxygen at position 5 of AA backbone (Brock et al., 1995). In
humans, the primary products of AA metabolism by LO are 5-, 12- and 15-
HPETE which, by the action of peroxidases, yield their hydroxy deriva-
tives (HETE). The 5- and 12-LO enzymes have a ubiquitous distribution,
while 15-LO is confined to eosinophils (Ivanov et al., 2010). The 5-LO in
neutrophils is translocated from the cytoplasm to the cell membrane in
the presence of raised intracellular calcium (Brock et al., 1995). 5-LO with
the help of 5-LO-activating protein (FLAP) converts AA to 5-HPETE,
which spontaneously reduces 5-HETE. 5-LO again acts on 5-HETE to
convert it to Leukotriene (LTA4) (Chiang et al., 2006). The biosynthetic
pathways involved in Lipoxin (LXA4) formation are complex, involve the
actions of at least two independent LOs and can occur through trans-
cellular cascades, particularly those involving platelets and leukocytes
(Chiang et al., 2006). However, the activity of 5-LO seems to be a common
step in LXA4 synthesis (Serhan et al., 1984). LXA4 is secreted by neutro-
phils and inhibited the activating effects of LTB4 on platelets (Serhan et al.,
1984); however, a growing list of anti-inflammatory/pro-resolving effects
have been associated with LXA4 resulting in the suggestion that they act
as ‘‘braking signals’’ in inflammation (Maderna and Godson, 2009). These
properties include limiting leukocyte trafficking and preventing endothe-
lial cell activation at the inflammatory site, stimulation of phagocytosis of
apoptotic cells by macrophages and are potential anti-fibrotic mediators
(Aliberti, 2005; Baker et al., 2009; Maderna and Godson, 2009; Ryan and
Godson, 2010).
LTs were first discovered in leukocytes but are now known to be
synthesized in many other cells and tissues including neutrophils,
monocytes, mast cells, macrophages, keratinocytes as well as in lung,
brain, spleen and heart. LTs are so named because it contains three
conjugated double bonds (trienes). LTA4 is the central metabolite from
which other LTs are derived. Cells expressing LTA4 hydrolase (neutro-
phils and monocytes) convert it to LTB4, while the cells that express LTC4
synthase convert LTA4 by the addition of tripeptide glutathione to LTC4.
Subsequent removal of the glutamic acid from the glutathione leads to the
formation of LTD4 with further degradation (by removal of the glycine
residue) leading to the formation of LTE4. LTC4, D4 and E4 contain a
cysteine residue in its structure; therefore, they are often referred to as

cysteinyl leukotrienes (cys-LTs). LTB4, 5-HETE and LTC4 are subse-
quently secreted from the cell (Lam et al., 1990).
1.2.3. The cytochrome P-450 pathway of AA metabolism

AA can also be metabolized by the cytochrome P-450 monooxygenases.
This pathway oxidizes AA to 19-hydroxy and 19-oxoeicosatetraenoic
acids (omega-1 oxidation) and 20-hydroxyeicosatetraenoic and eicosate-
traen-1,20-dioic acids (omega-oxidation). Hepatic and renal P-450
monooxygenases also produce a series of epoxides that are further
converted to diols. Moreover, omega-1 and omega-oxidation occur in
conjunction, and then trihydroxy-AA derivatives, or lipoxins, are formed.
The cytochrome P-450 monooxygenases are not only responsible for
eicosanoid synthesis but also for catabolism and have been shown to
inactivate several eicosanoids including PGE2 and LTB4. Thus, cyto-
chrome P-450 monooxygenases, in addition to PG 15-hydroxydehydro-
genase, provide a secondary pathway to catalyze the oxidation and
inactivation of these important bioactive lipids.
1.2.4. The COX pathway of AA metabolism

AA is hydrolyzed by the COX enzymes to PGH2 (Rouzer and Marnett,
2008). PGH2 is the central substrate for prostaglandin synthesis and is
further metabolized by specific terminal synthases to generate PGs and
TXA2 (Santovito et al., 2009). The relevance of these enzymes and the
bioactive lipids they produce are not well understood in parasitic disease.
1.2.4.1. The COX family of enzymes

Enzymes in the COX family are structurally and enzymatically similar but
have mechanistically different pathophysiological roles. There are two
COX isoenzymes (COX-1 and -2) in humans, sharing approximately
61% sequence homology (based on amino acid sequences) with the active
sites highly conserved. The two human COX enzymes share 61%
sequence homology (based on amino acid sequences) with highly con-
served active sites. COX-1 is constitutively expressed and mediates gas-
tric mucus production, platelet activation and vascular tone, while COX-
2 is inducible and functions in inflammation, cancer and tissue damage
(Haeggstrom et al., 2010; Rouzer and Marnett, 2008).
COX-1 is a 576-amino acid polypeptide (MW 66.2 kDa) that is consti-
tutively and ubiquitously expressed by many tissues, including gastric
mucosa, endothelial cells and platelets (Yokoyama and Tanabe, 1989).
COX-1 has a half-life of 10 min (Wu et al., 1988), indicating constant
synthesis is required to keep the PG/TX synthesis operative. This short

half-life is probably a reflection of the high degree of instability of the
COX-1 mRNA. It has been hypothesized that inactivation of an apopro-
tein moiety in the enzyme by free radicals formed during normal enzyme
function may be one of the reasons for its instability (Egan et al., 1976;
Ham et al., 1979). Indeed, inhibition of protein synthesis prevents the
release of prostacyclin (PGI2), a major protective factor for endothelial
cells. COX-2 is a 604-amino acid polypeptide (70 kDa) (Hla and Neilson,
1992) and is an inducible form of COX (with induction varying between
10- and 80-fold). Its activity has been reported to account for 40–60% of PG
synthesis in some tissues (Karim et al., 1996).
COX-1 and COX-2 are highly segregated in their intracellular com-
partmentalization prompting speculation that each isozyme is restricted
to a certain pool(s) of substrates and that COX-2 may not exist just to
augment the activity of COX-1 in times of physiological stress where
enhanced PG synthesis is required. Karim et al. (1996) found no evidence
for the hypothesis that COX-1 and -2 have separate synthetic pathways.
Indeed, the Vmax and Km of both enzymes for arachidonate are nearly
identical (Meade et al., 1993a,b). However, since COX-1 is constitutively
expressed and COX-2 is inducible, there arises a physiological division in
the function of these two enzymes with COX-1 mediating the effect of
prostanoids on normal cellular responses and COX-2 mediating
pathological responses in processes such as inflammation.
The COX isoforms use two sequential reactions to generate PGH2. The
COX reaction produces oxygenation and cyclization of a pentane ring in
AA leading to the formation of the unstable metabolite PGG2. The
peroxidase reaction then catalyses the reduction of PGG2 and the
endoperoxide PGH2 is formed (Miyamoto et al., 1976; Ohki et al., 1979).
The residues most important for the function of COX-1 and -2 are the two
heme-binding sites in exons 7 (His295) and 8 (His374), the active site
tyrosine in exon 8 (Tyr371) and the aspirin (ASA) acetylation site in exon
10 (Ser506) (DeWitt et al., 1990; Shimokawa and Smith, 1991; Shimokawa
et al., 1990; Smith et al., 1991). COX-1 contains four potential asparagine-
linked glycosylation sites at residues 67, 103, 143 and 409 and contains an
epidermal growth factor (EGF) homology domain (residues 33–71) which
is encoded by the whole of exon 2 (Toh, 1989; Yokoyama and Tanabe,
1989). These sites are well conserved in COX-2; however, the active site in
COX-2 varies by a substitution of a valine to an isoleucine residue at
position 509. This substitution appears to be solely responsible for the
ability of some classes of inhibitors to preferentially bind and inactivate
COX-2 over COX-1. The C-terminus of COX-2 also contains an 18-amino
acid sequence, not present in COX-1, giving rise to an additional N-linked
glycosylation site (Hla and Neilson, 1992).
1.2.4.2. Synthesis of PGs and thromboxane

PGH2 is the central substrate for the synthesis of all PGs and TXA2
(Haeggstrom et al., 2010; Rouzer and Marnett, 2008). PGI2 is derived
from PGH2 by the action of PGI2 synthase, a cytochrome P-450-like
membrane resident enzyme (Moncada and Vane, 1979). PGI2
spontaneously hydrolyzes to form the inactive metabolite 6-keto PGF1a
(t1/2 ¼ 3 min). PGF2a, PGD2 and PGE2 are derived from PGH2 by their
respective synthases. PGF2a is also formed by the degradation of PGE2 via
the action of the enzymes PG 15-hydroxydehydrogenase, cytochrome P-
450 monooxygenase or PGE9-ketoreductase (Hecker and Ullrich, 1989).
TXA2 is derived from PGH2 by the action of TXA2 synthase, a cytochrome
P-450-like heme-thiolate enzyme, which adds an oxan: oxetane ring to the
structure of PGH2, producing equimolar quantities of hydroxyheptadeca-
trienoic acid and malondialdehyde in the process. TXA2 (t1/2 ¼ 30 s)
spontaneously hydrolyzes to a stable but biologically inactive hemiacetal
form, TXB2 (Hecker and Ullrich, 1989). The determining factor in the
formation of prostanoid/TXA2 synthesis is the presence of the necessary
enzymes and not necessarily their regulation, which leads to tissue-spe-
cific expression patterns for many of these products, for example, TXA2 is
synthesized mainly by platelets and macrophages, while PGI2 is synthe-
sized predominantly by vascular smooth muscle and endothelial cells
(Hecker and Ullrich, 1989).
1.2.4.3. The biological responses to eicosanoids are mediated by cell

surface receptors
Prostaglandins and LTs both act through G-protein-coupled receptors
located on the plasma membrane of multiple cell types. Four LT receptors
are reported, B leukotriene receptor 1 and 2 (BLT1 and BLT2) and cys-LT
receptor 1 and 2 (Cyslt1 and Cyslt2). Prostaglandin receptors are
similarly named after the prostanoid that serves as the respective ligand,
‘‘D-, F-, E- and I-type’’ prostanoid receptor (DP, FP, EP and IP). TXA2
receptor is known as TXA2 prostanoid receptor (TP). LXA4 is thought to
bind to two types of receptor: (a) a surface seven-transmembrane G-
protein-coupled receptor, ALX/FPR2 (Brink et al., 2003; Chiang et al.,
2006; Devchand et al., 2003) and (b) a cytosolic nuclear ligand-activated
transcription factor, the aryl hydrocarbon receptor (AhR) (Schaldach
et al., 1999). LXA4–AhR translocates to the nucleus and binds with aryl
hydrocarbon receptor nuclear translocator, which interacts with regu-
latory regions of dioxin-responsive genes. This leads to the activation of
a series of genes to facilitate biotransformations and elimination of toxic
substances. The binding of ligands (e.g. LXA4) to AhR results in the
formation of an active transcription factor that binds to DNA domains—
dioxin-responsive elements—that activate the expression of a panel of

specific genes.
The receptors signal via heterotrimeric G-proteins to manifest their
biological effects that include modulation of immunity, vascular perme-
ability, tissue fibrosis, mucus secretion and vascular tone (Beller et al.,
2004; Hui et al., 2004; Tager and Luster, 2003).
1.3. LIPID METABOLISM AND EICOSANOID BIOSYNTHETIC

PATHWAYS IN TRYPANOSOMA CRUZI
Kinetoplasts have specialized, adaptable biosynthetic pathways for lipids

that reflect the extreme environments they must endure during their life
cycle, each with its own unique requirements for lipid synthesis. Prokary-
ote biosynthetic pathways synthesize fatty acids using a type I or type II
synthase (Cox, 1982; Lee et al., 2006). Unlike mammalian cells, T. cruzi and
Trypanosoma brucei utilize elongases for nearly all fatty acid synthesis (Lee
et al., 2006; Livore et al., 2007). Four novel elongase genes have been
identified in T. brucei and five in T. cruzi (Lee et al., 2006; Livore et al.,
2007). Of the elongases identified, ELO1 extends C4 to C10, ELO2 extends
C10 to C14, ELO3 elongates C14 to C18 and other elongases extend
beyond C18 (such as AAX69821 from T. brucei). In all cases, a preference
for n6 polyunsaturated fatty acids was observed (Lee et al., 2006). Thus,
fatty acid synthesis would appear modular to reflect the requirement for,
in the case of T. brucei, mostly stearate (C18) in the insect vector and
myristate (C14) in a mammalian host. As such, enzyme expression is
highly regulated. For example, ELO3 (that would prevent accumulation
of myristate) is downregulated in blood-borne parasite, and elevated
concentrations of exogenous fatty acids upregulate the entire pathway,
along with T. cruzi metacyclogenesis, allowing the needs of the parasite to
be highly adaptable to the surrounding environment (Lee et al., 2006;
Wainszelbaum et al., 2003). However, unlike Leishmania major, which has
a complete pathway for polyunsaturated fatty acid biosynthesis, trypano-
somes contain only Delta5 elongases and Delta4 desaturases (Livore et al.,
2007) which allow them to use eicosapentaenoic acid and AA, a precursor
that is relatively abundant in the host, for C22 polyunsaturated fatty acid
synthesis but have otherwise incomplete synthetic pathways for polyun-
saturated fatty acid synthesis.
Eicosanoid synthesis begins with liberation of AA from membrane
phospholipids via the activity of PLs that cleave the Sn-1 acyl chain.
PLA1- and PLA2-like activities have been reported in T. cruzi and T. brucei
(Belaunzaran et al., 2007; Opperdoes and van Roy, 1982; Sage et al., 1981;
Shuaibu et al., 2001). In all cases, activity was membrane associated and
Ca2þ dependent; however, activity was greatly enhanced in the infective
life stages of T. cruzi (trypomastigote and amastigote). Moreover, the

infective life stages release a PLA1-like activity with increased secretion
coinciding with metacyclogenesis (Belaunzaran et al., 2007). An addi-
tional non-PLA-dependent pathway, using sequential deacylation of
diacyl glycerophospholipids, has also identified (Ridgley and Ruben,
2001). The outcome of these activities is the liberation of lipid-based
second messengers from both the parasite (membrane bound form
PLA1) and the host (secreted form). Liberation of AA in such a fashion
results in activation of a plasma membrane localized Ca2þ channel and
mobilization of intracellular Ca2þ stores in T. cruzi and T. brucei (Catisti
et al., 2000; Eintracht et al., 1998). Responses were specific to AA as
shorter-chain lipids were without effect. Moreover, the liberation of dia-
cylglycerol and lysophosphatidylcholine from host membranes activates
kinases cascades that may be critical in parasite–host cell interactions that
precede invasion.
PGF2a is the dominant eicosanoid species produced in Leishmania and
T. brucei, along with smaller quantities of PGE2 and PGD2 (Kabututu et al.,
2003; Kubata et al., 2000; Opperdoes and van Roy, 1982). Importantly,
T. cruzi preferentially synthesizes TXA2 (Ashton et al., 2007) with smaller
amounts of PGF2a, and no significant levels of PGD2 produced. Eicosa-
noid synthesis relies on a series of terminal synthases, each specific to its
own species of lipid produced. Surprisingly, few homologues of the
mammalian eicosanoid biosynthetic enzymes have been identified and
characterized in kinetoplasts. PGF2a synthases have been identified only
in Old World Leishmania spp. and absent in New World Leishmania spp.
In T. cruzi, PGF2a synthase is similar to yeast old yellow enzyme (TcOYE)
and T. brucei (TbPGFS) (Kabututu et al., 2003; Kubata et al., 2000, 2002).
The primary sequence of TbPGFS and TcOYE is distinct from their mam-
malian counterparts (Kubata et al., 2000, 2002), and the enzymatic activity
is resistant to pharmacological agents (ASA or indomethacin) that inhibit
mammalian enzymes indicating that the active sites are also topographi-
cally or structurally different (Kabututu et al., 2003). The crystal structures
of TcOYE (Sugiyama et al., 2007) and TbPGFS (Kilunga et al., 2005; Okano
et al., 2002) have recently been solved. Both form barrel-like structures
with a central hydrophobic core, but TcOYE functions as a dimer
(Sugiyama et al., 2007; Yamaguchi et al., 2011) which is more analogous
to its mammalian homologue.
Both TcOYE and TbPGFS function in drug resistance. In T. cruzi,
TcOYE is essential for drug resistance (Kubata et al., 2002) and TcOYE
levels were recently found to be sixfold different between benznidazole-
sensitive and -resistant strains of T. cruzi (Murta et al., 2006). T. cruzi
possesses four copies of TcOYE demonstrating the importance of this
enzyme to parasite well-being. While differential expression of PGF2a
synthases has been reported in other studies (Andrade et al., 2008; Dost
et al., 2004), they have failed to corroborate the relationship between

PGF2a synthase expression and drug resistance. A reason for this may
be the presence of additional NADPH oxidoreductases from the
cytochrome P-450 family in T. cruzi (Portal et al., 2008). Three new
enzymes in this class were recently identified and displayed a role in
drug resistance as well as predicted roles in fatty acid/eicosanoid synthe-
sis (Portal et al., 2008). The enzymes possess conserved binding domains
for FMN, FAD and NADPH and are strongly inhibited by diphenyleneio-
donium, a classical flavoenzyme inhibitor. It is perhaps this last property
that distinguishes these new enzymes from TcOYE and TbPGFS, as they
appear more like their mammalian counterparts. However, the function
of these enzymes is largely undetermined.
The biological responses of T. cruzi to eicosanoids are likely to be
highly unconventional in nature. Neither orthologues of heterotrimeric
G proteins nor heptahelical G-protein-coupled receptors have been anno-
tated in the T. cruzi genome. Therefore, it is possible that the production of
these lipid mediators is exclusively to manipulate host responses to
infection and ensure parasite survival/transmission.
1.4. ENDOGENOUS REGULATION OF EICOSANOIDS DURING

EXPERIMENTAL CHAGAS DISEASE
It is now appreciated that the release of eicosanoids during infection with

T. cruzi regulates host responses and controls disease progression (Ashton
et al., 2007). The role of these bioactive lipids in acute and chronic Chagas
disease is largely unexplored. However, recent studies (see previous
section) have demonstrated that trypanosomes are capable of AA
metabolism. Thus, the interpretation of the importance of these bioactive
lipids to disease pathogenesis is potentially further complicated by
whether the host or the parasite is the primary source of synthesis
(Kabututu et al., 2003; Kubata et al., 2000, 2002). Despite this uncertainty,
it is clear that eicosanoids play essential and potent roles in the pathogen-
esis of experimental Chagas disease. Essential fatty acid deficiency
(including AA) results in up to 63% reduction in peripheral parasitaemia
and more than twice the usual survival rate during acute disease (Santos
et al., 1992). Moreover, rates of eicosanoid synthesis are higher in resistant
versus susceptible strains of mice (Cardoni and Antunez, 2004).
Eicosanoids released by T. cruzi may contribute to parasite differentiation,
phagocytosis (Freire-de-Lima et al., 2000) and host survival (Sterin-Borda
et al., 1996) by acting as immunomodulators to aid transition and mainte-
nance of the chronic phase of the disease. In support of this concept,
CD11bþ myeloid cells from infected mice have been shown to secrete
an unidentified PG that mediates the loss of immature B-cell populations
by apoptosis. This compromises host defence and favours chronic disease

(Zuniga et al., 2005).
1.4.1. Acute infection

Several species of eicosanoids have been implicated in both acute and
chronic Chagas disease. The question that remains is which AA
derivatives are most important for disease pathogenesis? Plasma from
infected mice displays increased levels of PGF2a, PGI2, TXA2 and PGE2
(Cardoni and Antunez, 2004; Pinge-Filho et al., 1999; Tanowitz et al., 1990)
compared to uninfected mice from 10 days post-infection onwards. We
have previously determined that the main PGs derived from T. cruzi are
TXA2 and PGF2a (Ashton et al., 2007), indicating that host is the likely
source of the elevated PGI2 and PGE2. PGE2 release is likely from
activated macrophages and CD8þ T-cells (Oliveira et al., 2010; Sterin-
Borda et al., 1996). No specific role has been delineated for the elevated
PGI2 and PGF2a observed in plasma from bona fide or experimental
Chagas disease. Minimal work on PGF2a indicates that PGF2a levels in
the TXA2 synthase null and wild-type mice were similar, indicating this
PG was likely not involved with the augmentation of parasitaemia
observed in the COX-1 null and ASA-treated mice or in the regulation
of mortality (Mukherjee et al., 2011). This leaves the potential role of
PGF2a in Chagas disease largely unexplored. However, the significant
amounts of PGF2a produced by T. cruzi and the fact that all members of
the kinetoplast family have an identifiable PGF2a synthases indicate that
this eicosanoid is of significant value to the parasite.
Most studies suggest the primary role of PGs/eicosanoids is to
manipulate the host response and enhance the likelihood of transition to
the chronic state (Kristensson et al., 2010; Pinge-Filho et al., 1999;
Sterin-Borda et al., 1996). During acute infection, PGE2 has been shown
to modulate the virulence of the T. cruzi strain. A non-lethal strain (K98)
provoked elevated circulating PGE2, while lethal strains (RA or K98-2)
did not (Celentano et al., 1995). Inhibition of COX activity (and therefore
PGE2 release) increased mortality in K98-strain-infected mice, but PGE2
infusion did not attenuate the virulence of the RA strain. The effects of
PGE2 may stem from a number of sources. PGE2 release from monocytes
drives Th1 immunity which has greater effect at controlling parasitaemia
(Oliveira et al., 2010). In addition, PGE2 is essential to the suppression of
TNF-a release and lymphoproliferation by the host during acute infection
in both patients and mice (Borges et al., 1998; de Barros-Mazon et al., 2004;
Pinge-Filho et al., 1999). Inhibition of PGE2 synthesis reduces number of
parasite nests, inflammatory infiltrates and cardiac fibrosis during acute
disease (Abdalla et al., 2008) all of which likely aids the transition to
chronic disease (Sterin-Borda et al., 1996).
Prostaglandin release from the parasite, primarily TXA2, also appears

to aid survival of the acute infection and transition to the chronic state.
Preventing host response to parasite-derived TXA2 augmented death and
parasitaemia (Ashton et al., 2007). Platelets exert a direct anti-trypanoso-
mal activity (Momi et al., 2000), and over the course of disease, there is a
generalized thrombocytopenia characterized by increased platelet
adherence and aggregation that likely limits the anti-parasitic action of
these cells (Tanowitz et al., 1990). TXA2 may regulate vasospasm,
thrombosis, vascular permeability and endothelial cell dysfunction
during acute disease; however, TXA2 also displays immunosuppressive
properties with the wild-type mice displaying minimal pathology, but
TXA2 receptor null mice exhibiting pronounced inflammation in the
myocardium with an almost threefold increased in parasite load in
cardiac tissue. There is also evidence that TXA2 signalling by the host
acts as a potential quorum-sensing mechanism for T. cruzi and regulates
amastigote proliferation to prevent overwhelming the host during acute
infection (Ashton et al., 2007). It is clear that TXA2 plays a prominent role
in acute T. cruzi infection; however, the previous belief that TXA2
manifests as a host response to infection, and not directly from the
parasite, suggests the role of T. cruzi-derived mediators have been
undervalued in disease pathogenesis.
In fact, quorum sensing may involve a variety of eicosanoids. These
short-lived auto/paracrine messengers are well suited to this role due to
quick inactivation and need for constant synthesis. Quorum sensing in
T. brucei involves release of PGD2 which slows proliferation largely
through the induction of apoptosis (Figarella et al., 2005). However,
PGD2 is metabolised to J-series PGs (PGJ2 and 12D-PGJ2) in the presence
of albumin, and these subsequent ‘‘metabolic’’ products are more potent
than PGD2 in regulating survival in T. brucei (Figarella et al., 2005, 2006).
Similar effects are also likely in T. cruzi, despite the fact that this parasite
produces no discernable PGD2. Administration of 15D-PGJ2 during the
acute stage of T. cruzi infection reduced the density of amastigote nests in
cardiac muscle (Rodrigues et al., 2010). However, 15D-PGJ2 also displays
immunosuppressive properties and reduced inflammation at the site of
infection and peripheral leukopenia via increased levels of IL-10 release
which clouds the interpretation of the results as to whether this molecule
may act as a quorum sensor during Chagas disease.
During the acute inflammatory phase of the T. cruzi infection, high-
level expression of proinflammatory cytokines and other mediators is
prevalent. The inflammatory cytokines and lipid mediators are essential
for host survival during acute infection (Borges et al., 2009; Hideko
Tatakihara et al., 2008). 5-LO has received the most attention in inflamma-
tion research involved in T. cruzi infection due to its involvement in LT
synthesis. LTs are known to participate actively in the control of infections
by protozoa, as demonstrated in several studies (Henderson and Chi, 1998;

Machado et al., 2005). LTB4 and LTC4 enhance association with and uptake
of T. cruzi by monocytes by stimulation of phagocytosis (Wirth and
Kierszenbaum, 1985a,b). Conversely, since LTC4 treatment increased asso-
ciation with non-phagocytic cells, LTC4 may facilitate parasite invasion, not
uptake, as a part of the enhanced parasite clearance. However, the role of
these molecules in host resistance and induction of myocarditis during
infection remains unclear. LTB4 promotes recruitment of inflammatory
cells (Tager et al., 2003); however, unlike LTB4, cys-LTs (LTC4) do not
induce leukocyte migration into inflamed tissue but increase vascular
permeability and subsequent oedema (Dahlen et al., 1981). Moreover,
they have also been described as detrimental factors to heart contractility
(Gorelik et al., 1992). Thus, the absence of 5-LO seems to prevent the
harmful effects of these mediators on heart contractile function. Many of
the effects of LTC4 are mediated by guanylate cyclase/NO inhibition. NO is
an important mediator of parasite killing in experimental T. cruzi infection
(Vespa et al., 1994) and is potently regulated by LTs.
While some lipid mediators drive acute inflammation, other endoge-
nous mediators counter-balance these proinflammatory events. In addition
to LTs, lipoxins (LXAs) play a pro-resolving role in inflammatory reactions
(Maderna and Godson, 2009; Ryan and Godson, 2010; Stables and Gilroy,
2010). To date, the role of LXAs in myocardial inflammation and modula-
tion of the immune response during T. cruzi infection remains unresolved,
but the action of LXAs in the regulation of Toxoplasma gondii infection
allows us to extrapolate the potential roles that this eicosanoid may play
during T. cruzi infection. The T. gondii model demonstrates that challenge
with parasite triggers endogenous LXA4 release that down-modulates
dendritic cell activation in vivo and in vitro (Aliberti et al., 2002a). T. gondii
infection in 5-LO null mice resulted in more extensive tissue pathology,
mainly due to lack of LXA4 production, as treatment with LXAs analogs
restored the resistance to tissue pathology with no mortality associated
with uncontrolled proinflammatory responses, in a similar manner as for
wild-type mice (Aliberti et al., 2002b). Moreover, there is evidence that the
AhR mediates the bioactions of LXAs during T. gondii infection. This
receptor is a ligand-activated transcription factor that regulates many of
the biologic actions of LXAs, including increasing the expression of
suppressor of cytokine signalling 2 (SOCS-2) (Machado et al., 2006). Ongo-
ing work in our laboratories on inflammation during T. cruzi infection has
revealed that SOCS-2 is important in the modulation of inflammation
during this infection (Mukherjee et al., 2011).
Thus, it appears that the eicosanoids present during acute infection
largely act as immunomodulators that aid in the transition to and main-
tenance of the chronic phase of the disease (Sterin-Borda et al., 1996). It is
unclear whether T. cruzi generates PGs as a defence against host immune
system or whether it hijacks the host PG metabolic pathway in its favour.

To this end, further studies using null mice missing biosynthetic enzymes
or receptors are required to fully elucidate the role of the identified PGs in
Chagas disease.
1.4.2. Chronic infection

In contrast to acute infection, where plasma levels of multiple prostanoids
are elevated, only increased levels of TXA2 are observed in chronic disease
(> 180 days post-infection) (Cardoni and Antunez, 2004). In chronic dis-
ease, 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. In support of this hypothesis,
treatment of mice with chronic T. cruzi infection with ASA may result in
improvement of cardiac function which likely results from negating the
detrimental effects of TXA2 on myocyte contractility, platelet function and
vascular tone. In addition to the maelstrom of changes that TXA2 mediates
during acute infection, the secretion of TXA2 would prevent the initiation
of an adaptive immune response by the host (Kabashima et al., 2003),
enabling maintenance of the chronic phase of the disease. Finally, the
role for TXA2 in chronic disease is made more complicated by its control
of parasite proliferation. We have suggested that parasite-derived TXA2 is
a possible quorum sensor for the parasite (Ashton et al., 2007); however,
parasite-derived TXA2 release is insufficient to suppress peripheral para-
sitaemia in chronic disease. This indicates a need for host-derived TXA2
for control of the severity of the chronic disease.
Despite the fact that TXA2 is the chief PG detected in plasma, other
eicosanoids may play a significant role during chronic disease. T-lym-
phocytes from patients with chronic Chagas disease affect cardiac func-
tion and remodelling in a rat model (de Bracco et al., 1984). Similarly,
lymphocytes derived from acute and chronic infection of mice display
negative and positive inotrophism, respectively (Gorelik et al., 1992). LO
products (primarily LTC4) released from the lymphocytes were shown to
positively affect cardiac function, while COX products (principally PGE2)
exerted a depressor inotropic action. Thus, eicosanoid release during
chronic disease appears to be more focused on damage to the host than
during the acute phase of infection.
1.4.3. Insect vectors

While several groups have investigated the impact of eicosanoids on
mammalian hosts, little has been done to determine their potential role
in insect vectors. Prostaglandin biosynthesis and release occur in all three
life stages of T. cruzi (Ashton et al., 2007; Kabututu et al., 2003). While it is
clear that in the trypomastigote and amastigote forms of the parasite,
multiple functions exist for bioactive lipids, almost nothing is known
about the role of these mediators in the epimastigote stage. Insects fed
on blood treated with inhibitors to COX, LO and PLA2 enhance parasi-
taemia and mortality due to parasite challenge with T. rangeli (Garcia
et al., 2004), leading to a hypothesis that parasite-derived PGs suppress
immunity and permit the chronic habitation of the vector (Azambuja and
Garcia, 2005; Azambuja et al., 2005). Eicosanoids such as TXA2 may aid in
the colonization of the gut by producing mucosal injury (Walt et al., 1987)
and increase the potential spread of the parasite through increasing gut
motility (Schultheiss and Diener, 1999). The survival of T. cruzi in the gut
of its insect vector is largely dependent upon nutritional status (Azambuja
and Garcia, 2005; Azambuja et al., 2005). Thus, the same scavenging
mechanism that operates to provide lipid precursors in the mammalian
host (see next section for a discussion of these mechanisms) may also
apply to the insect vector.
1.5. LESSONS FROM PHARMACOLOGICAL MANIPULATION

AND FROM NULL MICE
1.5.1. Pharmacological intervention

Given the increasing interest in the role of eicosanoids in T. cruzi infection, it
is not unexpected that there should be interest in pharmacological manipu-
lation of eicosanoid biosynthesis in the pathogenesis and clinical manage-
ment of this infection. Previous studies have attempted to document the role
of eicosanoids in early infection using pharmacological intervention with
mixed results (Celentano et al., 1995; Freire-de-Lima et al., 2000; Hideko
Tatakihara et al., 2008; Michelin et al., 2005; Mukherjee et al., 2011;
Pinge-Filho et al., 1999). Pharmacological antagonists selective for COX-1
(ASA), COX-2 (celecoxib) or both (indomethacin) increase mortality and
parasitaemia (both peripheral blood counts and cardiac parasite nests)
regardless of mouse or parasite strain used (Celentano et al., 1995; Hideko
Tatakihara et al., 2008; Mukherjee et al., 2011; Pinge-Filho et al., 1999; Sterin-
Borda et al., 1996). Moreover, administration of non-steroidal anti-inflam-
matory drugs (NSAIDs) may enhance mortality in patients (Celentano et al.,
1995; Sterin-Borda et al., 1996). Conversely, others have found inhibition of
PG synthesis/release ablates parasitaemia and extend survival in mice
infected with T. cruzi (Abdalla et al., 2008; Freire-de-Lima et al., 2000;
Michelin et al., 2005; Paiva et al., 2007). This was usually associated with a
decrease in the circulating levels of inflammatory cytokines (such as TNF-a,
IFN-g and IL-10) (Michelin et al., 2005). These outcomes are not unexpected
with the use of such general inhibitors and highlight the need for specific
receptor blockers and terminal synthase antagonists to be applied to identi-
fying the PGs most relevant to the pathogenesis of Chagas disease.
The dichotomy of the effects seen with NSAIDs in acute disease might
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 by immunoblotting (S. Mukherjee, unpublished data).
While the role of COX-2 in T. cruzi infection is largely undefined, both
COX-1 and COX-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 (Hideko
Tatakihara et al., 2008). Inhibition of COX-1 and -2 reciprocally regulates
NO release from M1 and M2 macrophages which may correlate with
resistance to infection. Consistent with this observation, COX-2-derived
PGs mediate most of the immunosuppressive effects during the initial
phase of T. cruzi infection (Michelin et al., 2005). This may result from the
observations that PGI2 and PGE2 are more closely linked to COX-2 metab-
olism, while COX-1 is responsible for TXA2 synthesis (Parente and
Perretti, 2003; Smith et al., 1997). In addition, timing of onset of treatment
is important; that is, administration of ASA early in disease, 5 days
post-infection increased parasitaemia and mortality (Mukherjee et al.,
2011). This observation suggests that caution should be exercised when
employing COX inhibitors for controlling fever and pain in the setting of
acute Chagas disease. Conversely, use of ASA during chronic disease had
no effect on mortality or parasitaemia but improved 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 stage.
The selectivity of the NSAIDs used may also determine whether
parasite or host production of PGs is the primary target of the treatment
regimen used. Many of the biosynthetic enzymes in trypanosomes appear
to be unaffected by inhibitors of their mammalian counterparts (Kabututu
et al., 2003). Conversely, indomethacin-amides were recently shown to
have anti-T. cruzi activity (Konkle et al., 2009). Although these com-
pounds were tested for inhibition of steroid biosynthesis in T. cruzi, they
are uniquely specific to COX-2 inhibition in mammals. Thus, a logical
hypothesis is that free fatty acid, eicosanoid and sterol biosynthesis may
be linked in T. cruzi through the use of enzymes whose biosynthetic
capabilities allow them to participate in more than one pathway.
LTs are necessary for control of parasitaemia and survival in the acute
T. cruzi infection (Borges et al., 2009) due to modulation of NO and
cytokine release. The treatment of T. cruzi-infected mice with a BLT1
receptor antagonist or the 5-LO inhibitor nordihydroguaiaretic acid was

accompanied by increased parasitaemia and tissue parasitism but not
lethality (Talvani et al., 2002). NO is a major effector molecule of
trypanocidal activity in mice (Malvezi et al., 2004; Petray et al., 1994;
Vespa et al., 1994) and is also a significant target for LTs in vivo. Therefore,
the enhanced parasitaemia is likely due to a reduction in NO activity with
5-LO/BLT1 antagonism; however, studies have shown that elevated NO
production occurred in the absence of 5-LO activity suggesting that
mechanisms independent of LTs may be operative. Indeed, 5-LO was
recently demonstrated to modulate the severity of myocardial inflamma-
tion during T. cruzi infection likely through the same mechanism
(Pavanelli et al., 2010). The impairment of LT synthesis clearly resulted
in increased parasite persistence most likely due to a combination of low
leukocyte infiltration and NO production during acute disease (Borges
et al., 2009).
Aside from studies suggesting that LT production is necessary for
efficient effector mechanisms during T. cruzi infection, a growing body
of the literature indicates that other bioactive lipids, such as the chemoat-
tractant platelet-activating factor (1-o-alkyl-2-acetyl-sn-glyceryl-3-
phosphorocholine) (PAF), may also activate NO-dependent trypanocidal
activity in macrophages. PAF is a non-AA-derived membrane phospho-
lipid mediator with widely recognized proinflammatory activities. PAF is
produced by and exerts its biological actions on a variety of mononuclear
cells and is implicated in several systemic inflammatory disorders
(Braquet et al., 1987; Chao and Olson, 1993; Chignard et al., 1979; Im
et al., 1996, 1997; Ishii et al., 1998). In vitro, PAF induced inhibition of
parasite growth via NO secretion by T. cruzi-infected macrophages. Addi-
tion of a PAF antagonist, WEB 2170, inhibited both NO biosynthesis and
trypanocidal activity which appear to be dependent on TNF-a production.
Treatment of T. cruzi-infected mice with PAF antagonist (WEB 2170)
promoted higher parasitaemia and earlier mortality, when compared
with controls, suggesting that PAF may help coordinate mechanisms of
resistance to T. cruzi infection. PAF also plays a role in acute myocarditis in
T. cruzi-infected mice (Chandrasekar et al., 1998). PAF secretion during
infection likely acts as a chemoattractant for several leukocyte populations
resulting in inflammatory cell infiltration and production of proinflamma-
tory cytokines that bring about damage in this tissue. Exacerbation of
parasitaemia and mortality with PAF receptor blockade suggests that
these molecules might regulate effector responses to the parasite.
In addition to these effects on the host, PAF may regulate T. cruzi
differentiation and growth (Rodrigues et al., 1996). PAF failed to signifi-
cantly alter T. cruzi growth; however, parasite growth in the presence of
PAF was significantly more differentiated than in its absence. Blockade
of the PAF receptor abrogated the PAF effect on cell differentiation. T. cruzi
produce a PAF-like lipid with all the biological properties of mammalian-

PAF (Tc-PAF) (Gomes et al., 2006). This Tc-PAF augments infection of host
cells by T. cruzi and triggers the differentiation of epimastigotes into
metacyclic trypomastigotes (Gomes et al., 2006). These effects were abro-
gated by WEB 2086, and antibodies against murine PAF receptor labelled
epimastigotes, suggesting that T. cruzi expresses an orthologue of the PAF
receptor. Moreover, these data suggest that T. cruzi contains the compo-
nents of an autocrine PAF-like ligand–receptor system that modulates cell
differentiation towards the infectious stage (Gomes et al., 2006). It remains
to be seen if T. cruzi infection in vivo can modulate host response by
secreting a PAF-like mediator.
1.5.2. Phenotypes of transgenic/knockout mice

It appears that multiple eicosanoids contribute significantly to the patho-
genesis of Chagas disease. Yet little is known of the role played by each.
The pharmacological approaches used to date have been in general to
decipher the role of individual eicosanoids; for instance, a COX-2 inhibitor
would decrease PGI2 and PGE2 production without identifying which
species might be causal. Further, pharmacological inhibition of one path-
way (COX) may shunt the precursor AA into other metabolic pathways
(such as LOX) along with increased synthesis of downstream metabolites.
Thus, it becomes difficult to identify the bioactive lipid(s) responsible for
the action of these generalized inhibitors. A cornucopia of transgenic and
knockout mouse lines has been generated in the biosynthetic pathway
and receptors for eicosanoids. Little work has been done using these
models to identify specific roles for individual eicosanoid species; how-
ever, the work that has been performed has yielded surprising results.
The inconsistency over the nature of COX inhibitors as modulators of
Chagas disease was recently resolved with the first use of COX-1 null
mice to model the changes in the course of experimental T. cruzi infection
(Mukherjee et al., 2011). This report confirmed that PGs derived from
COX-1-mediated biosynthesis of the host contribute to the suppression of
parasite proliferation, but not mortality in acute disease. Like in the
COX-1 null mice, ASA ablated the release of PGF2a and TXA2 in response
to T. cruzi infection. However, infection of COX-1 null mice only
mimicked the effects of ASA on parasitaemia, indicating control of para-
site proliferation, but not mortality, was due to regulation of PGs.
The mechanism for the enhanced mortality with NSAID treatment
during acute disease may lie with ‘‘off-target’’ effects of these agents
(Claria and Serhan, 1995). Aside from preventing PG synthesis,
ASA-mediated acetylation of COX-2 induces the synthesis of aspirin-
triggered lipoxin (ATL, or 15-epi-LXA4) (Serhan et al., 1984). ATL has
the same activity but is more metabolically stable than LXA4. Multiple
studies by several groups have shown that ATL regulates cytokine pro-
duction and release (Maderna and Godson, 2009; Ryan and Godson,
2010), induces SOCS-2 and suppresses TNF receptor-associated factor-6
(TRAF-6) to silence cytokine signalling (Machado et al., 2006). Impor-
tantly, Machado et al. (2006) demonstrated that ASA-treated SOCS-2
null mice given LPS by the intra-peritoneal route could not inhibit neu-
trophil migration and TNF-a. It is therefore possible that mortality may
have more to do with modulation of the impending cytokine storm
during acute disease than actual PG production. Thus, the effects of
ASA in T. cruzi infection may be via dual mechanisms that operate during
different phases of disease.
Pharmacological inhibition may not distinguish between the potential
for the host and parasite to function as the source of eicosanoid synthesis
during disease. Importantly, the differences between pharmacological inhi-
bition of COX-1 and the COX-1 null mice indicated that there was an
unrecognized and essential host–parasite interdependence that dictates
the biosynthetic activity of the parasite. The basis for this relationship
appears to be the requirement for host-derived PGH2 for PG synthesis
throughout infection. A reduction in PGF2a release in COX-1 null, but not
TXA2 synthase null, mice was observed. As TXA2 synthase null mice have
normal COX activity, the data indicate that COX activity in the host likely
provides precursor molecules required for the biosynthetic pathways of this
parasite. This ‘‘scavenging’’ hypothesis is confirmed by the inability of the
parasite (the primary source of TXA2 during infection) to sustain TXA2
release in the COX-1 null mice. Moreover, the fatty acid synthesis pathways
in trypanosomes are defective regarding synthesis of polyunsaturated lipids
(Livore et al., 2007) which makes scavenging precursors from the host not
just energetically favourable but a requirement. Secretion of PLA-like activ-
ity into the host cell from intracellular amastigotes (Belaunzaran et al., 2007)
would liberate AA from the inner layer of the host cytoplasmic membrane.
This variation on transcellular synthesis would ensure a constant supply of
precursors for parasite biosynthetic pathways.
If the parasite were scavenging precursors from the host, then they
would only need the terminal synthases to produce bioactive lipids. The
fatty acid biosynthetic pathways in trypanosomes are poorly defined, and
little homology is reported between the mammalian biosynthetic enzymes
and their trypanosomal homologues (Kubata et al., 2000). The PGF2a
synthase identified in the parasite is more similar to yeast old yellow
enzyme than to mammalian enzyme (Kubata et al., 2002), however, the
recent report of anti-parasitic activity of indomethacin-amide derivatives
indicates that the active site of some parasite enzymes, if not their primary
sequences, is sufficiently homologous to their mammalian counterparts
(Konkle et al., 2009). Interestingly, no enzyme other than COX has been
identified as being sensitive to indomethacin. However, it remains to be
determined whether the target gene (TcCYP51) of these indomethacin-

amide derivatives is an integral component of the eicosanoid biosynthetic
pathway of T. cruzi (Konkle et al., 2009). These data highlight, for the first
time, an interdependence of the parasite on host metabolism for prostanoid
biosynthesis, which would never have been identified using inhibitor/
pharmacological studies alone, and reveal a deeper understanding of
host–parasite relationships with potential new avenue for therapeutic
options.
The interdependence between host and parasite for endogenous
precursors begs the question whether the host or parasite is the primary
source of the lipid mediators regulating the pathogenesis of disease. TXB2
levels are elevated in mice infected with T. cruzi (Ashton et al., 2007;
Mukherjee et al., 2011; Tanowitz et al., 1990) and levels were maintained
in acute infection in the TXA2 synthase null mice (Ashton et al., 2007).
These experiments identified TXA2 as a parasite-derived molecule that
modulates survival and disease progression. However, the primary
source of TXA2 had always been assumed to manifest through a host
response to infection, such as inflammation and platelet activation. These
data dispelled this myth and firmly indicated that parasite-derived
eicosanoids are primary modulators of disease. Further, the use of TXA2
receptor (TP) null mice revealed a second interdependence in the
host–parasite relationship. The feedback from the TP on host cells initiates
a signalling cascade that controls parasite growth and permits parasite
replication at a rate that enables continued survival of the host.
Conversely, T. cruzi-derived TXA2 elicits a robust response from host
cells that appears to be largely anti-inflammatory. TP null mice had an
increased mortality, and their coronary artery endothelial cells had a
higher intracellular parasitism and degree of dysfunction compared
with wild-type mice that displayed minimal pathology. These data sug-
gest that parasite-derived TXA2 is sufficient to stimulate host TP to ensure
normal disease progression, parasitaemia and host survival. The combi-
nation of these effects allows for a balance between the needs of the
parasite (proliferation and survival through evading the host immune
response) and the host (to survive the initial infection and largely limit
collateral damage to organs during acute infection). Thus, the proposed
interdependence of the parasite on host precursors and host on parasite-
derived TXA2 for controlled disease pathogenesis is unique as it inter-
twines host biochemistry and parasite biology.
Recently, the regulatory role of endogenous LTs and lipoxins in exper-
imental Chagas disease was determined in mice with targeted deletion of
the 5-LO gene. The deficiency of 5-LO during T. cruzi infection enhances
peripheral parasite load and the number of myocardial parasite nests
compared to wild-type mice. Despite these observations, infected 5-LO
null mice controlled parasite burden and survived acute infection. Similar
results were obtained in mice treated with MK886, an inhibitor of LT

biosynthesis. These studies show that the endogenous LT production is
an important regulator of iNOS expression in the heart, which in turn
appears to help control parasite burden during early phase of infection in
mice. The high levels of NO observed in T. cruzi-infected wild-type mice
may impact the efficiency of immune response, allowing the proliferation
and persistence of parasite in the tissue. The lower NO production
observed in infected 5-LO null mice could be sufficient to control parasite
replication while avoiding extensive myocardial damage. Indeed, it was
recently demonstrated that 5-LO products are responsible for oxidative
stress in erythrocytes during this infection (Borges et al., 2009). Thus, the
lipid mediators derived from 5-LO metabolism, especially LTB4, may
induce inflammatory damage into parasitized cardiac tissues as a conse-
quence of controlling initial parasite load.
5-LO null mice displayed reduced leukocyte migration to the myocar-
dium with increased circulating IL-6 and IL-10 (Chandrasekar et al., 1996;
Saavedra et al., 1999; Truyens et al., 1994) and decreased levels of TNF-a,
IFN-g and NOS in the hearts of 5-LO null compared to WT mice (Pavanelli
et al.); however, the functional significance of these changes in vivo is not
clear. The diminished migration of CD4þ and CD8þ T cells into the
myocardium of 5-LO null mice is likely a product of diminished cyto-
kine/chemokine production combined with lower NO release. 5-LO null
mice present reduced indices of myocardial fibrosis at a late stage of acute
infection, which is a clear consequence of decreased tissue destruction
due to reduced leukocyte infiltration. In addition, the reduction in release
of fibrosis promoting cytokines, such as IL-4, TNF-a and TGF-b (Piguet
et al., 1989; Rossi, 1998), in the 5-LO null mice during infection would also
ameliorate the cardiac damage during infection. Further investigations
focused on the chronic heart pathology should demonstrate if absence of
5-LO could lead to reduce matrix remodelling and perhaps reduce
chronic morbidity. Thus, all the above factors indicate that host 5-LO
and its enzymatic products are essential for controlling T. cruzi replication
and pathogenesis of disease during T. cruzi infection. These data highlight
the insights and profound understanding about the pathogenesis of
Chagas disease that can be gained from the use of knockout mouse
models and the opportunities that yet exist to further define the roles on
individual eicosanoids on the pathogenesis of Chagas disease.
ACKNOWLEDGEMENTS
This work was supported by grants from the United States National Institutes of Health
(H. B. T. [AI-76248]), the National Health and Medical Research Council of Australia
(A. W. A. [512154]) and a Scientist Development Grant from the American Heart Association
(S. M. [0735252N]). This work was also supported by Career Development Awards from the
National Health and Medical Research Council of Australia (A. W. A. [402847]), Conselho
Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq) (F. S. M. [576200/2008-5;
473670/2008-9]) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais
(FAPEMIG) (F. S. M. [14916]).
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CHAPTER 2
Mechanisms of Host Cell
Invasion by Trypanosoma cruzi
Kacey L. Caradonna and Barbara A. Burleigh

2.2. General Features of Trypanosoma cruzi Invasion 36
2.2.1. Trypanosoma cruzi trypomastigotes actively
invade host cells 36
2.2.2. Host recognition and adhesion 37
2.2.3. Binding host cell extracellular matrix 38
2.2.4. Role of gp85/TS in tissue-specific homing 38
2.2.5. Role of gp82/gp90 in signal transduction and
regulation of oral transmission route by
metacyclic trypomastigotes 40
2.3. To the Lysosome . . . and Beyond 41
2.3.1. Establishment of cytosolic residence is critical
for Trypanosoma cruzi survival and replication 41
2.3.2. Pathways to the lysosome 42
2.3.3. Role of host cell phosphatidylinositol-3-kinases
in Trypanosoma cruzi invasion 48
2.4. Disruption of the Parasitophorous Vacuole
Membrane and Cytosolic Localization of Parasites 49
2.5. The Role of the Host Cell Cytoskeleton in
Trypanosoma cruzi Trypomastigote Invasion of
Non-Phagocytic Cells 51
2.5.1. Actin 51
2.5.2. Microtubules 52
2.6. The Discovery of Reversible Invasion 53
2.7. Concluding Remarks 54
References 54
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston,
Massachusetts, USA

33
34 Kacey L. Caradonna and Barbara A. Burleigh
Abstract One of the more accepted concepts in our understanding of the

biology of early Trypanosoma cruzi–host cell interactions is that
the mammalian-infective trypomastigote forms of the parasite
must transit the host cell lysosomal compartment in order to
establish a productive intracellular infection. The acidic environ-
ment of the lysosome provides the appropriate conditions for
parasite-mediated disruption of the parasitophorous vacuole and
release of T. cruzi into the host cell cytosol, where replication of
intracellular amastigotes occurs. Recent findings indicate a level of
redundancy in the lysosome-targeting process where T. cruzi try-
pomastigotes exploit different cellular pathways to access host
cell lysosomes in non-professional phagocytic cells. In addition,
the reversible nature of the host cell penetration process was
recently demonstrated when conditions for fusion of the nascent
parasite vacuole with the host endosomal–lysosomal system were
not met. Thus, the concept of parasite retention as a critical
component of the T. cruzi invasion process was introduced.
Although it is clear that host cell recognition, attachment and
signalling are required to initiate invasion, integration of this knowl-
edge with our understanding of the different routes of parasite
entry is largely lacking. In this chapter, we focus on current knowl-
edge of the cellular pathways exploited by T. cruzi trypomastigotes
to invade non-professional phagocytic cells and to gain access to
the host cell lysosome compartment.
2.1. INTRODUCTION
Trypanosoma cruzi, the protozoan parasite that causes human Chagas
disease, has a digenetic life cycle involving both vertebrate and invertebrate
hosts within which, distinct developmental stages of the parasite arise
(Fig. 2.1). As an obligate intracellular parasite in the vertebrate host, intracel-
lular localization is critical for establishment and maintenance of T. cruzi
infection. Host cell invasion is accomplished by trypomastigotes, both meta-
cyclic and bloodstream forms, which are highly specialized, non-dividing
forms of T. cruzi that can penetrate a wide variety of mammalian cell types.
Once inside the host cell, trypomastigotes undergo a developmental process
that culminates in the formation of replicative amastigotes that proliferate in
the host cell cytoplasm for 5–6 days until they occupy most of the cell
volume. At this stage, amastigote division ceases and differentiation to
trypomastigotes occurs followed by rupture of the host cell plasma
membrane (Costales and Rowland, 2007) releasing trypomastigotes that
disseminate infection (De Souza, 2002; Fig. 2.1).
Mechanisms of Host Cell Invasion by Trypanosoma cruzi 35
Metacyclic
Epimastigote
trypomastigote
Reduviid host
Mammalian host
Metacyclic/
bloodstream
trypomastigote
Intracellular
amastigote
FIGURE 2.1 Trypanosoma cruzi life cycle. Metacyclic trypomastigotes arising from
epimastigotes in the reduviid host are transmitted to mammalian host in the faeces of
the insect vector. Inside the host, trypomastigotes invade cells and are rapidly targeted
to a lysosome-derived vacuole. Within the vacuole, trypomastigotes begin the trans-
formation to amastigotes (2–8 h) after which the vacuole is gradually disrupted and
parasites localize to the host cell cytoplasm (8–16 h). Cytosolic amastigotes begin to
divide at 24 h post-invasion and continue to divide every 12 h for 5–6 days, then
differentiate back into trypomastigotes, rupture the host cell, enter the host circulation
and disseminate infection.
Cardiomyocytes are clearly one of the most important target cell types
for T. cruzi infection in vivo, where early establishment of infection and
parasite persistence in the heart correlate with disease progression in
chagasic cardiomyopathy (Benvenuti et al., 2008; Jones et al., 1993;
Monteon-Padilla et al., 2001; Mortara et al., 1999; Zhang and Tarleton,
1999). Tissues harvested from rare autopsies performed on acute Chagas
patients reveal the presence of intracellular T. cruzi amastigotes not only
in cardiomyocytes but also in smooth muscle of the oesophagus, larynx
and bladder with concomitant inflammatory infiltrate (e.g. Bittencourt
et al., 1984; Montalvo-Hicks et al., 1980). In cases of reactivation of acute
Chagas disease in immunocompromised individuals, parasites have also
been noted in the central nervous system (Marchiori et al., 2007; Mortara
et al., 1999; Sartori et al., 1995). Much of our knowledge of the T. cruzi
infection process has been generated from experimental animal models,
from which it is clear that T. cruzi is able to infect a variety of tissues
during the acute stage of infection (Barbabosa-Pliego et al., 2009; Barr
et al., 1991; Buckner et al., 1999; Caradonna and PereiraPerrin, 2009;

Zhang et al., 1999). However, with the exception of cardiomyocytes and
gastric mucosal cells (that are targeted by T. cruzi upon infection via the
oral route; Staquicini et al., 2010), knowledge of the specific cell types
infected within different tissues during acute and chronic infection is still
lacking. Because T. cruzi can infect most nucleated cell types in culture,
there has been no consistency in the use of model cell types for invasion
studies. Coupled with the genetic and biological diversity that exists
among different T. cruzi strains (Miles et al., 2009), cell type-specific
requirements introduced with different parasite strain–host cell combina-
tions are likely to complicate the ability to arrive at a unifying model for
T. cruzi invasion. Despite this obvious limitation, significant advances
have been made in the past decade towards delineation of the
mechanisms of host cell recognition, signalling and invasion by T. cruzi
trypomastigotes. In this chapter, we highlight several features of the host
cell recognition process and describe three cellular pathways that are
exploited in the host by T. cruzi trypomastigotes to facilitate the establish-
ment of intracellular infection in non-professional phagocytic cells.
2.2. GENERAL FEATURES OF TRYPANOSOMA CRUZI

INVASION
2.2.1. Trypanosoma cruzi trypomastigotes actively invade

host cells
The extent of the molecular interactions between T. cruzi trypomastigote
and the host cell plasma membrane that are required to initiate invasion is
largely unknown; however, it is generally accepted that a committed
attachment step precedes invasion. The invasive trypomastigote forms
of T. cruzi are long and slender ( 20 mm long; 2 mm wide), highly motile
organisms that attach and invade cells in energy-dependent fashion
(Martins et al., 2009; Schenkman et al., 1991). Observations from live cell
imaging studies reveal that trypomastigotes can spend several minutes at
a particular region of the host cell surface, where they appear to be
probing for appropriate binding partners or to receive specific signals,
before committing to invasion. As many of these interactions appear to
fail to promote stable attachment or entry, it is common to observe
parasites moving to other cells even after several minutes of probing at
a particular site. Thus, it appears that a complex set of conditions must be
met at the host cell surface for T. cruzi trypomastigotes to initiate the
active attachment and invasion process. While this behaviour complicates
the study of the early events associated with host cell penetration by
T. cruzi trypomastigotes, as invasion is asynchronous and few parasites
manage to enter cells within the first 5–10 min of contact with host cells,
studies that have incorporated live cell imaging techniques, as well as

pulse–chase types of invasion experiments, have provided insights into
the earliest events transpiring at the parasite–host interface (Caler et al.,
2000; Rodriguez et al., 1996; Romano et al., 2009; Tardieux et al., 1994;
Tyler et al., 2005; Woolsey et al., 2003).
2.2.2. Host recognition and adhesion

The expansion of several large polymorphic gene families in the T. cruzi
genome (containing between 500 and 3000 genes/family; El-Sayed et al.,
2005) encoding surface-expressed glycoproteins (Acosta-Serrano et al.,
2001; Bartholomeu et al., 2009; Cross and Takle, 1993; Kawashita et al.,
2009; Schenkman et al., 1992), coupled with the genetic diversity that exists
among parasite isolates (Miles et al., 2009), likely imparts the ability of this
pathogen to infect a wide range of vertebrate hosts and cell types. Unlike
the mono-allelic expression of major surface antigens observed with some
protozoan parasites (Horn, 2004; Voss et al., 2006), a population of mam-
malian-infective T. cruzi trypomastigotes co-express several variants
within individual gene families (Atwood et al., 2005; Bartholomeu et al.,
2009; Cordero et al., 2009; Kahn et al., 1990; Minning et al., 2009). While
studies suggest that individual trypomastigote populations are heteroge-
neous with respect to their expression of surface glycoproteins
(Bartholomeu et al., 2009; Pereira et al., 1996), the lack of specific tools
needed to explore co-expression of surface antigens at the level of individ-
ual parasites is presently limiting. Further, how differential expression of
surface glycoprotein genes is achieved during the long course of T. cruzi
infection in the vertebrate host (Weston et al., 1999) is not known, but it is
predicted that some level of switching occurs between family members to
expose new surface protein variants to the host immune system, as seen
with other protozoan parasites (Dzikowski and Deitsch, 2009; Morrison,
2009; Ropolo and Touz, 2010). While superfamilies such as the mucins,
mucin-associated proteins (MASPs), and smaller families such as dis-
persed gene family-1 (DGF-1) proteins and gp63s (El-Sayed et al., 2005)
are likely important in early host interactions, less is known about their
involvement in mediating host cell attachment and invasion and will not be
covered in detail here. With respect to their role in host recognition,
signalling and invasion, the best-characterized surface antigens are mem-
bers of the polymorphic gp85/TS superfamily that are expressed by mam-
malian-infective stages of T. cruzi including bloodstream and tissue
culture-derived trypomastigotes, metacyclic trypomastigotes and amasti-
gotes (Alves and Colli, 2007). The Tc-85 subfamily is specifically expressed
in bloodstream/tissue culture trypomastigotes, whereas the gp82 and
gp90 proteins are specific to metacyclic trypomastigotes. Despite the ques-
tions that remain regarding the regulation of surface antigen expression,
fundamental mechanistic insights into the host recognition and cell attach-
ment process, as mediated by members of the gp85/TS superfamily, have
recently been achieved (Alves and Colli, 2008; de Melo-Jorge and
PereiraPerrin, 2007; Scharfstein and Lima, 2008; Yoshida and Cortez,
2008) as outlined briefly below.
2.2.3. Binding host cell extracellular matrix

T. cruzi trypomastigotes are highly specialized stages of the parasite life cycle
that are capable of disseminating infection in the host. They circulate in
blood and are able to infect a range of tissues during the acute stage of
infection (Buckner et al., 1999). Not surprisingly, the ability to bind to and
exploit components of the extracellular matrix (ECM) appears to be an
important feature in the establishment of T. cruzi infection in the host. The
ability of T. cruzi trypomastigotes to bind to host ECM components is a
property of at least some of the members of the gp85/TS superfamily,
which have been shown to bind to laminin, collagen, fibronectin and heparin
sulphate proteoglycans (Calvet et al., 2004; Giordano et al., 1994, 1999;
Ouaissi et al., 1984; Ulrich et al., 2002). Some members of the g85 family
contain an RGD (arginine-glycine-aspartic acid) tripeptide, a well-charac-
terized binding motif that is recognized by integrins (Alves and Colli, 2008).
Members of the less studied T. cruzi DGF-1 superfamily also contain RGD
sequences, as well as integrin-like domains and putative carbohydrate-
binding sequences (Kawashita et al., 2009), indicating a role for DGF-1
family members in attachment to host ECM components and/or to other
host cell components as well. RNA aptamers that bind to the surface of T.
cruzi trypomastigotes were generated and selected for their displacement by
specific ECM components (Ulrich et al., 2002). Using these highly specific
tools, it was demonstrated that laminin-, fibronectin-, heparan sulphate- and
thrombospondin-displaceable aptamers used individually were able to
inhibit T. cruzi invasion of mammalian host cells (Ulrich et al., 2002). When
the laminin- and fibronectin-displaceable aptamers were combined, inva-
sion was further compromised suggesting that multiple interactions
between T. cruzi trypomastigotes and ECM components mediate stable
binding to the host cell and invasion (Ulrich et al., 2002).
2.2.4. Role of gp85/TS in tissue-specific homing

The FLY domain (VTVXNVFLYNR) is a conserved cell adhesive domain
of gp85 family members that is located in the C-terminal region of the
protein (Cross and Takle, 1993) and distinct from the laminin-binding
region of the molecule (Magdesian et al., 2001). A peptide containing the
FLY domain binds to the surface of epithelial cells in a saturable manner
and affinity isolation experiments recovered a 45-kDa host cell surface
protein identified as cytokeratin-18 (CK-18) by mass spectrometry which

was demonstrated to bind FLY specifically (Magdesian et al., 2001).
Pretreatment of epithelial cells with antibodies to CK-18 inhibited
T. cruzi invasion suggesting that the FLY-dependent binding of T. cruzi
to host cell surface CK-18 is an important mediator of invasion
(Magdesian et al., 2001). In subsequent studies, it was shown that
treatment of cells with FLY enhanced T. cruzi entry in a MAP kinase-
dependent fashion (Magdesian et al., 2007). Bacteriophage, engineered to
display the T. cruzi FLY peptide, was used to probe the potential for FLY
to mediate binding to endothelial cells originating from different mouse
tissues. In cell culture, the highest level of FLY-phage binding occurred to
isolated heart endothelial cells and when injected intravenously into mice,
FLY-phage was highly enriched in the vascular beds of the heart (Tonelli
et al., 2010). Homing of FLY-phage to the vasculature of the bladder and
oesophagus was also observed in this study, where differential homing to
tissues correlated with tissue-specific surface expression of cytokeratins
and the intermediate filament protein, vimentin (Tonelli et al., 2010).
Collectively, these studies suggest that the conserved FLY domain of
gp85/TS family members mediates binding to host cells via surface-
exposed intermediate filament proteins. Given the varying expression
pattern of cytokeratins in different cells and tissues, the property of
gp85 FLY domain to bind different intermediate filament proteins may
permit access of T. cruzi to a broader range of hosts and host cell types.
Moreover, given that intracellular amastigotes also express members of
the gp85/TS superfamily (Santos et al., 1997), it is tempting to speculate
that cytosolically localized amastigotes might also bind to host cell inter-
mediate filament networks via FLY. Interestingly, knockdown of CK-18
expression in HeLa cells was shown to exert a negative impact on intra-
cellular growth of T. cruzi amastigotes, with no effect on parasite invasion
(Claser et al., 2008). While the mechanistic basis of this observation has
not been determined, it suggests that host intermediate filaments may
provide structural or signalling platforms needed to support intracellular
T. cruzi growth. While there is clearly much to discover regarding the
molecular interactions occurring at the T. cruzi–host interface, the studies
outlined above provide evidence for a conserved region of members of
the large polymorphic gene family, gp85/TS in mediating attachment to a
number of host cell types via interactions with cytokeratins, which may
provide the first molecular explanation for the observed tissue tropism
exhibited by T. cruzi.
Another area of research centers on the ability of TS family members to
specifically bind and activate neurotrophin receptors TrkA and TrKC,
found primarily on neuronal cells, to facilitate parasite entry (de Melo-
Jorge and PereiraPerrin, 2007) as well as neuroprotection (Chuenkova and
PereiraPerrin, 2004, 2009; Weinkauf and Pereiraperrin, 2009) of the host
cell. At least some neuroprotective abilities lie within a specific 21 amino

acid region of the molecule called Y21 (Chuenkova and PereiraPerrin,
2009). It would be interesting to determine if similarly, the binding/inva-
sion phenotype is also related to Y21 region or other domains within TS.
2.2.5. Role of gp82/gp90 in signal transduction and regulation

of oral transmission route by metacyclic trypomastigotes
By exploiting inherent differences in infectivity of different T. cruzi strains,
the studies carried out primarily by Yoshida and colleagues have provided a
comprehensive view of the molecular basis for metacyclic trypomastigote
invasion of host cells (Staquicini et al., 2010; Yoshida and Cortez, 2008).
Metacyclic trypomastigotes from the CL Brener strain of T. cruzi exhibit a
greater capacity to invade cells than those from the G strain, where their
differential invasion capacity correlates with the differential expression of
several key surface glycoproteins. Gp82, a developmentally regulated mem-
ber of the gp85/TS superfamily, has a clear role in metacyclic trypomastigote
invasion where it is involved in transducing signals in both the parasite and
host cell to facilitate the invasion process (Dorta et al., 1995; Ramirez et al.,
1993). Although differences in invasive capacity among T. cruzi strains do
not correlate with differences in the levels of gp82 expression, early studies
using monoclonal antibodies revealed that the highly invasive CL strain
expresses low levels of the mucin-like protein, gp35/50 and negligible gp90,
whereas the poorly invasive G strain parasites express a relatively high
amount of gp35/50 and high levels of gp90 (Ruiz et al., 1998). This inverse
correlation between infectivity and expression of gp90 and gp35/50 was
upheld in several different T. cruzi strains. Knockdown of gp90 expression
using antisense oligonucleotides resulted in reduced expression of gp90 at
the parasite surface with a concomitant increase in parasite infectivity
(Malaga and Yoshida, 2001). Thus, it was proposed that expression of gp90
impairs the ability of gp82 to bind host cells which would negatively impact
invasion (Yoshida, 2006, 2009). More recent studies have substantiated the
regulatory role for gp90 in establishment of metacyclic trypomastigote
infection by the oral route (Yoshida, 2009). Different gp90 isoforms exhibit
different susceptibilities to pepsin, a major proteolytic enzyme in the stom-
ach, and parasites expressing pepsin-sensitive gp90, including an isolate
responsible for a recent outbreak of orally transmitted Chagas disease,
become highly invasive upon contact with gastric juice (Staquicini et al.,
2010; Yoshida, 2009). As selective binding of gp82 to gastric mucin has also
been demonstrated, the emerging model for oral transmission of T. cruzi
suggests that pepsin-sensitive gp90, which shields gp82 and prevents its
interaction with the host cell surface, is cleaved by pepsin in the gastric juice,
thereby liberating gp82 which binds to gastric mucin to initiate invasion of
the stomach mucosal epithelium (Staquicini et al., 2010).
In summary, studies of gp85/TS family members have provided

important insights towards our understanding of the role of this super-
family in mediating early host recognition and signalling. The ability of
some family members to negatively regulate host cell recognition by other
family members, as exemplified by the gp90–gp82 relationship (Yoshida,
2009), provides a glimpse into the potential for more sophisticated
mechanisms of regulating early trypomastigote–host cell interactions.
Given that T. cruzi trypomastigotes express multiple members of several
polymorphic surface glycoprotein families simultaneously, it will be
imperative to develop more specific tools for future analysis of expression
patterns in individual parasites as related to their behaviours with regard
to host selection, tissue tropism and invasion.
2.3. TO THE LYSOSOME . . . AND BEYOND
2.3.1. Establishment of cytosolic residence is critical for

Trypanosoma cruzi survival and replication
It is well established that host cell lysosomes are exploited by T. cruzi as
the gateway to the host cell cytoplasm, where the intracellular replicative
cycle of T. cruzi takes place. This general pathway was first pieced
together from a series of observations beginning with ultrastructural
studies conducted in the 1970s and 1980s that were instrumental in
defining the first steps of the intracellular life cycle of T. cruzi in mamma-
lian host cells. Electron micrographs revealed that shortly after invasion,
T. cruzi trypomastigotes are housed within tight-fitting, membrane-
bound vacuoles that fuse with host cell lysosomes (de Carvalho and de
Souza, 1989; de Meirelles Mde et al., 1987; Nogueira and Cohn, 1976;
Tanowitz et al., 1975). As intracellular infection progresses, trypomasti-
gotes differentiate into amastigotes which divide in the host cell
cytoplasm (Ley et al., 1990; Nogueira and Cohn, 1976). Evidence for
disruption of the parasitophorous vacuole membrane prior to cytosolic
localization of the parasite was also evident in thin sections suggesting
that the parasite could escape the vacuole (Nogueira and Cohn, 1976), but
it was not until the work of Andrews and Ley (Ley et al., 1990) that a
critical piece of the puzzle was solved. They demonstrated that the para-
sitophorous vacuole was rapidly acidified and that acidification was
essential for vacuole membrane disruption and release of parasites into
the cytoplasm (Ley et al., 1990). These observations coincided with the
discovery of a secreted T. cruzi lytic activity (TC-TOX) that is released into
the lumen of the parasitophorous vacuole where it is optimally active at
low pH (Andrews and Whitlow, 1989). It is now generally accepted that
failure of internalized parasites to traffic to lysosomes or failure to acidify
this compartment once the parasite enters results in poor T. cruzi infectiv-
ity (Andrade and Andrews, 2005; Andrews et al., 1990; Woolsey and
Burleigh, 2004). Studies over the past decade or so suggest that T. cruzi
trypomastigotes can access the host cell lysosomal compartment by
several different routes. In the following sections, we outline the
studies that have led to this hypothesis as well as current knowledge of
the molecular basis for vacuole egress and cytosolic localization of
the parasites.
2.3.2. Pathways to the lysosome

Early ultrastructural studies provided evidence that the T. cruzi vacuole
could fuse with lysosomes in both phagocytic and non-professional phago-
cytic host cell types (Nogueira and Cohn, 1976). However, contrasting the
fate of many microbes that find themselves within phagolysosomes, T. cruzi
could clearly withstand the harsh conditions of the lysosomes and eventu-
ally break free of this compartment and establish residence in the host cell
cytoplasm. As the mechanism of trypomastigote entry into non-professional
phagocytes was emerging as clearly distinct from phagocytosis (Schenkman
et al., 1991), one of the obvious questions that arose from these early observa-
tions was the mechanism by which T. cruzi could gain access to host cell
lysosomes in non-professional phagocytic cells. Below we describe three
cellular pathways that are exploited by T. cruzi trypomastigotes to gain
access to host cell lysosomes (Fig. 2.2).
2.3.2.1. The lysosome exocytosis pathway

The seminal work of Tardieux et al. (1992) launched the novel concept
that the T. cruzi vacuole is generated by the localized fusion of host cell
lysosomes with the plasma membrane at the parasite attachment site
(Fig. 2.2, Pathway 1). Perturbations that result in a reduction of peripheral
lysosomes or that inhibit the ability of lysosomes to move or fuse with the
plasma membrane in the host cell result in significantly less cell invasion
by T. cruzi (Rodriguez et al., 1996; Tardieux et al., 1992). Live cell imaging
studies demonstrated the directional movement of peripheral lysosomes
towards the plasma membrane at the parasite attachment site, where
clustering and apparent fusion occurred over the course of several
minutes as the trypomastigote entered the cell (Rodriguez et al., 1996).
Collectively, these findings were quite remarkable as they suggested that
T. cruzi had solved the dilemma of trafficking to the host cell lysosome in
non-professional phagocytic cells by targeting this compartment directly.
Moreover, these studies provided an important clue towards the discov-
ery of an unrecognized property of lysosomes to undergo regulated
exocytosis (Andrews, 2002).
1. Exocytosis 2. Endocytosis
Ca2+-dependent
exocytosis
Kinesin-based
movement on
Rab5
microtubules
Early
endosomes
EEA1
?
Autophagosome Lysosome
targeting Late
endosomes
Lysosome
3. Autophagy
Trans golgi network
vesicles
Golgi
Endoplasmic
N
reticulum
FIGURE 2.2 Pathways for host cell invasion by T. cruzi. En route to the host cell
lysosomal compartment in non-professional phagocytic cells, T. cruzi trypomastigotes
can engage three major cellular pathways. Pathway 1 involves recruitment and targeted
exocytosis of host cell lysosomes with the host plasma membrane at the parasite
attachment site. Kinesin motors propel lysosomes along microtubules to the host
plasma membrane where Ca2þ-dependent fusion takes place. Exploiting this pathway,
trypomastigotes bypass other cellular pathways to directly target lysosomes. Pathway 2
involves formation of a plasma membrane-derived vacuole around T. cruzi that fuses
with early endosomes prior to fusion with lysosomes. Pathway 3 involves both direct
targeting of autophagosomes, which are derived from lysosome fusion with early
autophagic compartments, to the nascent parasite vacuole and/or fusion with T. cruzi
vacuoles formed following entry via the other routes. The contribution of the autop-
hagic route is enhanced following nutrient starvation of cells.
The ability of lysosomes to respond rapidly to elevated intracellular

Ca2þ levels and to undergo regulated fusion with the plasma membrane
was later shown to be an important plasma membrane repair pathway
regulated by intracellular calcium levels (Reddy et al., 2001). A key
regulator of this process is a ubiquitously expressed lysosomal membrane

protein, synaptotagmin VIII (SytVII) which functions as a Ca2þ-sensing
protein and mediator of regulated lysosome–plasma membrane fusion
(Martinez et al., 2000). Introduction of antibodies to one of the
Ca2þ-binding domains of SytVII into the cytosol of mammalian host
cells results in almost complete abolition of lysosome exocytosis in NRK
cells (Martinez et al., 2000) and 50% reduction of T. cruzi invasion in
CHO cells (Caler et al., 2001). Consistent with these earlier findings,
embryonic fibroblasts from SytVII-deficient mice exhibited defective
lysosomal exocytosis and plasma membrane repair in response to wound-
ing, and were less susceptible to T. cruzi infection ( 50% that of cells from
WT mice; Chakrabarti et al., 2003). Although no studies were reported in
which the course of T. cruzi infection in SytVII knockout mice was
examined, the fact that these mice demonstrate the presence of inflamma-
tory infiltrate in skeletal muscle suggests a level of immune dysregulation
(Chakrabarti et al., 2003) that could complicate the interpretation of
experimental T. cruzi infection.
2.3.2.1.1. Role of Ca2þ signalling in lysosome exocytosis and Trypanosoma

cruzi invasion Consistent with the need for elevated intracellu-
lar calcium levels to promote lysosome–plasma membrane fusion, T. cruzi
trypomastigotes activate cellular signalling pathways that result in the
mobilization of intracellular Ca2þ stores in a phospholipase C and inositol
1,4,5-trisphosphate (IP3)-dependent manner (Rodriguez et al., 1995). Ca2þ
transients triggered in mammalian cells by tissue culture-derived trypo-
mastigotes (Scharfstein et al., 2000; Tardieux et al., 1994) or metacyclic
trypomastigotes (Dorta et al., 1995) are required for efficient invasion by
T. cruzi. In the context of the lysosome recruitment model of T. cruzi
invasion, the ability of live trypomastigotes to trigger repetitive [Ca2þ]i-
transients in mammalian host cells (Scharfstein et al., 2000; Tardieux et al.,
1994) in a localized fashion (Caler et al., 2000) is consistent with the
gradual and localized Ca2þ-dependent fusion of host cell lysosomes at
the parasite attachment site. In addition to Ca2þ signalling, trypomasti-
gotes induce elevated cAMP levels in host cells, which correlate with
enhanced lysosome exocytosis and T. cruzi invasion (Rodriguez et al.,
1999). Similar to tissue culture-derived trypomastigotes, metacyclic try-
pomastigotes trigger the rapid mobilization of intracellular Ca2þ stores in
both the host cell and parasite, where bidirectional signalling is critical for
efficient invasion (Dorta et al., 1995; Moreno et al., 1994; Tardieux et al.,
1994). Although the host cell signalling pathways activated by metacyclic
trypomastigotes are not as well established as those for tissue culture
trypomastigotes, it appears that metacyclics may engage a distinct set of
signalling receptors to mobilize intracellular Ca2þ stores in mammalian
cells (Dorta et al., 1995).
T. cruzi binding and internalization activates various host cell recep-

tors and signalling pathways (Maganto-Garcia et al., 2008; Melo-Jorge
and Pereira, 2007; Ming et al., 1995; Todorov et al., 2003). Current data
suggest that T. cruzi trypomastigotes binding to various host cell surface
receptors trigger the mobilization of intracellular Ca2þ stores where
G-protein coupled receptors (GPCRs) have been implicated in T. cruzi-
triggered Ca2þ responses and invasion (Caler et al., 2000; Leite et al.,
1998; Scharfstein et al., 2000; Tardieux et al., 1994). The inhibition of
trypomastigote-induced Ca2þ transients and invasion of fibroblasts by
pertussis toxin implicate the involvement of GaI/o-coupled receptor(s) in
parasite-triggered signalling pathways in fibroblasts (Caler et al., 2000;
Tardieux et al., 1994). In contrast, bradykinin B2 receptors regulate para-
site uptake into human umbilical vein endothelial cells (HUVEC) and
B2R-expressing CHO cells (Scharfstein et al., 2000) where signalling
through GaI/o does not occur in transfected CHO cells (Taketo et al.,
1996). Recently, evidence for the involvement of host cell microdomains
in the T. cruzi invasion process was reported (Fernandes et al., 2007)
consistent with the idea that GPCRs and their associated signalling
components are clustered into lipid rafts (Insel et al., 2005). The para-
site-derived activities that stimulate host cell GPCR have been partially
described, where roles for two different peptidolytic enzymes have been
implicated. T. cruzi oligopeptidase B (OPB) is a cytosolic serine endopep-
tidase that was identified in parasite lysates for its indirect role in trig-
gering Ca2þ transients in mammalian cells (Burleigh and Andrews, 1995;
Burleigh et al., 1997) presumably via the generation of a peptide ligand
that binds a host cell GPCR (Leite et al., 1998). Although the phenotype of
the T. cruzi OBP/ mutants is consistent with the proposed mechanism
of action of OBP in host cell Ca2þ signalling (Caler et al., 1998), an OBP-
generated peptide agonist was never identified. Interestingly, the pro-
posed mechanism of action of OBP is similar to that demonstrated for
cruzipain, a secreted T. cruzi cysteine protease that enhances host cell
invasion by generating short-lived kinins via the proteolytic cleavage of
high-molecular-weight kininogen which engage bradykinin receptors
(Scharfstein et al., 2000) (reviewed in this issue). In summary, parasite-
induced Ca2þ transients are critical to facilitate trypomastigote entry into
a variety of mammalian cell types and for inducing Ca2þ-regulated
lysosome exocytosis. As treatments that abolish lysosome exocytosis
result in a significant, but partial reduction of parasite invasion, para-
site-elicited Ca2þ signalling is predicted to have additional roles in host
cell invasion, such as actin remodelling (Rodriguez et al., 1995). While
additional studies are needed to identify the range of signalling receptors
engaged by T. cruzi trypomastigotes to facilitate invasion, current data
suggest that these parasites have evolved mechanisms to engage differ-
ent host cell receptors that signal through GPCR to elicit Ca2þ responses.
It was recently reported that the T. cruzi can utilize the low-density
lipoprotein receptor (LDLr) in its invasion and for the subsequent fusion
of the parasitophorous vacuole with host lysosomes (Nagajyothi et al.,
2011). Endocytosis of LDLr in association with calcium mobilization, its
subsequent trafficking to lysosomes and the release of ligands at low pH
are processes reminiscent of those involved in T. cruzi invasion.
Nagajyothi et al. (2011) demonstrated that T. cruzi directly binds to
LDLr, and inhibition or disruption of LDLr significantly decreases para-
site entry. Additionally, immunofluorescence analysis demonstrated an
association of phosphotidylinositol phosphates to LDLr cross-linked
parasites in clathrin-coated pits, which may initiate a signalling cascade
that results in the recruitment of lysosomes, possibly via the sorting motif
in the cytoplasmic tail of LDLr, to the site of adhesion/invasion. The
results were supported by the earlier reports that demonstrated that
parasite invasion was perturbed in dynasore-treated cells (Barrias et al.,
2010) and that there was increased rate of invasion in the presence of
lipoproteins (Prioli et al., 1990). Studies of infected CD1 mice demon-
strated that LDLr expression is upregulated in infected mice hearts and
that both LDL and LDLr were associated with amastigotes (pseudocysts)
in the heart tissue of infected mice. The accumulation of LDL and LDLr in
the heart is likely a contributing factor in the pathogenesis of chagasic
heart disease (Nagajyothi et al., 2011).
2.3.2.2. Plasma membrane invagination/endocytic pathway

The idea that T. cruzi had neatly solved the problem of access to the host
cell lysosomal compartment by exploiting a ubiquitous plasma
membrane repair pathway in mammalian cells is highly compelling and
elegant. As outlined above, there is a substantial amount of data to
support the lysosome exocytosis pathway as the mechanism of T. cruzi
invasion. It turns out, however, that T. cruzi trypomastigotes also use a
less elegant pathway to gain access to mammalian host cells, which does
not rely on direct lysosome–plasma membrane fusion (Burleigh, 2005;
Fig. 2.2, Pathway 2). This pathway, originally termed the ‘lysosome-inde-
pendent’ pathway, involves initial entry into a plasma membrane-derived
vacuole that subsequently fuses with early endosomes and lysosomes
(Woolsey et al., 2003). An ‘alternate’ T. cruzi invasion pathway was first
recognized when quantitative studies were carried out to determine the
impact of small molecule inhibitors on parasite internalization and
lysosome association. It was noted that at early time points of infection
(10–20 min), only a fraction of parasite vacuoles ( 20–30%) contained
markers for host lysosomes under control conditions, but that lysosomal
marker accumulation increased over time, with kinetics similar to that
for latex bead phagosomes (Woolsey et al., 2003). This pattern was
observed in several different mammalian cell types, including primary
cardiomyocytes (Woolsey et al., 2003), indicating that T. cruzi

trypomastigotes exploit both lysosome-dependent and -independent
entry pathways to infect a variety of cultured mammalian cells types.
While this lag in the accumulation of lysosome markers by the T. cruzi
vacuole had been noted in a prior study (Caler et al., 2000), the link to an
alternate invasion route was not made.
Several lines of evidence support the idea that T. cruzi trypomastigotes
can successfully invade cells via a route that is not initiated by lysosome
exocytosis. Infection of cells expressing plasma membrane-targeted GFP
constructs clearly reveals intimate contact between trypomastigotes and
the host cell plasma membrane as parasites penetrate host cells (Woolsey
et al., 2003). Time course studies revealed that 50% of recently inter-
nalized trypomastigotes (15-min time point) were housed within vacuoles
that were positive for plasma membrane–GFP markers. Co-staining these
cells with antibodies for LAMP-1 revealed the lack of association of
lysosomal markers with the plasma membrane-derived vacuoles, but
that 20–30% of recently formed parasite vacuoles stained were LAMP-
1 positive. Further, immunostaining of early endosomes with an antibody
to endogenous EEA1 or by transfection of the FYVE-domain-GFP (which
binds phosphatidylinositol 3-phosphate formed on early endosomes or
autophagosomes) clearly revealed that 20–30% of parasite vacuoles
were enriched in markers for early endosomes (Woolsey et al., 2003).
Both plasma membrane and early endosome association with the
T. cruzi vacuole were transient and maximal in the early course of infec-
tion. As the gradual loss of EEA1 from the parasitophorous vacuole was
observed, an increasing proportion of vacuoles became positive for
LAMP1 (or lysosomally targeted fluorescent dextran) suggestive of a
maturation process. However, given that only 20–30% of early T. cruzi
vacuoles stained with early endosomal markers, whereas 80% of latex
bead phagosomes were EEA1 positive early on, also suggests that the
process of T. cruzi vacuole maturation may be fundamentally different to
that of phagosomes. Either the transition between early endosomes and
lysosomes is more rapid for T. cruzi vacuoles or a proportion of plasma
membrane (EEA1-negative) vacuoles fuse directly with lysosomes. While
these details have yet to be resolved, the study of Woolsey et al. (2003) was
the first to clearly show that invading T. cruzi trypomastigotes can take an
indirect route to the host cell lysosomal compartment, which does not rely
on lysosome exocytosis at the host cell plasma membrane.
2.3.2.3. Autophagy
Autophagy is a conserved catabolic process that operates in the cytoplasm
of eukaryotic cells to degrade excess or damaged cellular organelles and
proteins via the formation of autophagosomes that fuse with lysosomes
(Yang and Klionsky, 2010). The hallmarks of early autophagosome
formation are the required recruitment of Atg12–Atg5 complex to the

initiation membrane and the generation and recruitment of the membrane
form of Atg8 or LC3-II which is conjugated to phosphatidylethanolamine
and can therefore be distinguished from the unconjugated cytosolic form
of LC3 (Tanida, 2011). Autophagosome formation can be readily
visualized in cells expressing LC3-GFP where starvation or rapamycin
treatment of cells results in characteristic LC3-GFP-positive autophagic
bodies (Tanida, 2011). T. cruzi invasion of CHO cells stably expressing
LC3-GFP demonstrated that autophagosomes associate with 30% of the
parasite-containing vacuoles following a 1-h infection and that LC3–GFP
association is maintained until parasites egress from the vacuole (Romano
et al., 2009). Starvation-induced activation of the autophagic pathway
enhances parasite uptake into cells in a manner that is inhibited by the
pan-PI-3 kinase inhibitor, wortmannin, as well as the class III PI-3 kinase
selective inhibitor, 3-MA (Romano et al., 2009). The role of autophagy in
the T. cruzi infection process is supported by observations of reduced
infection of Atg5/ or beclin-1 knockdown cells as compared to controls
(Romano et al., 2009). Time-lapse imaging carried out in this study
suggests that parasites may associate with LC3-GFP-labelled autophago-
somes during the entry process (Romano et al., 2009); however, this is
difficult to see clearly in the movies. While additional studies are needed
to understand the role of autophagy in T. cruzi invasion process, and how
this pathway integrates with other lysosome-targeting routes, this study
(Romano et al., 2009) is the first to provide evidence that autophagosomes
play a role in the early establishment of T. cruzi infection in mammalian
cells (Fig. 2.2, Pathway 3).
2.3.3. Role of host cell phosphatidylinositol-3-kinases in

Trypanosoma cruzi invasion
It has been known for a number of years that host cell phosphatidylino-
sitol-3-kinases (PI3 kinases) play a role in the invasion of non-professional
phagocytic cells by T. cruzi trypomastigotes (Chuenkova and
PereiraPerrin, 2004; Wilkowsky et al., 2001; Woolsey et al., 2003). Using
fluorescent probes such as the pleckstrin homology (PH) domain of Akt
coupled to GFP (Akt-PH-GFP), it was demonstrated that class I PI3
kinases are activated at the host cell plasma membrane by T. cruzi
trypomastigotes prior to, and during, the invasion process (Woolsey
et al., 2003). Pretreatment of cells with wortmannin (which inhibits both
class I and class III PI3 kinases) inhibits T. cruzi entry of cells (50–60%
reduction) and blocks Akt-PH-GFP association with the host plasma
membrane at the parasite entry site (Woolsey et al., 2003). Although the
recruitment of Akt-PH-GFP to the host cell plasma membrane and
nascent parasite vacuole clearly marked the plasma membrane
invagination pathway of invasion which is distinct from the lysosome

exocytosis pathway (Fig. 2.2), wortmannin pretreatment of cells
completely abolishes early lysosome association with the nascent
T. cruzi vacuole (Woolsey et al., 2003), suggesting a role for PI3 kinase
signalling (via class I or class III) in regulating the lysosome exocytosis
pathway. Interestingly, infection of mouse embryonic fibroblasts lacking
the main regulatory subunits that activate the class I PI3 kinases (p85a/
b/) showed a significant reduction in overall invasion by T. cruzi as
compared to WT control cells, whereas the relative proportion of early
lysosome-positive parasite vacuoles (indicative of the lysosome-depen-
dent entry pathway) was unchanged in the p85a/b/cells (Woolsey
et al., 2003). Thus, dampening of class I PI3 kinase signalling appears to
impact the lysosome-dependent (Fig. 2.2, Pathway 1) and lysosome-
independent (Fig. 2.2, Pathway 2) invasion pathways to a similar degree,
suggesting the loss of a more general early requirement to initiate these
pathways. Further, these findings suggest that the exquisite sensitivity
of the lysosome-dependent invasion pathway to wortmannin may be due
to inhibition of the class III PI3 kinase (VPS34) which also functions in
early steps of endosome fusion and autophagosome formation (Simonsen
and Tooze, 2009).
2.4. DISRUPTION OF THE PARASITOPHOROUS VACUOLE

MEMBRANE AND CYTOSOLIC LOCALIZATION OF
PARASITES
Targeting of T. cruzi to the lysosomal compartment of mammalian host

cells and the disruption of the parasitophorous vacuole membrane is a
critical step in establishment of intracellular infection. Before any
molecular identification had been made, it was hypothesized that
T. cruzi might produce a pore-forming molecule that could insert into
the vacuole membrane and initiate its disruption. In 1989, Andrews and
Whitlow demonstrated that T. cruzi trypomastigotes and amastigotes
secrete/release a hemolysin that is optimally active at pH 5.5, an excellent
candidate for the putative vacuole-lysing activity (Andrews and Whitlow,
1989) as it was known that blocking acidification of the parasite vacuole
prevented cytosolic localization by T. cruzi (Ley et al., 1990). The T. cruzi
hemolysin, named TC-TOX, was enriched by liquid column chromatog-
raphy where it co-fractionated with a 60- to 75-kDa protein that cross-
reacted with antibodies to complement component C9 (Andrews et al.,
1990). Antibodies raised to TC-TOX labelled the parasite Golgi mem-
branes and the flagellar pocket as well as the phagosome membrane in
infected macrophages as determined by cryoimmunoelectron microscopy
(Andrews et al., 1990). Using anti-C9 antibodies to screen a T. cruzi
amastigote expression library, a single copy gene, LYT1, was identified

where recombinant LYT1 exhibited hemolytic activity (Manning-Cela
et al., 2001). LYT1 null mutants display an accelerated differentiation
phenotype in vitro, reduced hemolytic activity and reduced infectivity
suggesting that LYT1 plays an important role in the establishment of
T. cruzi infection (Manning-Cela et al., 2001). Two isoforms of the LYT1
protein have been identified that arise from alternative trans-splicing of
the LYT1 transcript (Manning-Cela et al., 2002). The products, termed
mLYT1 and kLYT1, exhibit differential localization in the parasite
(Benabdellah et al., 2007). The plasma membrane-targeted protein
mLYT1 is responsible for the hemolytic activity, and expression of this
isoform on the LYT1/ background rescues the reduced infectivity phe-
notype, whereas the kinetoplast-localized kLYT1 isoform is associated
with the differentiation phenotype but not infection (Benabdellah et al.,
2007). Collectively, these studies provide strong evidence for the role of
the membrane-targeted hemolytic protein, mLYT1 in the T. cruzi infection
process; however, its direct role in vacuole disruption during the estab-
lishment of intracellular infection has not been demonstrated.
In addition to the action of a T. cruzi hemolysin on the parasitophorous
vacuole membrane, there is evidence for the participation of the parasite
surface-expressed neurominidase/trans-sialidase (TS) enzymes in facil-
itating the process of vacuole disruption (Hall et al., 1992). The luminal
face of the lysosomal membrane is a dense glycocalyx, rich in sialic acid
containing proteins such as the lysosome-associated membrane proteins
(LAMPs; Kornfeld and Mellman, 1989). T. cruzi TS is shed from the
parasite surface, is active at low pH and can desialylate isolated LAMP
molecules (Hall et al., 1992). Therefore, TS has all the characteristics to
function in the parasitophorous vacuole and may target LAMPs for
desialylation in this setting. Consistent with this idea, over-expression
of TS in metacyclics (which normally express relatively low levels of TS)
enhances parasite escape from the vacuole (Rubin-de-Celis et al., 2006).
Similarly, parasites escape the parasitophorous vacuole more rapidly in
sialic acid-deficient or LAMP-deficient cells (Albertti et al., 2010; Hall
et al., 1992). Further, surface expression of LAMP-1 on host cells enhances
T. cruzi invasion (Kima et al., 2000) and the lack of sialic acid-containing
glycoproteins in cells results in reduced levels of T. cruzi invasion (Ming
et al., 1993). Thus, while host sialic acids and T. cruzi TS have been
implicated in several aspects of the T. cruzi invasion process (Pereira-
Chioccola et al., 2000), the proposed function of TS in vacuole egress is to
facilitate access of TC-TOX/LYT1 to the lysosome membrane by the
desialylation of the lysosomal membrane and parasite release into the
host cell cytosol (Andrade and Andrews, 2004; Andrews, 2002; Andrews
et al., 1990; Manning-Cela et al., 2001). Given that all of the tools are
currently available, this model should be straightforward to test.
2.5. THE ROLE OF THE HOST CELL CYTOSKELETON IN

TRYPANOSOMA CRUZI TRYPOMASTIGOTE INVASION
OF NON-PHAGOCYTIC CELLS
2.5.1. Actin
Early studies addressing the mechanism of T. cruzi trypomastigote
invasion into non-professional phagocytic cells quickly revealed that
the entry process was distinct from the actin-dependent macropinocy-
totic or phagocytic processes that are engaged by a number of bacterial
or protozoan pathogens in order to induce uptake into host cells
(Schenkman et al., 1991). Rather than inhibiting T. cruzi uptake,
actin-depolymerizing drugs enhanced trypomastigote entry into
non-professional phagocytic cells, whereas similar treatments block
phagocytic uptake of latex beads or bacteria. When cells were exposed
to live trypomastigotes or lysate that trigger IP3-dependent increases in
intracellular Ca2þ levels (Burleigh and Andrews, 1995; Rodriguez et al.,
1995; Tardieux et al., 1994), rapid and transient reorganization of the
host cell actin cytoskeleton occurs. Because the [Ca2þ]i-transients that
were triggered in cells by parasites or other agonists produced a tran-
sient decrease in cortical F-actin content, these data were interpreted
within the context of the existing T. cruzi invasion model: the lysosome
recruitment model. It was postulated that the cortical actin cytoskeleton
acts as a barrier to prevent docking and fusion of lysosomes with the
plasma membrane, and that transient depolymerization of the cytoskel-
eton with each [Ca2þ]i-transient would provide greater access of lyso-
somes to the plasma membrane for fusion (Rodriguez et al., 1995;
Tardieux et al., 1992, 1994). This assumption turned out to be incorrect
as cytochalasin D pretreatment of cells completely abolished early lyso-
some association with nascent T. cruzi vacuoles while enhancing entry
via the alternate, plasma membrane invagination pathway (Woolsey
and Burleigh, 2004). The ability to uncouple trypomastigote entry of
cells from early lysosome association with the vacuole with cytochalasin
D provided a convenient way to probe the kinetics of organelle marker
accumulation by the T. cruzi vacuole during and shortly after parasite
entry. In drug treatment and washout experiments, it was noted that as
host cells recovered from cytochalasin D pretreatment, the parasite
vacuole accumulated the early endosome marker, EEA1, before the
lysosomal marker, LAMP1 (Woolsey and Burleigh, 2004). This observa-
tion provides strong experimental support for the existence of a viable
T. cruzi invasion pathway that does not immediately rely on lysosome–
plasma membrane fusion. Secondly, these experiments revealed that
actin dynamics are required for lysosome fusion with the plasma
membrane/parasite vacuole (Woolsey and Burleigh, 2004).
Given that the Rho family of GTPases are important regulators of actin
polymerization that can be targeted by bacterial effector proteins in order
to modulate the uptake of bacterial pathogens (Bulgin et al., 2010), the
activation state of the main Rho GTPases, Rac, RhoA and Cdc42, in host
cells was investigated in the context of T. cruzi infection. Exposure of
fibroblasts to T. cruzi trypomastigotes resulted in a rapid and transient
reduction in the level of GTP-bound RhoA, with no change in the
activation state of Rac1 or Cdc42, suggesting that RhoA may play a role
in parasite entry and association with the endosomal/lysosomal compart-
ment. In support of this hypothesis, it was shown that while T. cruzi
trypomastigote entry was initially greatly enhanced in CHO cells expres-
sing a dominant negative (DN)-RhoA most of the internalized parasites
failed to traffic to lysosomes and were not retained in the cells. These
experiments, therefore, were the first to introduce the concept of cellular
retention of T. cruzi following internalization (see Section 2.6).
2.5.2. Microtubules
Experimental evidence suggests that host cell microtubules play a signifi-
cant role in the T. cruzi invasion process (Rodriguez et al., 1996; Tyler
et al., 2005). First, disruption of microtubule dynamics within mammalian
host cells using a variety of microtubule/tubulin-binding drugs reduces
the overall efficiency of T. cruzi invasion of fibroblast and myoblast cell
lines (Rodriguez et al., 1996). The localized accumulation of tubulin at the
point of parasite contact with the host cell in cells stably expressing
GFP-a-tubulin, as well as the recruitment of tubulins to nascent T. cruzi
vacuoles, supports the idea that host microtubules participate in the
parasite entry process (Tyler et al., 2005). While colchicine treatment of
recently invaded cells does not prevent the recruitment of GFP-a-tubulin
to the parasite vacuole, subsequent removal of drug permits the forma-
tion of microtubules, which can be seen radiating out from the parasito-
phorous vacuole membrane (Tyler et al., 2005). Given the clear role for
endosomal/lysosomal fusion with membrane surrounding invading and
recently internalized parasites, a likely function of microtubules is the
transport of endosomes, lysosomes or autophagosomes which contribute
to vacuole biogenesis, as discussed above. Lysosomes are transported
along microtubules, moving in both anterograde and retrograde
directions guided by the action of kinesins and cytoplasmic dyneins,
respectively (Harada et al., 1998; Nakata and Hirokawa, 1995). As the
motor-based movements of lysosomes are susceptible to changes in cyto-
solic pH (Heuser, 1989), this property was exploited to demonstrate
that cells with an increase in the number of peripherally localized
lysosomes are more readily infected by T. cruzi than control cells and,
conversely, cells exhibiting perinuclear aggregation of lysosomes were
less susceptible to parasite infection (Tardieux et al., 1992). In addition,

T. cruzi entry is inhibited following microinjection of antibodies to kinesin
heavy chain, KIF5B, (Rodriguez et al., 1996), a plus-end microtubule
motor protein that is involved in lysosome transport and autophagosome
formation (Cardoso et al., 2009). Collectively, these studies reveal a critical
role for microtubule- and kinesin-based transport of host cell lysosomes
towards the biogenesis of the T. cruzi vacuole. As our view of the T. cruzi
invasion process has evolved over the past 5–10 years, it will be critical to
investigate the role of microtubules, and other host cell regulators of
T. cruzi infection in the context of the different routes the parasite takes
from the extracellular space to the host cell lysosome.
2.6. THE DISCOVERY OF REVERSIBLE INVASION
Infection of mammalian cells expressing plasma membrane-targeted GFP

constructs provided the first visual evidence that T. cruzi trypomastigotes
could reverse the cellular invasion process (Woolsey and Burleigh, 2004).
A relatively low number of reversible invasion events were noted under
control conditions; however, in cytochalasin D-treated cells, this number
increased substantially (Woolsey and Burleigh, 2004). To follow reversible
invasion in a more quantitative manner, host cells were pulsed with
parasites for several minutes and extracellular parasites were removed
by washing and infected cells were fixed at time points thereafter. Under
control conditions, the number of intracellular parasites remained fairly
constant over time; however, in the presence of cytochalasin D or in DN-
RhoA expressing cells, this number dropped dramatically over time. By
1-h post-invasion, the only parasites that are retained by cells are those
that can rapidly associate with lysosomes following invasion. Given that
actin dynamics/polymerization are required to facilitate fusion of the
nascent parasite vacuole with the endosomal/lysosomal system, these
observations suggested that even fully internalized parasites (as judged
by an outside/inside staining method) can leave a cell if fusion with early
endosomes and lysosomes fails to occur. A similar outcome was noted in
wortmannin-treated cells (Andrade and Andrews, 2004). Because wort-
mannin blocks early lysosome association with invading T. cruzi
trypomastigotes and retards parasite vacuole maturation (Andrade and
Andrews, 2004; Woolsey et al., 2003), the decreased parasite retention
observed in wortmannin-treated cells was interpreted solely within the
context of the lysosome exocytosis pathway, that is, parasites that could
not engage the lysosome exocytosis pathway and entered cells via an
alternate pathway, would abort the invasion attempt. It was postulated
that these, parasites, now extracellular, would continue to undergo failed
invasion attempts until such time as the effects of wortmannin, were
overcome (> 8-h post-treatment), at which point parasites could gain

access to cells via the lysosome exocytosis pathway (Andrade and
Andrews, 2004). However, given that wortmannin inhibits class III PI3
kinase, VPS34, which is required for endosome fusion, vacuole
maturation and autophagosome formation, all the invasion/maturation
pathways outlined in Fig. 2.2 would be affected by wortmannin. Clearly
what is needed to clarify this issue are time-lapse imaging studies of
parasite invasion under conditions of recovery from cytochalasin D or
wortmannin treatment.
2.7. CONCLUDING REMARKS
Host cell lysosomes have clearly emerged as the interim target of at least
three cellular pathways/processes that T. cruzi trypomastigotes exploit to
infect non-professional phagocytic cells. The parasite can enter lysosomes
directly by inducing Ca2þ-dependent lysosome exocytosis at the site of
trypomastigote attachment at the plasma membrane or can target this
compartment indirectly by intersecting with the endocytic or autophagic
pathways. The ability of T. cruzi to engage different organellar/vesicular
trafficking pathways within mammalian cells to attain their goal of
transient lysosome residence may increase options for host cell selection
aiding in the overall success of the pathogen in establishment of infection.
Although it is clear that the activation of cellular signalling pathways via
parasite–host cell interactions at the cell surface, our ability to integrate
particular T. cruzi-triggered signalling events with the different routes of
parasite entry is currently limited and further study is warranted. Further,
there is still much to learn regarding the role of surface-expressed glyco-
protein families and in mediating early host cell interactions. Looking
ahead, increasing access to new genome-scale technologies will permit
the application of functional genomics approaches for more rapid discov-
ery of important regulators of the T. cruzi infection process that have the
potential to be exploited as targets for drug or vaccine development.
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CHAPTER 3
Gap Junctions and Chagas
Disease
Daniel Adesse,*,† Regina Coeli Goldenberg,*
Fabio S. Fortes,‡ Jasmin,*,§ Dumitru A. Iacobas,§
Sanda Iacobas,§ Antonio Carlos Campos de
Carvalho,*,§ Maria de Narareth Meirelles,† Huan
Huang,} Milena B. Soares,k Herbert B. Tanowitz,}
Luciana Ribeiro Garzoni,† and David C. Spray§

3.2. Loss of Gap Junctions and Coupling in Rodent
Chagasic Cardiomyopathy 66
3.3. Not All Junctional Proteins are Affected by
Trypanosoma cruzi Infection 74
3.4. Microarray Experiments have Revealed Profound
Changes in Gene Expression in the Chagasic Mouse,
Both in Acute and in Chronic Disease Phases 74
3.5. Conclusions 76
References 79
Abstract Gap junction channels provide intercellular communication

between cells. In the heart, these channels coordinate impulse
propagation along the conduction system and through the con-
tractile musculature, thereby providing synchronous and optimal
* Instituto de Biofı́sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil
{
{
Colegiado de Ciencias Biologicas e da Saude (CCBS), Centro Universitario Stadual da Zona Oeste (UEZO),
Rio de Janeiro, Brazil
}
Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx,
New York, USA
}
k
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil

63
64 Daniel Adesse et al.
cardiac output. As in other arrhythmogenic cardiac diseases, cha-

gasic cardiomyopathy is associated with decreased expression of
the gap junction protein connexin43 (Cx43) and its gene. Our
studies of cardiac myocytes infected with Trypanosoma cruzi
have revealed that synchronous contraction is greatly impaired
and gap junction immunoreactivity is lost in infected cells. Such
changes are not seen for molecules forming tight junctions, another
component of the intercalated disc in cardiac myocytes. Transcrip-
tomic studies of hearts from mouse models of Chagas disease and
from acutely infected cardiac myocytes in vitro indicate profound
remodelling of gene expression patterns involving heart rhythm
determinant genes, suggesting underlying mechanisms of the func-
tional pathology. One curious feature of the altered expression of
Cx43 and its gene expression is that it is limited in both extent and
location, suggesting that the more global deterioration in cardiac
function may result in part from spread of damage signals from
more seriously compromised cells to healthier ones.
3.1. INTRODUCTION
Gap junction channels are composed of the connexin family of transmem-

brane proteins that assemble as end-to-end alignments of hexameric
connexin subunits (Fig. 3.1). These structures form intercellular conduits
that are permeable both to current-carrying ions (primarily Kþ) and to
second messenger molecules with molecular mass (Mr) < 1 kDa such as
Ca2þ, IP3 and cyclic AMP. The connexin gene family in mammals includes
more than 20 isoforms encoded by separate genes (Söhl and Willecke,
2004), and such isoforms are named according to the molecular weight (in
kDa) of the protein predicted from its cDNA (Goodenough et al., 1996);
genes encoding connexins follow a different nomenclature, where they
are divided into several subfamilies and identified according to the order
of their discovery. Gap junction channels are critical in the heart, where
they mediate synchronized rhythmic contractions and maintain cardiac
homeostasis by allowing the free diffusion of metabolites between cardiac
myocytes. Connexin43 (Cx43, encoded by the Gja1 gene in rodents and by
GJA1 in humans) is the most abundant gap junction protein in ventricular
myocytes, being localized at intercalated discs in normal myocardium
(see Duffy et al., 2006 for review). In addition, Cx40 (encoded by Gja5) and
connexin45 (Cx45, Gjc1) are found more prominently in atrium in the
working myocardium and in the conduction system, and connexin37
(Cx37, Gja4) is a major component of gap junctions between endothelial
cells in the vessel wall.
Gap Junctions and Chagas Disease 65
Gap junction protein

(connexons)
Gap junction channel
(paired connexons)
Cell 1
Membrane
Gap
Membrane
Cell 2
Amino
Terminus
Carboxyl
Terminus
FIGURE 3.1 Schematic representation of gap junction and connexin structures. Two cells
are coupled through connexon hemichannels, each composed of six subunits of con-
nexin (Cx). Gap junction channels connect two cells’ cytoplasms, allowing free exchange
of small metabolites such as Ca2þ, IP3 and cAMP, which in heart tissue are important for
maintaining synchronous contraction. Gating properties of gap junctions can be regu-
lated by Cx structure, which consists of four primarily a-helical transmembrane domains,
cytoplasmic amino and carboxyl termini and a cytoplasmic loop, all of which contain
some regions of a-helix, and extracellular loops that are primarily b-structure. A number
of proteins that bind to cardiac connexins are known, and more are certain to be
discovered, linking the connexin into an intercellular signalling complex, the Nexus.
Binding sites may either correspond to structured regions within the connexin molecules
or be unstructured, leading to presumably low-affinity and dynamic interactions.
Gap junction function and distribution within and between cells are
affected by phosphorylation state of the connexins that form them and by
other factors including intracellular pH and protein–protein interactions
(see Hervé et al., 2004; Spray et al., 2001 for reviews). In diverse cardiac
disease states, such as myocardial infarction and ischaemia, significant
remodelling of the distribution of Cx43 occurs in ventricles, resulting in
disorganization of normal microconduction pathways and arrhythmias
(Severs, 2001); similarly, altered Cx40 distribution has been associated
with atrial fibrillation (see Chaldoupi et al., 2009 for review).
Studies of alterations in cardiac myocytes during in vitro infection with
Trypanosoma cruzi indicate that the parasite is capable of impairing host
cell functioning through alterations in cell–cell communication (de
Carvalho et al., 1992). Such an effect is expected to be of particular
importance in the heart, where maintenance of synchronous contractions
requires functional gap junctions (see Duffy et al., 2006; Severs et al., 2006
for reviews). Because gap junctional communication is important in

normal cardiac conduction, and because chagasic cardiomyopathy result-
ing from infection with T. cruzi is associated with arrhythmias, a major
focus of our laboratories has been the examination of the expression and
distribution of Cx43 in widely used in vivo and in vitro models of infection.
Chagas disease has both acute and chronic stages, and in both, there
may be cardiac involvement. While myocarditis may be observed during
acute infection, chronic infection may result in arrhythmias, congestive
heart failure or thromboembolic events (see Tanowitz et al., 1992, 2009 for
review). Dilated cardiomyopathy usually occurs many years after the
initial infection. There is little tissue parasitism in the chronic stage, but
conduction pathways are damaged with resultant disturbed heart
rhythm. Mechanisms postulated by various authors to explain the
development of chronic chagasic heart disease include autoimmunity,
microvascular disturbances and autonomic nervous system derange-
ments (see Marin-Neto et al., 2007 for review). Clearly, there is evidence
for each: inflammation is present in the absence of appreciable parasite
burden; verapamil is therapeutically useful because it alleviates vasos-
pasms; and there is destruction of autonomic ganglia in chronic disease.
However, mechanisms responsible for the arrhythmogenic nature of the
disease have received little attention.
3.2. LOSS OF GAP JUNCTIONS AND COUPLING IN RODENT

CHAGASIC CARDIOMYOPATHY
In early studies, our laboratory groups examined spontaneous beating

rate of cultures of T. cruzi-infected mouse and rat cardiac myocytes,
finding opposite effects in the two murine species (Aprigliano et al.,
1993; Bergdolt et al., 1994; de Carvalho et al., 1992; see de Carvalho
et al., 1994 for review): in infected mouse cells, beat rate was higher,
whereas in infected rat myocytes, beat rate was slower and less rhythmic.
Studies also revealed that variability in interbeat interval was much
higher in infected neonatal rat cardiac myocytes (de Carvalho et al.,
1992); mouse myocytes exhibited decreased chronotropic response to
norepinephrine (Aprigliano et al., 1993), whereas rat heart cells showed
the opposite effect (Bergdolt et al., 1994). The extent to which these
differences reflect parasite strain is unknown, but the strikingly different
effects that parasite strain can cause are illustrated by a recent gene
profiling experiment performed on L6E9 myoblasts infected with four
different T. cruzi species that identified distinct transcriptomic finger-
prints caused by each parasite (Adesse et al., 2010).
Studies in both rat (de Carvalho et al., 1992; see Fig. 3.2) and mouse
cardiac myocytes following infection with Y or Tulahuan strains of
A C
B D
Tc
20 µm
FIGURE 3.2 Trypanosoma cruzi infection impairs cell–cell coupling. The micrographs
depict the pioneer experiment that tried to understand the basis of arrhythmogenesis in
T. cruzi infection. Cultured cardiac myocytes were injected with the dye lucifer yellow
(LY) that spreads to adjacent cells through gap junctions. Non-infected cells (A,B) were
capable of transmitting LY to up to six cells, whereas when the injection was done in a
highly infected cell (C,D), dye spread was abolished. Asterisks indicate the cells that were
injected. Bars ¼ 20 mm. From de Carvalho et al. (1992).
T. cruzi (Adesse et al., 2008; de Carvalho et al., 1992) demonstrated that

Cx43 immunofluorescence in infected cell pairs was substantially lower at
72 h after infection; coupling between non-parasitized cells in infected
dishes was not affected, indicating that factors secreted into the medium
are unlikely to be responsible for the decrease in Cx43 abundance by
infected myocytes. Junctional conductance and dye transfer were also
measured electrophysiologically at 72 h after infection in the rat myocytes
(de Carvalho et al., 1992), and those results indicated a strong decrease in
coupling, but only in cases where both cells were infected.
We also have examined expression of the major cardiac gap junction
protein Cx43 employing immunoblots and RNA measurement. In both
rat and mouse, Western blots of Cx43 protein and Northern blots of Gja1
mRNA indicated only slightly decreased levels in rat astrocytes and
leptomeningeal cells at up to 72 h after infection in culture (Campos de
Carvalho et al., 1998). This only slight decrease in Cx43 mRNA at later
time points in culture is consistent with recent microarray studies
described below. However, immunofluorescence has uniformly detected
strikingly less Cx43 in junctional regions of infected rat cells. Such a
dramatic decrease in Cx43 immunostaining without major change in total

Cx43 protein might indicate that an antigenic site became ‘‘cryptic’’ as a
consequence of parasitic infection (as was reported to occur between CNS
glial cells in kainate-induced excitotoxicity; Hossain et al., 1994). How-
ever, similar results were obtained using other Cx43 antibodies, and we
suggested initially that the infection altered Cx43 distribution within the
cell, presumably as a result of altered trafficking and retention in non-
junctional compartments (de Carvalho et al., 1992, 1994).
Another peculiarity with regard to Cx43 immunostaining arose in
initial studies using an antibody (181A) raised by Dr. Elliot Hertzberg
against an epitope on the carboxyl terminus of Cx43. This antibody
seemed to stain the intracellular parasite, raising the possibility that
T. cruzi might either express Cx43 or highjack it intracellularly. However,
the use of antibodies directed to different residues of the cytoplasmic
Cx43 C-terminal tail demonstrated that a T. cruzi surface protein actually
cross-reacts with certain Cx43 antibodies. As illustrated in Fig. 3.3, immu-
nogold analysis using the 181A antibody (residues 346–360) displayed
typical localization at cell–cell contacts in non-infected cardiac myocytes
(Fig. 3.3A, arrows). However, in T. cruzi-infected cells, membrane Cx43
immunolocalization was absent and there was consistent staining of the
amastigotes (Fig. 3.3B, arrowheads). Confocal microscopy showed that
using a commercially available anti-Cx43 antibody (Sigma), recognizing
an adjacent region (residues 363–382) of the C-tail, there was no staining
of the intracellular parasites (Fig. 3.3C). Highly infected cardiac cells lose
Cx43 immunoreactivity for both Sigma and 181A antibodies (Fig. 3.3C
and D, stars), but non-parasitized cells displayed normal Cx43 plaques
(Fig. 3.3C and D, arrows). Thus, staining of T. cruzi by 181A antiserum is
likely due to the recognition of a homologous protein, present in all three
life forms of the parasite (Fig. 3.3F), showing a different molecular weight
from Cx43 as compared to mouse heart lysates used as positive controls.
Staining of a 120-kDa band was observed in immunoblots using the 181A
antibody (Fig. 3.3F) but not with the Sigma antibody (Fig. 3.3E).
Our previous observations (Bergdolt et al., 1994; Garzoni et al., 2003)
and those of others (Rodriguez et al., 1995) demonstrated that intracellu-
lar calcium concentrations in the host cell were increased during initial
times of infection with trypomastigotes and that these calcium waves
could propagate in a cluster of neighbouring cells (Garzoni et al., 2003).
To re-examine the issue of whether T. cruzi invasion could modulate gap
junctional communication, we examined Cx43 expression/distribution in
mouse cardiac myocytes during initial infection with the Y strain. In
cultured mouse myocytes, the infection with the Y strain of T. cruzi had
an interesting effect on Cx43 expression. At the first hour of infection,
which corresponds to initial steps of this interaction, Cx43 protein levels
were substantially increased, whereas Cx43 transcripts were unaltered
A B
F
ER
SR
MF
C D
50 mm 50 mm
E F
E A T E A T
150 kDa 150 kDa
100 kDa 100 kDa
75 kDa 75 kDa
Cx43 Cx43
Sigma (363–382) 181A (346–360)
FIGURE 3.3 Does Trypanosoma cruzi express connexin43? The use of specific antibo-
dies directed to different residues of connexin43 C-terminal tail demonstrated that Cx43
shares a homologous residue with a T. cruzi surface protein. (A and B) Immunogold
analysis using the 181A antibody (residues 346–360) displayed typical localization at
cell–cell contacts in non-infected cardiac myocytes (A, arrows). However, in T. cruzi
infected cells, membrane Cx43 immunolocalization was absent and there was a consis-
tent staining of the amastigotes (B, arrowheads). Confocal microscopy using a com-
mercially available anti-Cx43 antibody (Sigma), recognizing residues 363–382 of the
C-tail, reveals no staining of the intracellular parasites (C). Highly infected cardiac cells
lost Cx43 immunoreactivity for both Sigma and 181A antibodies (C and D, stars), but
non-parasitized cells displayed normal Cx43 plaques (C and D, arrows). Staining of
T. cruzi by 181A antiserum is likely due to the recognition of a homologous protein,
(Fig. 3.4). These observations suggest an effect of parasite invasion on

connexin trafficking: As T. cruzi depends on lysosome recruitment for the
formation of the parasitophorous vacuole (Andrade and Andrews, 2004),
Cx43 removal from the plasma membrane could be impaired. Further
analysis at 24–72 hours post-infection (hpi) revealed a 61% decrease in
protein levels (Fig. 3.4A) and a 20% decrease in mRNA levels at the final
time point (Fig. 3.4), when cells are highly parasitized, with abundant
intracellular amastigotes and no Cx43 staining (Fig. 3.3). The changes in
Cx43 levels detected in mouse cardiac myocytes infected with the Y strain
are in contrast with previous observations on rat glial cells infected with
Tulahuan strain, which showed no significant alteration in levels of either
Cx43 or its phosphorylation state at 72 hpi (Campos de Carvalho et al.,
1998). These discrepancies could be explained by the differences in
A 1 hpi 72 hpi
Cont. T. cruzi Cont. T. cruzi
C´43 (43 kDa)
GAPDH (36 kDa)
B 1 hpi 72 hpi
Cont. T. cruzi Cont. T. cruzi
C´43 (407 bp)

18S (324 bp)
FIGURE 3.4 Connexin43 protein and mRNA expression during in vitro infection with
T. cruzi. Mouse cardiac myocytes were cultivated and infected with the Y strain of
T. cruzi. Protein analysis showed that infection induces a bidirectional effect on Cx43,
starting with a significant increase at 1 hour post-infection (hpi), followed by a normal-
ization in protein levels until 72 hpi, when there is a drop of 61% in protein levels. Semi-
quantitative RT-PCR showed no alteration on Cx43 mRNA at 1 hpi, but a significant
decrease in Cx43 transcripts at 72 h.
present in all three evolutive forms of the parasite (F), showing a different molecular
weight from Cx43 as compared to mouse heart lysates used as positive controls. Staining
of a 120-kDa band was observed in immunoblots using the 181A antibody (F) but not with
the Sigma antibody (E). (E, epimastigote; A, amastigote; T, trypomastigote; SR, sarco-
plasmic reticulum; ER, endoplasmic reticulum; F, fibroblast; P, parasite; MF, myofibril.)
parasitaemia (which was considerably lower in the glial study) or in

parasite strain pathogenicity in vivo (Caetano et al., 2010) or in vitro, as
recently demonstrated in our study that used oligonucleotide microarrays
to compare the infection of rat myoblasts with four reference strains of
T. cruzi (Adesse et al., 2010).
Thus, there seems to exist a direct relationship between host cell
parasitism and Cx43 downregulation in vitro, as demonstrated in
Figs. 3.3 and 3.4, in which highly infected cells display decreased Cx43
protein and mRNA levels. This hypothesis is further supported by a
recent study that showed recovery of infected myocytes after treatment
with amiodarone, a potent anti-arrhythmic that is commonly prescribed
for chagasic patients with severe cardiac compromise (Dubner et al.,
2008). Interestingly, this compound is also an inhibitor of sterol biosyn-
thesis in fungi (Courchesne, 2002; Courchesne et al., 2009) and trypano-
somatids (Benaim et al., 2006; Serrano-Martı́n et al., 2009). When used in
micromolar concentrations (1.6–6.5 mg/ml) in infected myocyte cultures,
amiodarone induced profound morphological alterations to amastigotes
that led to clearance of the parasitism and host cell recovery. Among the
changes observed was a marked recovery of Cx43 immunoreactivity and
spontaneous contractility rate (Adesse et al., 2011). These observations,
combined with that of minimal change in Cx43 transcripts during in vitro
infection (Fig. 3.4), reinforce the idea that the Cx43 protein downregula-
tion that follows in vitro infection results from impaired protein synthesis,
rather than altered Cx43 gene expression. Amiodarone is a promising
compound, as it has a strong bioavailability, is highly lipophilic
(Van Herendael and Dorian, 2010) and selectively eradicates T. cruzi
infection in doses much lower than what is recommended by the
American Heart Association for daily dosage in cases of atrial fibrillation
(200–400 mg/day) with no side effects (Schweizer et al., 2011).
The murine models of chagasic infection have brought important con-
tributions to the understanding of the arrhythmogenic impact of Cx43
remodelling. During acute infection with T. cruzi (11 days post-infection
with the Y strain or 30 days post-infection with the Brazil strain), Cx43
protein levels are decreased in atria and ventricles, consistent with what
was observed in the in vitro infection (Adesse et al., 2008). Such a reduction
in overall Cx43 abundance in the infected heart presumably reflects inho-
mogeneous distribution, a condition that is a prominent feature of ventric-
ular conduction disorders (see Severs, 2001 for review). The reduction in
the expression of Cx43 levels in the infected heart may be induced by the
parasite per se. However, the effects of soluble factors present in the serum
and the inflammatory response on Cx43 expression cannot be entirely
ruled out. When serum from chagasic cardiomyopathic patients was
added to cultured cardiomyocytes or isolated rabbit hearts, there was a
substantial impairment of dye spread through gap junctions, atrioventricular
conduction block and changes in heart rate (Costa et al., 2000). This
observation seems to contradict the results described above, in which
we observed substantial impairment in the coupling and Cx43 expression
of infected cells but not in non-parasitized cells in infected dishes (Adesse
et al., 2008, de Carvalho et al., 1992). The difference between these data
could be in part explained by the high concentrations of serum proin-
flammatory cytokines and chemokines found during chronic infection in
which parasite load is much reduced. It has been shown that growth
factors, such as transforming growth factor-b (TGF-b), can regulate gap
junction intercellular communication (Chandross et al., 1995; see Chanson
et al., 2005 for review). TGF-b is required for the invasion of host cells and
is produced early upon infection, and constantly throughout the acute
and chronic phases (see Araújo-Jorge et al., 2008 for review). Recently, it
was demonstrated that the addition of 2 ng/mL TGF-b in cardiomyocytes
in vitro downregulated Cx43 protein expression in non-infected myocytes,
resulting in reduced organization of gap junctions similar to the pattern
observed in infected cultures. These results were further reinforced when
the TGF-b receptor type 1 (ALK-5) was inhibited by SB-431542, which
completely reversed the effect of TGF-b and T. cruzi infection on Cx43
expression. The authors suggested that TGF-b produced in infected cul-
ture could affect both infected and non-infected cells and affect the pat-
tern of Cx43. In addition, because TGF-b regulates a diverse array of
cellular processes, including tissue development and repair (see Ramos-
Mondragón et al., 2008, Yarnold and Brotons, 2010 for reviews), the high
levels of TGF-b and consequent disorganized expression of Cx43 could
both act in synergy to promote dysrhythmias in chagasic patients
(Waghabi et al., 2009).
Confocal microscopy experiments revealed that acute infection
(30 days post-infection with the Brazil strain) induces connexin remodel-
ling with lateralization of Cx43 plaques, that is, delocalization from the
intercalated discs (Fig. 3.5A–B). Such remodelling is commonly observed
in cardiac diseases such as hypertrophic cardiomyopathy (Seidel et al.,
2010), myocardial infarction (Wang et al., 2010) and heart failure (Akar
et al., 2004) and contributes to impairment of impulse propagation.
In a murine model of chronic T. cruzi infection (Y strain), we observed
structural damage to the myofibrils, mitochondria and sarcoplasmic
reticulum with intercalated disc discontinuity, as shown in the electron
micrographs in Fig. 3.5C and D. Interestingly, using oligonucleotide
microarrays, we have previously described that both in in vitro and in
in vivo models of infection, there are marked changes in the expression of
genes related to contractile proteins as well as to the intercalated
disc (Adesse et al., 2010; Goldenberg et al., 2009; Mukherjee et al., 2008).
An important recent report indicated that in human chagasic cardiomyo-
pathic hearts, Cx43 distribution is altered in areas of fibrosis and this
FIGURE 3.5 Cardiac Chagas disease affects connexins and intercalated discs morphol-
ogy. Hearts from acutely infected mice (30 days post-infection with the Brazil strain)
were harvested and processed for immunohistochemistry for Cx43 (red) and F-actin
(green) (A–B). Non-infected animals (A) displayed abundant Cx43 staining (red) in
cell–cell contacts, mainly in the intercalated discs (arrows). Acutely infected myocar-
dium (B) presented amastigotes pseudo-cysts (*), as revealed by DAPI staining in blue and
lateralization of Cx43 in neighbour cells (arrowheads). Transmission electron microscopy
revealed that during chronic Chagas disease (180 days post-infection with the Y strain),
there are foci of severe damage to myocytes (D) in which cells are hypertrophied and
display mitochondria swelling and disarray of contractile elements as compared to
age-matched uninfected mouse hearts (C). The arrows point to a region where myofibrils
anchor to intercalated discs, indicating substantial cellular disorganization. Original
magnification: 8000 (C) and 10,000 (D). M, mitochondria; MF, myofibril; T, T-tubule.
observation was most prominent in patients with cardiomegaly (Waghabi

et al., 2009). These findings highlight an evolving concept that many types
of cardiomyopathy target expression or involve mutations in molecular
components of the intercalated disc (see Saffitz et al., 2007 for review and
Celes et al., 2007 for changes in sepsis). Thus, as pointed out in recent
commentary (Spray and Tanowitz, 2007), cardiomyopathies, including

chronic chagasic cardiomyopathy, may be considered to be ‘‘junctiono-
pathies’’. Taken together, these data may explain in part the dysrhythmias
and conduction abnormalities that attend this infection (see Section 3.4).
3.3. NOT ALL JUNCTIONAL PROTEINS ARE AFFECTED BY

TRYPANOSOMA CRUZI INFECTION
As emphasized above, our studies have consistently demonstrated that

T. cruzi infection decreases Cx43 expression at appositional membranes in
cultured cardiac myocytes. In order to investigate effects of T. cruzi
infection on other protein families, we used Mardin-Darby canine kidney
(MDCK) cells to evaluate Cx43 and the tight junction protein zona
occludens-1 (ZO-1) expression after T. cruzi infection (48 h). Immunocy-
tochemistry with antibodies specific for Cx43 (Fig. 3.6A) demonstrated
that gap junctional staining was significantly reduced in most of the
infected cells, although uninfected neighbours could display normal
Cx43 abundance and distribution (Fig. 3.6A). Although a number of
connexins (including Cx43) are associated with ZO proteins (Giepmans
and Moolenaar, 1998; Sorgen et al., 2004; Toyofuku et al., 1998), the
infection did not affect the ZO-1 tight junction proteins in MDCK cells
(Fig. 3.6B). These results demonstrate that T. cruzi infection disrupts
specifically Cx43 gap junction protein and spares several proteins
that form tight junctions (F. Fortes, A.C. Campos de Carvalho and
R. Goldenberg, unpublished observations).
3.4. MICROARRAY EXPERIMENTS HAVE REVEALED

PROFOUND CHANGES IN GENE EXPRESSION IN THE
CHAGASIC MOUSE, BOTH IN ACUTE AND IN CHRONIC
DISEASE PHASES
We have published several papers characterizing the impact of chagasic
cardiomyopathy and acute infection of neonatal rodent myocytes on gene
expression by host tissue or cells (Adesse et al., 2010; Goldenberg et al.,
2009; Manque et al., 2011; Mukherjee et al., 2003; 2008; Soares et al., 2010).
Our most extensive data set involved hybridization on Duke oligonucleo-
tide arrays of RNA extracted from four biological replicas of hearts from
control and C57Bl6 mice infected with the Colombian strain of T. cruzi for
8 months (i.e. chronic stage) (Soares et al., 2010). For this chapter, we
further analyzed those data with respect to heart rhythm determinant
(HRD) genes selected using our prominent gene expression analysis
(PGA; Iacobas et al., 2010a). In this data set, Cx43 (Gja1) was slightly but
FIGURE 3.6 Trypanosoma cruzi infection affects Cx43 but not other junctional proteins.
MDCK2 cells were cultured and infected with T. cruzi (Brazil strain) for 72 h. Immuno-
fluorescence for Cx43 (A, in red) and Zona Occludens-1 (B, in green) showed that despite
the drastic decrease in Cx43 immunoreactivity in most of the highly infected cells (*),
ZO-1 distribution was maintained intact (arrows mark regions where Cx43 was lost but
ZO-1 was still present, arrowhead where Cx43 was still present in nonparasitized cells).
With nucleic acid staining with TOPRO3, is possible to visualize host cell nuclei and also
kinetoplastid DNA from intracellular amastigotes (small spots in (C)). In (D) the merged
image is displayed. Bars ¼ 20 mm.
not significantly reduced (1.8-fold reduction, p ¼ 0.07), whereas Cx37, the

major gap junction protein in endothelial cells, was strongly upregulated
(3.3-fold, p ¼ 0.02). Of the cadherins (Cdh) for which there were adequate
data (Cdh4, 5 and 13), only Cdh13 was regulated ( 1.7-fold, p ¼ 0.02).
Other HRD genes downregulated include those encoding the inwardly
rectifying potassium channel J8 ( 1.6, p < 0.04) and Pklaa ( 1.7,
p ¼ 0.01). Upregulated HRD genes included Lamin A (1.7-fold,
p ¼ 0.04), Myh7 (2.7-fold, p ¼ 0.006) and TGFb2 (1.9-fold, p ¼ 0.005).
Some other genes encoding proteins that have been associated with
Cx43 were not significantly affected, including Cx40 and Cx45, catenins,
CAR and plakophilin. Another metric by which to examine the impact of
disease on experimental intervention is to use coordination analysis to

determine the degree to which network interlinkage is altered. Results of
such a network analysis are shown in Fig. 3.7, where we have compared
the extent to which HRD genes are (p < 0.05) synergistically (Pearson
coefficients > 0.9) or antagonistically (Pearson coefficients < 0.9) coor-
dinately expressed among themselves (Fig. 3.7A–D) and with connexins.
Inspection of the networks in normal heart reveals that there are many
synergistic interlinkages and very few antagonistic connections (Fig. 3.7A
and B); in the infected heart, synergistic connections are reduced, whereas
antagonistic ones are greatly increased (Fig. 3.7C and D). This network
remodelling is especially evident when the interlinkages are focused on
connexin genes in control and infected hearts (Fig. 3.7E). With respect to
individual gap junction genes, Cx43 (Gja1) is synergistically expressed
with five genes (Gja4, Gjc1, Cav3, Ctmd1, Scr5a), Gja4 with eight (Gja1,
Cdh5, Ctnna2, Dsg2, Epas1, Pcdh18, Scn5a and Tjap1), Gja5 with six (Atplal,
Dsc2, Pcd1, Pcdhga12, Tgfb2 and Tjap1) and Gjc1 with four (Gja1, Ctnnd1,
Cxad5, Pcdh7). By contrast, these interlinkages are radically different in T.
cruzi-infected heart. Gja1 is synergistically expressed with six genes (Gja5,
Ank3, Pdch18, Pcdhga12, Ryr2 and Tjp1) and antagonistically with three
genes (Gjc1, Cdh5 and Lmma1). Gja4 becomes synergistically expressed
with only two genes (Adrbk1 and Ctnnal1) and antagonistically with eight
(Abcc9, Casq2, Cdhl2, Csrp3, Cxddr, Gaa, Kcnj8, Pbp2). Gja5 becomes
synergistically expressed with three genes (Gja1, Tjp1 and Ttn) and antag-
onistically with only two (Gjc2 and Pcdh1). Gjc1 becomes synergistically
expressed with five genes (Cay3, Cdh5, tmnd1, Hand2 and Pcdh1) and
antagonistically with six (Gja1, Gja5, Atpla1, Epas1, Tjap1 and Tjp1).
These pairwise interconnections among HRD genes are illustrated in
Fig. 3.7A–B, showing the remarkable extent of alteration in response to
parasitic infection. Although the resulting altered topology is specific for
Chagas disease, profound remodelling of the HRD genomic fabric was
also reported in other arrhythmogenic conditions such as chronic
constant and intermittent hypoxia (Iacobas et al., 2010b).
3.5. CONCLUSIONS
Chagas disease is the result of infection with T. cruzi. Acute infection is

accompanied by an acute myocarditis with myonecrosis, inflammation
and intracellular parasites. Despite the small number of cells that are
infected, nevertheless, there is often functional deterioration, manifested
both as arrhythmias and as compromised cardiac output. Infection of
cultured cardiac myocytes can be viewed as an in vitro correlate of acute
infection of the heart. These infected cultured cells display altered
chronotropy and synchronous activity even though few cells are infected.
FIGURE 3.7 Remodelling of heart rhythm determinant (HRD) gene interlinkages in

the chagasic heart and impact on connexin-dependent HRD networks. (A) Synergistic
coordinations (red lines) are very common between HRD gene pairs in normal heart.
(B) Antagonistic coordinations (blue lines) between HRD gene pairs are rare in normal
heart. (C and D) In infected heart, synergistic coordinations decrease and antagonistic
coordinations greatly increase, indicating profound network remodelling (E and F).
Expression coordinations of connexin genes with HRD genes are substantially altered by
T. cruzi infection, with different HRD genes showing synergistic coordination (red lines)
and the appearance of numerous antagonistic coordinations.
This suggests that there are signals sent out from infected cell to
non-infected cells that may alter the physiological responses of cells
within the whole culture dish. This is likely similar to what occurs in
the heart with alterations in gap junctions as a result of infection. Chronic
chagasic heart disease is associated with profound conduction distur-
bances associated with fibrosis, lipid accumulation and cellular and tissue
level hypertrophy. We now appreciate that even during the chronic phase
of the disease, there is a persistence of the parasite with a low-level
continuous infection that is associated with fibrosis and vasculopathy.
In part, this manifestation of dysfunction as a consequence of only a small
number of cells being affected may reflect the anatomy of the tissue that is
targeted. The heart is composed of specialized conduction and contrac-
tion myocytes, and optimized output depends upon the progressive
synchronized activation of the contractile myocardium. Thus, reducing
gap junction expression in only a small number of cells could provide
focal slowing of conduction or focal compromise of chamber contraction.
Infection of cardiac myocytes and more globally, infection of the
animal, leads to functional uncoupling of cardiac myocytes, as a conse-
quence of reduced expression of Cx43 and its gene. A variety of methods
have been used to evaluate the changes in gap junction expression in the
chagasic heart. These methods include functional studies in which dye
coupling, junctional conductance or conduction synchrony were evalu-
ated, by immunostaining and Western blotting and measurements of
gene expression, either through Northern blots or, more recently, from
microarray analysis. The findings from these studies include the observa-
tion that the cardiac gap junction protein and the channels that it forms
are a target of infection. In a population of acutely infected cardiac
myocytes, gap junction abundance and immunoreactivity with certain
antibodies are severely compromised, as are functional coupling and
synchronous contraction. In adjacent non-infected cells, gap junction
expression and function are less affected so that there is a mosaic of
cells that are either connected or disconnected to their neighbours
depending on presence and extent of parasitaemia. In chronic chagasic
cardiomyopathy, the number of parasitized cells is low, but circulating
factors such as IL-1b and TGF-b are elevated in the chronically inflamed
myocardium, resulting in not only reduced expression of Cx43 but also
structural remodelling due to fibrosis.
In summary, the available data suggest that the effect on gap junctions
of small numbers of infected cells in both acute and chronic disease has a
critical role in the underlying pathophysiological processes which result
in clinical chagasic cardiomyopathy.
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CHAPTER 4
The Vasculature in Chagas
Disease
Cibele M. Prado,* Linda A. Jelicks,§
Louis M. Weiss,†,‡ Stephen M. Factor,†,‡ Herbert B.
Tanowitz,†,‡ and Marcos A. Rossi*
Contents 4.1. Historical Aspects 84

4.2. Small Animal Studies of the Microcirculation in
4.3. Studies in Dogs 88
4.4. Vasoactive Peptides and Eicosanoids 89
4.5. In Vitro Studies 91
4.6. Studies in Humans 92
4.7. Conclusions 94
Acknowledgements 95
References 95
Abstract The cardiovascular manifestations of Chagas disease are well

known. However, the contribution of the vasculature and
specifically the microvasculature has received little attention.
This chapter reviews the evidence supporting the notion that
alterations in the microvasculature especially in the heart
contribute to the pathogenesis of chagasic cardiomyopathy.
These data may also be important in understanding the contri-
butions of the microvasculature in the aetiologies of other
cardiomyopathies. The role of endothelin-1 and of thromboxane
* Department of Pathology, Laboratory of Cellular and Molecular Cardiology, Faculty of Medicine of Ribeirão
Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
{
{
}
Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, New York, USA

83
84 Cibele M. Prado et al.
A2 vascular spasm and platelet aggregation is also discussed.

Further, these observations may provide target(s) for
intervention.
4.1. HISTORICAL ASPECTS
Chagas disease, caused by infection with Trypanosoma cruzi, is a cause of

acute myocarditis and chronic cardiomyopathy and often associated with
a vasculitis. The involvement of the vasculature in the pathogenesis of
Chagas disease has not been generally appreciated. Although its involve-
ment was described in the early years following the initial description of
the parasite and the disease it caused, it remained for others to suggest an
aetiologic role for the vasculature in the development of chagasic heart
disease. The understanding of the contribution of vascular and, in partic-
ular, microvascular dysfunction in the pathogenesis of chagasic heart
disease is important in understanding not only chagasic disease but also
cardiomyopathies of other infectious and non-infectious aetiologies.
Vianna (1911) was the first to detail the pathology of Chagas disease.
From the earliest description of the disease, there was a fascination with
the heart so that Chagas disease became almost synonymous for chagasic
heart disease or chronic chagasic cardiomyopathy. Vianna stated that the
‘‘heart is one of the viscera for which the Schizotrypanosome shows
predilection both in man and in animals’’. In addition, Vianna first
reported vascular involvement in Chagas disease stating that ‘‘perivas-
cular inflammations exist, some of them quite pronounced, and others
barely incipient . . . (in the myocardium) . . . in many of the arterioles that
irrigate the nervous substance, overt phenomena of periarteritis are found
. . . (in the cerebellum)’’. Subsequently, in autopsy specimens, Torres
described alterations in the heart and considered them as unrelated to
tissue parasitism but rather a result of the disruption of the coronary
circulation. These alterations were also observed in experimental
T. cruzi infection (Torres, 1917). In 1941, Torres also observed cardiovas-
cular involvement and he defined these lesions as ‘‘the inflammatory cell
infiltrate in the interstitial tissue of the myocardium starts at the level of
and around the capillaries, and not near S. cruzi, whether the latter is
exudative myocarditis related to early vascular lesions’’. He also
described similar lesions in the coronary arterioles of T. cruzi-infected
monkeys that he considered to be ischaemic alterations in the myocar-
dium resulting from occlusion of vessels. In 1960, Torres (1960) examined
chagasic and non-chagasic human hearts and, in the former, identified
marked, constriction-type irregularities with extensive myocytolysis in
the intramyocardial arterioles. He suggested that the diffuse myocytolysis
was caused by metabolic changes in the myocytes resulting from
The Vasculature in Chagas Disease 85
circulatory disorders of low intensity or short duration. Further, he

suggested that the requirement for arterial blood supply was reduced
resulting in areas of marked diffuse myocytolysis and extensive destruc-
tion of myocardial cells. However, this does not completely account for
collapse of small arterial branches occasionally observed as these changes
could also be the result of a cycle that includes passive hyperaemia, local
anaemia, metabolic disturbances in myocardial fibres, myocytolysis.
Several other investigators of that era described vascular lesions in Cha-
gas disease. For example, Mazza and Benitez (1937) demonstrated amas-
tigotes in cells of the perivascular adventitia in the conjunctiva of patients
during acute infection, and Couceiro (1943) reported vascular lesions in
the sciatic nerve of infected dogs and Coelho (1944) observed coronary
arteriole lesions in 19 cases of chronic Chagas disease. These lesions were
also detected at autopsy in coronary circulation by Ramos and Tibiriça
(1945), Dias et al. (1956) and Koberle (1958).
Andrade and Andrade (1955) observed that the inflammation
observed in chronic chagasic cardiomyopathy could be ‘‘allergic’’,
provoking ischaemic lesions of myocardium by capillary involvement.
The microscopic infarctions could be responsible for alterations in the
conduction system, mainly in the right branch of bundle of His, due to
their preferential intramyocardial localization. They also suggested that
the fibrotic lesions frequently detected at apex of the left ventricle could
originate from vascular obstructions due to subendocardial parietal
thrombosis. Subsequently, the ‘‘allergic phenomena’’ became less empha-
sized and the vascular changes were considered to be only congestion and
marked dilatation of venules and capillaries. Brito and Vasconcelos
(1959), in a study of 19 cardiac biopsies from patients with megaesopha-
gus, detected necrotizing arteritis in nine and identified the inflammation
as an ‘‘allergic phenomenon’’. Vascular lesions in hearts of infected mice
were also observed by Macclure and Poche (1963) using electron micros-
copy, and by Lucena et al. (1962) and Alencar et al. (1968) using light
microscopy. Okumura et al. (1962) observed necrotizing arteritis in the
myocardium and digestive tract to which they attributed an ‘‘allergic’’
origin. This concept was expanded when these investigators detected a
parasitized endothelial cell (EC). They reported that ‘‘during the acute
phase the trypanosomes may cause a focal lesion with sensitization of the
vessels by an allergic mechanism, triggering hypersensitivity phenomena
reflected by necrotizing arteritis’’.
Jörg (1974) compared histological images obtained from a healthy
heart after vascular injection of an opaque substance with those obtained
in an injected heart of a patient who died of chagasic cardiomyopathy.
‘‘Decapillarization’’ was observed in those zones where the mesenchymal
reaction was more intense. He postulated that the ‘‘angioarchitectonic’’
anarchy was a result of intense mesenchymal reaction secondary to the
parasitic infection which led to a progressive decapillarization and a

destructive loss of many meshes of the capillary net resulting in myocy-
tolysis and destruction of cardiac ganglia. Subsequently, Jörg (1991)
described vascular lesions characterized by endothelial oedema, denuda-
tion, cell accumulation and platelet–fibrin aggregation in a collecting vein
of the left ventricle in a pig model of Chagas disease.
The observation that in chronic chagasic heart disease there was
chronic inflammation and fibrosis and a dearth of parasites led investi-
gators to search for a cause of the progressive pathological changes. In an
effort to explain the pathology, several avenues of research were devel-
oped. Microvascular lesions in chagasic heart disease were described in
the 1980s by Rossi et al. (1984) and Factor et al. (1985). The involvement of
microvasculature in the pathogenesis of chronic chagasic heart disease
was further underscored by Rossi (1990). It should be noted that at this
time, autoimmunity and disturbances in the cardiac anatomic nervous
system were being intensely investigated (Acosta and Santos-Buch, 1985;
Koberle, 1968; Oliveira, 1985; Ribeiro-dos-Santos and Rossi, 1985). In this
regard, a relationship between cardiac autonomic nervous system
abnormalities and sudden cardiac death has been demonstrated (Rossi
and Bestetti, 1995). A relationship between cardiac autonomic nervous
system abnormities and sudden death has been demonstrated. Malignant
ventricular tachyarrhythmias such as ventricular tachycardia and fibrilla-
tion are major causes of sudden death among patients with chronic
chagasic cardiomyopathy. We are now aware that parasite persistence is
present in the cardiovascular system and in other organs even though it is
not obvious by histological examination (Combs et al., 2005; Zhang and
Tarleton, 1999) and that this is a major contributor to the chronic disease.
4.2. SMALL ANIMAL STUDIES OF THE MICROCIRCULATION

IN TRYPANOSOMA CRUZI INFECTION
BALB/c mice immunized with epimastigotes of the avirulent PF strain of
T. cruzi and challenged with trypomastigotes of the virulent Colombian
strain developed a cardiomyopathy similar to that observed in human
chronic chagasic cardiomyopathy including the development of an apical
aneurysm (Rossi et al., 1984). Histological examination revealed focal
areas of myocytolysis, necrosis and myocardial degeneration associated
with a lymphomononuclear inflammatory infiltrate accompanied by
interstitial fibrosis and occasional parasite pseudocysts. Additionally,
platelet aggregates forming transient occlusive thrombi were observed
in small epicardial and intramyocardial vessels. The focal nature of the
myocardial lesion and the type of myonecrosis indicated involvement of
the microcirculation.
A/J mice infected with the Brazil strain and perfused with silicone rubber
(Microfil) 15–17 days post-infection revealed numerous areas of focal
vascular constriction, microaneurysm formation, vascular dilatation and
proliferation of microvessel (Factor et al., 1985) which is similar to the
observations in the Syrian cardiomyopathic hamster and in human
cardiomyopathies of other aetiologies (Sonnenblick et al., 1985). In that
model, the administration of verapamil ameliorated the microvascular
alterations and the myocardial pathology. Similarly, in the Brazil strain-
infected CD-1 mouse, verapamil ameliorated the myocardial pathology
when verapamil was administered early but not late infection (Chandra
et al., 2002; De Souza et al., 2004; Morris et al., 1989). Verapamil increases
coronary blood flow, inhibits platelet aggregation and contributes to the
amelioration of the pathology. These observations were corroborated by
direct in vivo visualization utilizing a surrogate murine model, that is, the
cremaster microvascular bed (Tanowitz et al., 1996). Direct observation of
the effects of T. cruzi infection on microcirculatory flow in vivo and quantita-
tive measurement of parameters such as the velocity of red blood cell flow
(Vrbc) and vessel diameter were provided. When the cremaster model was
examined 20–25 days post-infection in male CD-1 mice infected with the
Brazil strain, a significant decrease in Vrbc, reversed by verapamil treatment,
was observed in the first- and third-order arterioles and venules accompa-
nied by an attenuation of the inflammation. The arterioles of the infected
mice exhibited segmental areas of vasospasm and dilatation, possibly the
initiating event in microaneurysm formation (Tanowitz et al., 1996; Fig. 4.1).
A B
C D
FIGURE 4.1 (A) Images obtained from T. cruzi-infected mouse. Perivascular inflamma-
tion. (B) Vasculitis of the pulmonary vasculature. (C) Endothelialitis of the subendocar-
dium. (D) Vasculitis of a blood vessel in the liver (images from Petkova et al., 2001).
The infection of mice with T. cruzi caused a vasculitis. There was a

gradual reduction in coronary flow in infected mice over time giving
further credence to the notion that there was vascular dysfunction in
experimental Chagas disease (Tanowitz, 1992b). Importantly, amastigotes
are evident in the coronary microvascular ECs early in infection before
parasitaemia can be detected, suggesting that the coronary endothelium
could be an initial target of infection (Factor et al., 1985). Acutely infected
rats developed changes in the endothelial layer characterized by EC
swelling and a few points of cytoplasmic discontinuity that appeared as
holes exposing the subendothelial collagen that is usually associated with
platelet–fibrin aggregates, which might affect the generation of vasoactive
substances, and impairs the equilibrium between opposing forces (Rossi,
1997). In vitro and in vivo studies indicate that infection of the endothelium
results in expression of both pro-inflammatory cytokines and vascular
adhesion molecules, which are important components of the inflamma-
tory response (Huang et al., 1999a,b; Tanowitz et al., 1992a,b). Infection of
ECs activates NF-kB and likely contributing to the induction of cytokine
and adhesion molecular expression in the endothelium (Huang et al.,
1999a). Further, in the myocardium obtained from T. cruzi-infected
humans and experimental animals, increased expression of cytokines,
nitric oxide synthases and adhesion molecules has been reported
(Huang et al., 1999a; Laucella et al., 1996; Reis et al., 1993; Fig. 4.2).
Taken together, all of aforementioned studies in experimental animals
strongly suggest that the vasospasm of the branches of the coronary
microcirculation leads to a reduction in blood flow and ischaemia to a
small area of the myocardium subserved by that microvessel which
resulted in a microinfarct. When this process is repeated over a period of
time in different areas of the heart, these areas may coalesce and lead to
falling out of cardiac myocytes and replacement by fibrous tissue. The focal
but widespread nature of the pathology supports, in part, this hypothesis.
4.3. STUDIES IN DOGS
Dogs have been used in investigations of Chagas disease because they are
a larger animal than the standard mouse model and may better recapitu-
late the human disease. Hearts obtained from dogs sacrificed 18–26 days
after intraperitoneal inoculation with the 12SF strain of T. cruzi demon-
strated myocarditis characterized by small focal areas of lesion and
myocytic necrosis associated with interstitial mononuclear infiltration.
Electron microscopic studies revealed degenerative changes in the ECs
in contact with T lymphocytes, as well as platelet aggregates and fibrin
thrombi in the intramyocardial capillaries. These alterations suggested
that a possible interaction between ECs and effector immune cells might
A B
Day 30 Day 15 Day 0
C D
FIGURE 4.2 (A) Coronary perfusion of mouse hearts as determined by autoradio-

graphic imaging utilizing the fatty acid analog 19-iodo-3,3,-dimethyl-18 nonadecenoic
acid (DMIVM). A: uninfected normal mouse with normal perfusion. B: perfusion in a
mouse infected for 15 days. Note the reduced perfusion C: perfusion in a mouse infected
for 30 days demonstrating a marked reduction in perfusion (taken from Tanowitz, 1992).
(B) Microfil injection of the coronary vasculature of an A/J mice 15 days post-infection
with the Tulahuen strain of T. cruzi demonstrating a section through the subendocardium
of the atrium showing saccular microaneurysms and vasospasm (Rossi et al., 2010).
(C) Pseudocyst in the wall of a blood vessel (Tanowitz et al., 2009). (D) Vasculitis of a large
blood vessel obtained from an infected mouse (Tanowitz et al., 2009).
play an important role in the pathogenesis of the myocellular lesion and

of the observed microangiopathy (Andrade et al., 1994). More recently,
Melo et al. (2011) demonstrated that the administration of simvastatin
ameliorated the cardiac remodelling in a canine model of chronic chagasic
heart disease by histological and functional criteria. Importantly, statins
have been demonstrated to inhibit platelet aggregation (Lee et al., 2010)
and reduce the inflammation in the vasculature (Liu et al., 2009), thus
increasing coronary blood flow in some studies (Brands et al., 1991).
4.4. VASOACTIVE PEPTIDES AND EICOSANOIDS

Endothelin-1 (ET-1), a 21-amino acid peptide (Yanagisawa et al., 1988),
was originally described as a powerful vasoconstrictor secreted by endo-
thelial cells (ECs). T. cruzi infection of ECs results in a dramatic increase in
biologically active ET-1. However, other cell types have found to be
sources of ET-1 such as cardiac myocytes, fibroblasts, astrocytes and

macrophages (Kedzierski and Yanagisawa, 2001). The synthesis of ET-1
is mediated by endothelin-converting enzyme (ECE) which converts Big
ET-1 (31 amino acids) to ET-1. The actions of ET-1 are mediated by the
G-protein-coupled endothelin receptors ETA and ETB. Although ET-1 is
constitutively expressed in many cells, increased synthesis has been
associated with many disease states such as malignant hypertension,
primary pulmonary hypertension, CHF, sepsis, meningitis, eclampsia
and subarachnoid haemorrhage (Kedzierski and Yanagisawa, 2001).
Increased expression/synthesis of ET-1 has been implicated in the patho-
genesis of cerebral malaria (Machado et al., 2006) and chagasic cardiomy-
opathy (Petkova et al., 2000, 2001; Tanowitz et al., 2005).
Eicosanoids are lipid mediators that participate in many biological
activities including vascular tone, inflammation, ischaemia and tissue
homeostasis (Haeggstrom et al., 2010). The biosynthetic pathways in
mammals for these important biological mediators are dependent upon
liberation of arachidonic acid for the inner leaflet of the plasma mem-
brane. Thromboxane A2 (TXA2), an eicosanoid generated during arachi-
donic acid metabolism, is the most potent vasoconstrictor known and acts
via its receptors TPa and its splice variant TPb, both of which are
expressed on human ECs. Several parasitic organisms produce eicosa-
noids which may modulate host response and the progress of an infection
(Belley and Chadee, 1995; Kubata et al., 1998, 2000; Liu and Weller, 1990;
Noverr et al., 2003).
Thus, the observation in experimental animals and humans regarding
vasospasm and platelet aggregation and thrombi in the coronary
microcirculation was reminiscent of the actions of TXA2. Tanowitz et al.
(1990) observed that there was increased platelet aggregation in infected
mice accompanied by an increase in plasma TXA2. The increased levels of
TXA2 could explain the vascular spasm and the platelet aggregation
(Tanowitz et al., 1990). Ashton et al. (2007), 17 years later, demonstrated
that T. cruzi was capable of synthesizing TXA2. It was further demon-
strated that the majority of TXA2 detected in the blood of infected mice is
parasite derived. These observations suggest that TXA2 could contribute
to the pathogenesis of chronic chagasic cardiomyopathy and its clinical
manifestations. More recently, on the basis of these observations,
Mukherjee et al. (2011) administered aspirin (ASA) to T. cruzi (Brazil
strain)-infected mice. There was a reduction in the plasma levels of
TXA2. ASA inhibits the mammalian COX-1 enzyme thus reducing the
levels of PGH2 available for the synthesis of TXA2. Thus, we believe that
ASA treatment of the infected host decreases the ability of the parasite to
scavenge PGH2 from the host to synthesize TXA2. In addition,
ASA-treated infected mice suffer a high parasitaemia and mortality.
This effect of ASA is a result of ‘‘off-target’’ factors unrelated to TXA2.
It may suggest that caution should be used in the treatment of fever and
pain with ASA during acute infection.
TXA2 and ET-1 share several important properties important in the
pathogenesis of Chagas disease. They both cause vasoconstriction and
platelet aggregation. Additionally, they are both pro-inflammatory. Mice
infected with T. cruzi display an increased expression of ET-1 protein and
mRNA in the myocardium and an increase in plasma ET-1 levels (Petkova
et al., 2000). Treatment of infected mice with phosphoramidon, an
inhibitor of ECE, reduced T. cruzi-infection-induced right ventricular
dilation (Tanowitz et al., 2005). T. cruzi infection of mice in which the
gene for ET-1 is deleted either in cardiac myocytes or in ECs ameliorated
cardiac remodelling as demonstrated by histopathology, echocardiogra-
phy and cardiac MRI (Tanowitz et al., 2005). Elevated plasma levels of
ET-1 have been demonstrated in patients with chronic chagasic cardio-
myopathy (Salomone et al., 2001). However, it is unclear if this is a result
of congestive heart failure in general or chagasic cardiomyopathy in
particular. It is important to note that Hassan et al. (2006) found increased
expression of ET-1 in the carotid arteries of infected mice. This observa-
tion clearly demonstrated the importance of ET-1 in the vasculature of
infected mice and by implication in infected humans. The release of
platelet-activating factor by macrophages in this infection causes transient
ischaemia and myocytolytic necrosis (Talvani et al., 2003; see Chapter 1
for a discussion of eicosanoids and Chapter 5 for a discussion of the role of
bradykinin and bradykinin receptors).
4.5. IN VITRO STUDIES
Direct infection of human ECs in culture with T. cruzi resulted in the

alteration of various critical biochemical processes responsible for the
maintenance of microvascular perfusion, such as calcium homeostasis
and generation of inositol trisphosphate (IP3), ET-1, TXA2 and prostacy-
clin (PG12) which is a vasodilator and inhibits platelet aggregation
(Morris et al., 1988). EC infection also resulted in alterations of cyclic
AMP metabolism, which plays a protective role against the direct and/
or indirect lesion caused by the adhesion and aggregation of circulating
platelets to ECs (Morris et al., 1992).
Inflammatory cells contribute to microvascular hypoperfusion by
secreting cytokines and other factors known to affect platelets and ECs.
Infection of cultured ECs results in increased synthesis of interleukin-1b
(IL-1b), IL-6 and colony-stimulating factor 1 (CSF-1) which may result in
altered function (Tanowitz et al., 1992a). IL-1b is elaborated by activated
macrophages and by peripheral blood mononuclear cells, including those
infected with T. cruzi, and by a variety of other cell types, such as ECs
(Van Voorhis, 1992). The antithrombotic properties of ECs may be altered

by IL-1b. This cytokine may reduce tissue production of the plasminogen
activator and increase production of the inhibitor of this activator, which
may result in thrombus formation (Bevilacqua et al., 1984; Nachman et al.,
1986). CSF-1 is an important growth factor needed for the proliferation
and maturation of cells of the mononuclear lineage (Mantovani et al.,
1990). It is also important in recruitment, possibly acting in conjunction
with IL-1b. High CSF-1 levels have been detected in infected cultured
ECs. These observations may reflect the growth of the monocyte popula-
tion in the microvasculature resulting in the synthesis of pro-inflamma-
tory cytokines (Mantovani et al., 1990; Tanowitz et al., 1992a). In addition,
trypomastigotes have been demonstrated to produce neuraminidase
(trans-sialidase) that may be involved in the removal of sialic acid from
the surface of mammalian myocardial cells and ECs, facilitating thrombin
binding. The loss of this endothelial surface protector molecule could
contribute to platelet aggregation and thrombosis within the small coro-
nary vessels (Libby et al., 1986). These factors acting together may ulti-
mately result in spasm and thrombosis in the small coronary vessels,
inducing focal myocardial damage.
Mukherjee et al. (2004) examined infected human ECs which resulted
in activation of extracellular signal-regulated kinases 1and 2 (ERK1/2)
but not c-Jun N-terminal kinase or p38 MAPK. Treatment of these cells
with the MAPK kinase inhibitor PD98059 prior to infection blocked the
increase in phosphorylated ERK1/2 observed with infection. Transfection
with dominant-negative Raf(301) or Ras(N17) constructs reduced the
infection-associated levels of phospho-ERK1/2, indicating that the activa-
tion of ERK1/2 involved the Ras–Raf–ERK pathway. Infection also
resulted in an increase in activator protein 1 (AP-1) activity, which was
inhibited by transfection with a dominant-negative Raf(301) construct.
Infected ECs were found to synthesize ET-1 and IL-1b, which activated
ERK1/2 and induced cyclin D1 expression in uninfected smooth muscle
cells. More recently, Tonelli et al. (2010) demonstrated that T. cruzi gp85/
trans-sialidase surface protein family is important in the attachment of the
parasite to the host cells.
Taken together, these data suggest a possible molecular paradigm for
the pathogenesis of the vasculopathy in this infection.
4.6. STUDIES IN HUMANS

Anatomical studies have shown structural derangement and rarefied
microvasculature in the left ventricular myocardium. A histotopographi-
cal study comparing the microcirculatory system after injection of an
opaque medium into chagasic and control human hearts demonstrated
focal decapillarization in chronic Chagas disease due to extraluminal

compression, suggesting that this might be the cause of focal myocytolytic
necrosis ( Jörg, 1974). Similarly, a post-mortem radiological study of
chagasic hearts revealed vascular changes at the heart apex characterized
by distorted and/or scarce vessels associated with decreased arterial
density, presumably related to the pathogenesis of apical aneurysm
(Ferreira et al., 1980).
Patients with Chagas disease may exhibit symptoms that are atypical
for classic angina pectoris. Although symptoms suggestive of myocardial
ischaemia are present, coronary angiographic studies show normal or
nearly normal coronary arteries in more than 90% of patients studied
(Marin-Neto et al., 1992). Patients specifically selected on the basis of
chest pain did show perfusion abnormalities detectable by thallium-201
scintigraphy, suggesting that myocardial ischaemia may be due to altera-
tions in the microvasculature. Abnormal perfusion in different groups of
chagasic patients has been confirmed using isonitrile-99m-technetium
(Castro et al., 1988) or thallium-201 (Hagar and Rahimtoola, 1991;
Marin-Neto et al., 1992). Myocardial capillary blood flow in chronic
chagasic patients with no significant clinical or electrocardiographic
manifestations proved to be markedly reduced when evaluated with
rubidium-86, while the major coronary vessels appeared normal. The
reduction observed is comparable to that exhibited by a group of
non-chagasic patients with obstructive coronary disease (Kuschnir et al.,
1974a,b). Vasospasm has been proposed in the genesis of myocardial
ischaemia in patients with chronic chagasic cardiomyopathy (Vianna
et al., 1979). For example, it was demonstrated that in patients with
chagasic cardiomyopathy, there is an abnormal, endothelium-dependent,
coronary-vasodilating mechanism as demonstrated by acetylcholine and
adenosine infusion into the left coronary artery, suggesting that epicardial
and microvascular coronary reactivity may be altered in these patients.
The clinical importance of this alteration awaits elucidation. However,
this abnormality of the coronary microvasculature may contribute to the
genesis of the symptoms related to the ischaemic processes observed in
chronic chagasic patients and to acute myocardial infarction in the
absence of significant coronary damage (Torres et al., 1995).
Biopsies of chronic chagasic hearts revealed a marked thickening of
the basement membrane in most myocytes and capillaries (Ferrans et al.,
1988). These alterations are similar to the thickening reported for the
basement membranes of myocardial capillaries in other cardiomyopa-
thies (Factor et al., 1983). A very well developed capillary network has
been observed in chagasic human hearts using a cell-maceration scanning
electron microscopic method (Higuchi et al., 1999). This network may
result in reduced flow of blood thus contributing to the hypoxic changes
observed in chronic chagasic cardiomyopathy. Significant dilatations of
arterioles and capillaries in ventricular areas of chagasic hearts compared

to hearts with dilated cardiomyopathy were described. These microcircu-
latory dilatations could be responsible for a reduction in blood flow
distribution in the watershed area lying between the two main coronary
flow sources (the anterior- and posterior-descending arteries, and the
right and circumflex coronary arteries). These findings could result in
ischaemia and extensive fibrosis within the left ventricle apical and
posterior regions (Higuchi et al., 1999).
The relation of regional sympathetic denervation and myocardial
perfusion disturbance to wall motion impairment was described in patients
with chronic chagasic cardiomyopathy. Global left ventricular function,
segmental wall motion analysis and myocardial perfusion were evaluated
in 58 patients. There were myocardial perfusion defects in the absence of
epicardial coronary artery disease, and the extension and severity of perfu-
sion abnormalities paralleled the progression of myocardial damage. These
observations support the notion that perfusion disturbances in chronic
chagasic cardiomyopathy may be caused by transient disturbances of
coronary blood flow regulation at the microvascular level (Simoes et al.,
2000). The same group correlated the clinical, electrocardiographic,
angiographic, electrophysiologic and wall motion/myocardial perfusion
disturbances in chronic chagasic patients with either sustained or
non-sustained ventricular tachycardia. The fact that both fixed perfusion
defects (which reflect local fibrosis) and reversible and paradoxical defects
predominate in the arrhythmias in the left ventricular region is also com-
patible with the hypothesis that microvascular ischaemia is aetiologic.
Thus, several observations suggest that in human chagasic heart disease,
transient disturbances of coronary blood flow regulation at the level of the
microvasculature may result in regional myocardial degeneration, with a
consequent reparative fibrosis that ultimately constitutes the substrate for
re-entrant circuits and the appearance of both sustained and non-sustained
ventricular tachycardia (Sarabanda et al., 2005).
4.7. CONCLUSIONS
Abnormalities in the coronary circulations were observed since the

earliest studies by Vianna and Torres conducted soon after the discovery
by Carlos Chagas of the disease that bears his name. Since then, much
information has accumulated from attempts to define the physiopathol-
ogy of chagasic heart disease. The changes observed both on humans and
in experimental models of T. cruzi infection suggest that myocardial
lesions are multifactorial including parasite persistence, autoimmunity
and microvascular involvement. Importantly, they are not mutually
exclusive.
ACKNOWLEDGEMENTS
This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de
São Paulo (M. A. R.; FAPESP 09/17787-8; 10/19216-5) and National Institutes of Health
(NIH) Grants AI-076248 (H. B. T.) and CA-123334 (L. A. J.). C. M. P. was supported in part by
a grant from the Fogarty International Center–NIH (D43-TW007129). M. A. R. is senior
investigator of the Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq).
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CHAPTER 5
Infection-Associated
Vasculopathy in Experimental
Chagas Disease: Pathogenic
Roles of Endothelin and
Kinin Pathways
Julio Scharfstein and Daniele Andrade

5.2. A Brief Overview on the Immunopathogenesis of
Chagas Disease 103
5.2.1. Mechanisms underlying infection-associated
vasculopathy 105
5.2.2. Bradykinin receptors: A gate of entry for
Trypanosoma cruzi invasion of cardiovascular
cells 108
5.2.3. Interstitial oedema induced by
trypomastigotes: Role of the kinin system 112
5.2.4. ACE is a negative modulator of TH1 induction
by kinin danger signals released in peripheral
sites of infection 114
5.2.5. DCs activated by kinins induce
immunoprotective type-1 effector T cells in
mice systemically infected by Trypanosoma
cruzi 117
5.3. Future Directions 118
Acknowledgements 120
References 120
Instituto de Biofı́sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Laboratório de
Imunologia Molecular, Cidade Universitária Rio de Janeiro, Rio de Janeiro, Brazil

101
102 Julio Scharfstein and Daniele Andrade
Abstract Acting at the interface between microcirculation and immunity,

Trypanosoma cruzi induces modifications in peripheral tissues
which translate into mutual benefits to host/parasite balance. In
this chapter, we will review evidence linking infection-associated
vasculopathy to the proinflammatory activity of a small subset of
T. cruzi molecules, namely GPI-linked mucins, cysteine proteases
(cruzipain), surface glycoproteins of the trans-sialidase family and/
or parasite-derived eicosanoids (thromboxane A2). Initial insight into
pathogenesis came from research in animal models showing that
myocardial fibrosis is worsened as result of endothelin upregulation
by infected cardiovascular cells. Paralleling these studies, the kinin
system emerged as a proteolytic mechanism that links oedemato-
genic inflammation to immunity. Analyses of the dynamics of
inflammation revealed that tissue culture trypomastigotes elicit
interstitial oedema in peripheral sites of infection through synergis-
tic activation of toll-like 2 receptors (TLR2) and G-protein-coupled
bradykinin receptors, respectively, engaged by tGPI (TLR2 ligand) and
kinin peptides (bradykinin B2 receptors (BK2R) ligands) proteolytically
generated by cruzipain. Further downstream, kinins stimulate lymph
node dendritic cells via G-protein-coupled BK2R, thus converting
these specialized antigen-presenting cells into TH1 inducers. Tightly
regulated by angiotensin-converting enzyme, the intact kinins (BK2R
agonists) may be processed by carboxypeptidase M/N, generating
[des-Arg]-kinins, which activates BK1R, a subtype of GPCR that is
upregulated by cardiovascular cells during inflammation. Ongoing
studies may clarify if discrepancies between proinflammatory
phenotypes of T. cruzi strains may be ascribed, at least in part, to
variable expression of TLR2 ligands and cruzipain isoforms.
ABBREVIATIONS
ACE angiotensin-converting enzyme

BKRs bradykinin receptors
CCM chronic chagasic myocardiopathy
cruzipain major cysteine protease of T. cruzi
CTLs cytototoxic CD8þ T cells
DCs dendritic cells
ET endothelin
HCP hamster cheek pouch
HK high molecular weight kininogen
HUVECs human umbilical vein endothelial cells
KKS kallikrein–kinin system
PRR pattern-recognition receptors
Role of Kinins and Endothelins in Chagasic Vasculopathy 103
TCTs tissue culture trypomastigotes

tGPIm trypomastigote-derived glycosylphosphatidylinositol-
anchored mucin-like glycoproteins from T. cruzi
TLR2 toll-like 2 receptors
TS trans-sialidase
TXA2 thromboxane A(2)
5.1. INTRODUCTION
After decades of systematic investigations, the concept that low-grade

tissue parasitism is the primary mechanism leading to chronic chagasic
myocardiopathy (CCM) is firmly established. Although this hypothesis
predicts that Trypanosoma cruzi antigens and/or proinflammatory
molecules play a central role in CCM, there is growing awareness that
the clinical pleiomorphism of Chagas disease might result from the
interplay between the genetically diversified T. cruzi species and the
variable genetic make-up of the human host (Andrade, 1999; Macedo
and Pena, 1998; Vago et al., 2000; Venegas et al., 2009). In spite of early
evidences that T. cruzi diversification resulted from clonal evolution of
ancestor lineages (Tibayrenc et al., 1986), it was recently recognized that
genetic exchange has also produced hybrid ancestor lineages that further
contribute to the variability of currently circulating strains (De Freitas
et al., 2006; Sturm and Campbell, 2009; Westenberger et al., 2005). Follow-
ing recommendations made by an expert panel (Zingales et al., 2009), it
was recently proposed that the isolates/strains of T. cruzi should be
classified in six ancestor lineages, designated as TcI to TcVI. In the present
chapter, we will discuss in general terms the impact of genetic diversifica-
tion of T. cruzi on oedematogenic inflammation.
5.2. A BRIEF OVERVIEW ON THE IMMUNOPATHOGENESIS OF

CHAGAS DISEASE
In the early 1970s, pathologists were intrigued with the observation that
T. cruzi pseudocysts were rarely detected in myocardial specimens from
chronic chagasic patients, in spite of the presence of extensive inflamma-
tory infiltrates and tissue fibrosis (Andrade et al., 1994). For several years,
CCM was tentatively classified in textbooks as an autoimmune disease,
the underlying premise being that self-reactive (anti-heart) lymphocytes
were the principal effectors of cardiac inflammation. Although the con-
troversy is not definitively resolved (Teixeira et al., 2011), in the mid 90’s
the hypothesis that autoimmunity was the primary pathogenic mecha-
nism driving CCM was called into question by independent studies
showing presence of traces of DNA or T. cruzi antigens in heart tissues
of chagasic patients (Benvenuti et al., 2008; Higuchi et al., 1997; Palomino

et al., 2000). The notion that T. cruzi organisms are directly involved in
disease outcome was further substantiated by evidences that chronic
patients displaying the cardiac forms of Chagas disease bear parasite
DNA in the heart, but not in oesophageal tissues, whereas, reciprocally,
the patients that exclusively develop gastrointestinal abnormalities show
the presence of parasite DNA in oesophagus tissues, but not in the heart
(Jones et al., 1993; Vago et al., 1996). While these human pathological
studies were in progress, research in animal models showed that immune
control of T. cruzi infection depends on the integration between humoral
and the cellular (innate and adaptive) branch of anti-parasite immunity
(Tarleton et al., 1994). Concerning the T cell-dependent branch of immu-
nity, the analysis of the epitopes recognized by class I MHC-restricted
effector CD8þ T cells identified the trans-sialidase (TS) family of antigens
as dominant targets in both humans and mice (Garg and Tarleton, 2002;
Tzelepis et al., 2008; Wizel et al., 1997). Given the extensive polymorphism
observed in TS antigens, these results initially suggested that the immune
system is able to efficiently reduce parasite tissue burden in the acute
phase by focusing the effector CD8þ T cell responses on a limited range of
dominant TS peptides, consequently bringing the intracellular level of
infection to limits that are compatible with host survival (Wizel et al.,
1997). Adding complexity to this picture, subsequent studies revealed
that the hierarchy of immunodominant TS epitopes recognized by effec-
tor CD8þ T cells varies from one T. cruzi strain to another (Martin et al.,
2006; Tzelepis et al., 2008). Since naturally infected hosts are often exposed
to multiple T. cruzi clones, Martin et al. (2006) hypothesized that stochas-
tic expression of variant TS epitopes by intracellular amastigotes and/or
trypomastigotes may allow for parasite escape from the immune
response, thus providing a driving force for the evolutionary diversifica-
tion of TS family genes. More recently, Rosenberg et al. (2010) challenged
the concept that resistance to infection is critically dependent on the
generation of TS-specific effector CD8þ T cells recognizing dominant
TS-encoded epitopes. In an elegant study, they showed that mice previ-
ously tolerized by high-dose injections of dominant TS peptides were
resistant to an acute challenge, implying that the mice are able to effec-
tively combat T. cruzi by generating effector T cells that recognize sub-
dominant epitope specificities, not necessarily encoded by TS family
members.
Despite the wealth of information emerging from immunological
studies in animal models, it is not obvious why a small proportion of
T. cruzi organisms subvert clearance by effector CD8þ T cells. Initial
studies suggested that endogenous suppressive factors generated in the
inflamed muscle tissue may limit the efficacy of cytotoxicity mediated by
CD8þ T cells (Leavey and Tarleton, 2003). Additional studies suggested
that differentiation of effector cytototoxic CD8þ T cells (CTLs; Albareda

et al., 2006; Grisotto et al., 2001) may be hampered as a result of dysfunc-
tions occurring in the memory T cell compartment of TS-specific T cells.
Although the nature of the mechanisms underlying immune subver-
sion is still uncertain, there were mounting evidences linking the
low-grade myocardial parasitism to the presence of inflammatory infil-
trates enriched in TNF-a-producing CD8þ T cells in the heart of patients
with chronic myocardiopathy (Reis et al., 1993) or in experimentally
infected mice (Tarleton, 2003; Zhang and Tarleton, 1996). Given the tech-
nical obstacles to compare the antigen specificity and immune response
profiles of intracardiac T cells isolated from cardiac versus indeterminate
chagasic patients (Fonseca et al., 2007), immunologists relied on lympho-
cytes isolated from peripheral blood to analyze systematically the profile
of antigen-experienced T cells from chronic patients. Using epimastigote
antigens, Gomes et al. (2003) were able to categorize the immune respon-
sive profile of chagasic patients based on IFN-g production by CD4þ T
cells. In their study, the frequency of type-1 responders was significantly
higher among cardiac patients, whereas low type-1 responders predomi-
nated in patients with indeterminate disease. Interestingly, the low IFN-g
production observed in indeterminate patients was inversely correlated
with high frequencies of IL-10-producing monocytes (Gomes et al., 2003).
More recently, Souza et al. (2007) reported that patients with the indeter-
minate form of Chagas disease display a higher ratio of IL-10 over TNF-a-
producing monocytes. Along similar lines, Araujo et al. (2007) found that
indeterminate patients display a higher percentage of CD4þ CD25þ T cells
expressing FOXP3 and IL-10. Adding substance to these in vitro studies,
Costa et al. (2009) reported that patients exhibiting polymorphism of an
IL-10 promoter gene associated to lower expression levels of the IL-10
regulatory cytokine had a higher frequency of heart disease. Collectively,
the studies with peripheral blood cells suggest that patients with asymp-
tomatic/attenuated heart disease may rely on IL-10 producing macro-
phages and/or regulatory T cells to limit the collateral damage which is
otherwise inflicted by intracardiac TH1-type effector cells.
5.2.1. Mechanisms underlying infection-associated

vasculopathy
In the early 1990s, experts in vascular pathology advanced the proposi-
tion that infection-associated vasculopathy could induce cumulative
damage in the chronically parasitized myocardium, perhaps rendering
the heart tissues more vulnerable to antigen-induced immunopathology
(Morris et al., 1990; Rossi, 1990). Years later, refined histochemical studies
revealed a derangement of the microcirculation and abnormal interstitial
matrix patterns in the heart sections of CCM patients (Higuchi et al.,
1999). Paralleling these human studies, investigations carried out in the

mouse model of Chagas disease suggested that endothelin-1 (ET-1) could
contribute to infection-associated vasculopathy (Tanowitz et al. 1999).
Constituted by a family of three peptides (ET-1, ET-2 and ET-3) of 21
amino acids encoded by distinct genes, endothelins are expressed by
endothelial cells, cardiac myocytes and cardiac fibroblasts (Goto, 2001;
Kedzierski and Yanagisawa, 2001). Synthesized as prepro-endothelin,
these precursor proteins are cleaved by endothelin-converting enzymes
forming big-endothelin, which upon further processing yields peptides
that activate cells via G-protein-coupled receptors (GPCRs; for review, see
Dhaun et al., 2007). Endothelin is involved in a host of physiological
processes via the activation of two GPCR subtypes, ETA and ETB.
Endowed with powerful vasoconstrictor function, ET-1 is also able to
modulate the expression of leukocyte adhesion molecules on endothelial
cells and on fibroblast-like synovial cells (Schwarting et al., 1996), induces
plasma exudation and oedema formation (Filep et al., 1993; Sampaio et al.,
2000), stimulates cytokine production (Sampaio et al., 2000; Speciale et al.,
1998) and regulates neutrophil adhesion and migration (Sampaio et al.,
2000; Zouki et al., 1999).
After reporting that the plasma levels of ET-1 are increased both in
chagasic patients and in mice (Petkova et al., 2000; Salomone et al., 2001),
these authors documented that ET-1 (i) expression is upregulated in
T. cruzi-infected cardiovascular cells (endothelial cells and cardiac myo-
cytes; Petkova et al., 2000) and (ii) induces vasospasm in T. cruzi-infected
mice, hence contributing to the development of myocardial ischaemia and
myonecrosis (Tanowitz et al., 2005). These authors demonstrated that
cardiac remodelling was ameliorated in T. cruzi-infected mice in which
the ET-1 gene was deleted exclusively from cardiac myocytes (Tanowitz
et al., 2005). Based on these findings, the authors advanced the proposi-
tion that ETR antagonists might be considered in adjunctive therapy of
chagasic heart disease (Mukherjee et al., 2004; Tanowitz et al., 2005).
Further insight on infection-associated vasculopathy emerged from
studies of the pathogenic roles of T. cruzi prostanoids (Ashton et al.,
2007). These authors focused their attention on thromboxane (TXA2),
after pondering that the multiple vascular sequelae associated with
T. cruzi infection could relate to the upregulated function of this eicosa-
noid, for example, denudation of the endothelium (leading to increased
vascular permeability) and increased expression of leukocyte adhesion
molecules on the endothelium. In addition, TXA2 promotes proliferation
and migration of smooth muscle cells, thus contributing to neointima
formation (Ashton et al., 2007). Research focusing on TXA2 could also
shed light on dysfunctions in haemostasis, since this eicosanoid promotes
platelet activation/aggregation and degranulation. Importantly, Ashton
et al. (2007) reported that mice deficient in the thromboxane receptor (TP)
bear a highly susceptible phenotype, characterized by increased mortal-
ity, cardiac pathology and higher tissue parasitism. After showing that
TXA2 is the predominant eicosanoid lipid produced in the blood of
chagasic mice, the authors demonstrated that up to 90% of the circulating
levels of TXA2 were of parasite origin, rather than from the host. Interest-
ingly, the levels of TXA2 produced by amastigotes are significantly higher
than those of trypomastigotes or epimastigotes. Clues to understand the
potential significance of these findings emerged from analysis of the
outcome of infection in cultures of endothelial cells derived from
wild-type versus TP-deficient mice; the authors noted that the infection
index was markedly increased in the mutant mice. Based on these obser-
vations, the authors proposed that TP, most likely triggered by amasti-
gote-derived TXA2, may fine-tune the rate of intracellular parasite
growth, preventing dysregulated expansion of the intracellular load of
parasites within endothelial cells. Extending these studies to the in vivo
settings, Ashton et al. (2007) observed that T. cruzi-TP-null mice displayed
an increased mortality, parasite tissue load and cardiac pathology.
Infections employing bone marrow chimeric mice argued against the
possibility that TP deficiency in immune cells might account for the
susceptible phenotype of TP-null mice. These results, combined with
the culture studies performed with endothelial cells, suggest that the
TXA2/TP axis may limit parasite infectivity in somatic cells, through
mechanisms that remain unclear.
Another area of research linking T. cruzi activity to endothelium injury
emerged from studies on the pathogenic role of TS. Progress in this field
started with the observation that endothelial cells and cardiomyocytes
suffered de-sialylation upon treatment with T. cruzi neuraminidase
(Libby et al., 1986), the latter being described as a TS (Previato et al.,
1985; Zingales et al., 1987). More recently, Dias et al. (2008) used catalyti-
cally inactive recombinant TS to characterize in further details the
molecular basis of TS binding to endothelial cells. Their data showed
that TS binds to endothelial cell surface a2,3-linked sialic acid residues
through a lectin-binding site. Functional analysis of the outcome of the
lectin site of TS with the endothelium revealed that the interaction (i) led
to the activation of NF-kB, (ii) increased expression of adhesion molecules
and (iii) reduced apoptosis upon endothelial cell exposure to growth
factor deprivation (Dias et al., 2008). Focusing a novel aspect of TS
research, that is, the molecular mechanism involved in endothelium
transmigration and tissue tropism, Tonelli et al. (2010) postulated that
trypomastigotes might interact with microvascular beds through the
binding of a conserved peptide motif of TS shared by several members
of the polymorphic T. cruzi family. The presence of circulating antibodies
to TS (Duthie et al., 2005) may also account for the infection-associated
microangiopathy described in chagasic patients (Higuchi et al., 1999) and

experimentally infected mice (Andrade et al., 1994). For example, endo-
thelium decorated with TS molecules that are shed by trypomastigotes
might be injured as result of antibody-mediated cellular cytotoxicity,
reminiscent of the bystander mechanism of host cell death originally
envisaged by Ribeiro Dos Santos and Hudson (1981).
5.2.2. Bradykinin receptors: A gate of entry for

Trypanosoma cruzi invasion of cardiovascular cells
Given the low level of intracellular parasitism observed in the myocar-
dium of chronic patients, we may predict that the interstitial spaces of the
heart are only sporadically exposed to intracellular T. cruzi released from
‘‘pseudocysts’’, that is, the membrane-containing structures harbouring
parasites at the final stages of their intracellular life cycle. Once released
from pseudocysts, the trypomastigotes—which for operational
reasons will be henceforth designated as tissue culture trypomastigotes
(TCTs)—rapidly move away from the primary foci of infection, seeking
for a safer environment (i.e. non-inflamed) to efficiently propagate the
infection. As previously suggested (Scharfstein and Morrot, 1999), it is
possible that premature killing of parasitized target cells by amastigote-
specific MHC Class I restricted CTLs may lead to the release of amasti-
gotes to the heart interstitium. Devoid of a moving flagellum, the amas-
tigotes tend to cluster in the surroundings of the primary infection foci,
perhaps accounting for most, if not all, of the parasite antigens detected in
heart specimens of CCM patients (Higuchi et al., 1999). As reviewed
below, the immunohistochemical identification of cruzipain depots in
the myocardium of CCM patients (Morrot et al., 1997) suggested that
this major T. cruzi antigen could play a role in immunopathology
(Scharfstein, 2010). While these immunological studies were in progress,
Scharfstein and co-workers realized that enzymatically active cruzipain
may fuel inflammation through the activation of the kallikrein–kinin
system (KKS; Del Nery et al., 1997; Lima et al., 2002).
The term ‘‘kinin’’ refers to a small group of vasoactive metabolites
related to the bradykinin (BK), a nonapeptide proteolytically released
from an internal moiety of high (HK) or low (LK) molecular weight
kininogens (Bhoola et al., 1992). Although kinins are traditionally viewed
as classical mediators of acute inflammation (e.g. inducers of oedema
formation, vasodilation and pain sensations), it is now well established
that these short-lived peptide hormones may modulate the microcircula-
tion homeostasis (Bhoola et al., 1992; Schmaier, 2004). As discussed later
in this chapter, knowledge emerging from studies of the KKS role in
immunity has linked the role of kinins to the IL-12-dependent cytokine
circuitry that shapes T-cell development (Aliberti et al., 2003; Monteiro

et al., 2006, 2007, 2009).
Due to their short life (half-life of < 15 s in the plasma), kinins must
swiftly activate their cognate heterotrimeric GPCRs, that is, BK2R or
BK1R. BK2R is expressed by several cell types, such as pain-sensitive
neurons, vascular endothelial, smooth muscle cells (Leeb-Lundberg
et al., 2005) and conventional dendritic cells (DCs; Aliberti et al., 2003;
Bertram et al., 2007; Kaman et al., 2009). In order to maintain vascular and
tissue homeostasis, the adverse effects resulting from excess liberation of
kinins are usually attenuated by (i) BK2R downregulation and (ii) the
kinin-degrading activity of metallopeptidases, such as angiotensin-con-
verting enzyme (ACE)/kininase II, a transmembrane dipeptidyl carboxy-
peptidase (Skidgel and Erdos, 2004) that is highly expressed in the
endothelium lining and in other cell types, including monocytes and
DCs (Danilov et al., 2003). Besides degrading kinins, a vasodilator ACE
has a dual effect on vascular homeostasis because it generates angiotensin
II, a potent vasopressor octapeptide. Noteworthy, the presence of soluble
forms of ACE in plasma and other body fluids is due to cleavage of
membrane form of somatic ACE by desintegrin and metalloproteinase
(ADAM)-type ‘‘sheddase’’ (Parkin et al., 2004).
In contrast to the constitutive BK2R, whose expression is restricted to
‘‘steady-state’’ tissues, the expression of BK1R is strongly upregulated
during inflammation (Marceau and Bachvarov, 1998). While the intact
kinins (BK or LBK) activate BK2R, the generation of high-affinity ligands
for the inducible BK1R (i.e. [des-Arg]-BK/LBK) depends on removal of the
C-terminal Arg of the intact kinins (BK/LBK) by carboxypeptidase N/M
(kininase I). Apart from inducing pain sensations (Calixto et al., 2004;
Cunha et al., 2007), BK1R drives leukocyte transmigration through the
endothelium (McLean et al., 2001). In another interesting precedent,
research in experimental autoimmune encephalitis revealed that BK1R
suppression reduces recruitment of pathogenic T cells into the central
nervous system, presumably due to impaired expression of ICAM-I and
VCAM-I at the inflamed blood–brain barrier (Göbel et al., 2011).
Although kinins are commonly released from kininogens through the
activity of plasma and tissue kallikreins, there is growing evidence that
other proteolytic enzymes, whether acting alone or in cooperative fashion,
may act as ‘‘kininogenases’’. For example, in the settings of chronic
inflammation, oxidized forms of kininogens undergo processing by the
concerted action of neutrophil elastase and mast cell tryptase, leading to
the release of slightly larger kinin, Met-LBK (Kozik et al., 1998). In the
context of bacterial infection, kinins can be directly liberated from the
kininogens by the action of microbial cysteine proteases, such as gingi-
pain from Porphyromonas gingivalis (Imamura et al., 1994), staphopain A
from Staphylococcus aureus (Imamura et al., 2005) and streptopain from

Streptococcus pyogenes (Herwald et al., 1996).
The first clues indicating that T. cruzi activated the kinin system came
as a result of the studies by Del Nery et al. (1997) who analysed the
substrate specificity properties of the major cysteine protease of T. cruzi
(cruzipain). Classified as member of clan A of the C1 peptidase family
(Cazzulo et al., 1989), cruzipain is a well-characterized therapeutic
target in Chagas disease (Doyle et al., 2007). Substrate specificity studies
performed with intramolecularly quenched fluorogenic peptides span-
ning the N- and C-terminal flanking sites of the lysyl-BK sequence, Del
Nery et al. (1997) revealed that cruzipain resembles tissue kallikrein, that
is, both enzymes are able to cleave HK, releasing the internal lysyl-BK
moiety. Initially, the discovery that cruzipain is a kininogenase seemed
paradoxical because kininogens are members of the cystatin family of
cysteine protease inhibitors, hence rely on cystatin-like domains to
potently inactivate papain-like enzymes, including cruzipain (Stoka
et al., 1995). Noteworthy, however, the studies performed by Del Nery
et al. (1997) revealed that purified cruzipain was able to release bioactive
kinins from soluble forms of HK, but unlike tissue kallikrein, the reaction
occurred at slow rates. The conundrum was settled after considering that
HK binds to endothelial cells through two distinct domains: (i) a domain
(D3) that overlaps with the cystatin domain (Herwald et al., 1995) and (ii)
a histidine-rich positively charged motif (D5H) localized at the C-terminal
end of the BK (D4) sequence, which binds to negatively charged
sulphated proteoglycans, such as heparan or chondroitin sulphates
(Renne et al., 2000; Renne and Muller-Esterl, 2001). Based on this infor-
mation, Lima et al. (2002) hypothesized that the spatial orientation of cell-
bound HK docked to heparan sulphate proteoglycans was not suitable for
cruzipain binding and inactivation by the cystatin-like inhibitory domain.
Indeed, model studies performed with cruzipain and HK in the test tube
offered circumstantial support to this hypothesis: the addition of heparan
sulphate (tested at optimal concentrations) drastically reduced the cyste-
ine inhibitory activity of soluble HK on cruzipain, while reciprocally
increasing the catalytic efficiency (sixfold) of the parasite protease. Con-
sistent with these findings, the addition of heparan sulphate increased the
efficiency of the kinin-releasing activity of cruzipain (albeit only at rela-
tively narrow concentration range) and resulted in the formation of mul-
tiple HK breakdown products. Combined, these biochemical studies
suggested that the substrate specificity of the parasite protease was redir-
ected as result of reciprocal interactions between sulphated proteoglycans
with the substrate (HK) and protease (cruzipain) molecules, hence
increasing the efficiency of the kinin release reaction (Lima et al., 2002).
While these biochemical studies were in progress, Scharfstein et al.
(2000) demonstrated that living TCTs (Dm28c) rely on the kinin-releasing
activity of cruzipain to infect cells that overexpress BK2Rs, such as human

umbilical vein endothelial cells (HUVECs) or CHO-transfected cell lines
overexpressing BK2R. After showing that TCTs induce strong [Ca2þ]i
transients via the cruzipain/BK2R pathway, the authors suggested that
parasite uptake involved the [Ca2þ]i/lysosomal pathway originally
described by Tardieux et al. (1992). Evidence linking the processing of
kininogens to cruzipain-dependent generation of the BK2R agonist was
obtained in invasion assays performed in the presence of exogenous HK.
These studies showed that parasite uptake by CHO-BK2R was enhanced
upon addition of purified HK or, alternatively, by addition of physiologi-
cal concentration of BK (i.e. the BK2R agonist) into the serum-free
medium. Further, mAbs directed to kininogens blocked invasion on
CHO-BK2R but did not interfere with the baseline levels of infection of
CHO mock, further suggesting that cell-bound kininogens serve as
precursors for the BK2R agonist(s) released by cruzipain (Scharfstein
et al., 2000). Another interesting revelation of this study was the evidence
that ACE/kininase II, a metallopeptidase that is strongly upregulated in
HUVECs, limits the ability of the parasite to invade this particular cell
type via the BK2R pathway.
In view of the technical obstacles to ablate the multiples cruzipain
genes, invasion assays were carried out with active-site directed cysteine
protease inhibitors. Unexpectedly, the results revealed that membrane-
permeable cruzipain inhibitors markedly reduced extent of parasite inva-
sion via the BK2R pathway, while addition of soluble inhibitors such as
cystatin C or E-64 did not interfere at all with parasite infectivity
(Scharfstein et al., 2000). Given that trypomastigotes are poorly endocytic
(De Souza, 1995) and that these flagellates accumulate cruzipain in the
flagellar pocket (Murta et al., 1990; Souto-Padron et al., 1990), Scharfstein
and co-workers reasoned that the kinin-releasing reaction may occur in
enclosed areas formed by juxtaposition of host cell and parasite plasma
membranes, perhaps equivalent to a ‘‘synapse’’ (Tyler et al., 2005).
This mechanistic model predicts that the lysosomal-like cruzipain mole-
cules might diffuse from the parasites’ flagellar pocket into this
intercellular space, being thus spared from physiological inactivation by
soluble forms of plasma protease inhibitors (e.g. cystatins, kininogens,
a2-macroglobulin) present in extracellular body fluids. Although not
directly demonstrated, this concept also implies that surface-bound
kininogens, along with bradykinin receptors (BKRs), are actively
recruited to such signalling centres (Scharfstein et al., 2000).
Although BK2R was the first GPCR with defined pharmacological
specificity to be implicated in the [Ca2þ]/lysosomal pathway of T. cruzi
invasion (Andrews, 2000; Burleigh and Woolsey, 2002; Leite et al., 1998),
in vitro studies subsequently showed that the inducible BK1R may serve
as gateway for infective trypomastigotes (Todorov et al., 2003). In order to
simulate the settings of inflammation, the authors examined the outcome

of T. cruzi interaction with (i) HUVECs pre-activated, or not, with
lipopolysaccharides (LPS) (TLR4 ligand) and (ii) neonatal cardiomyo-
cytes, which spontaneously express BK1R. Assays performed in the pres-
ence of BK1R antagonists or kininase I inhibitors revealed that parasite
uptake was markedly reduced. Noteworthy, measurements of intracellu-
lar amastigotes several days after the onset of infection confirmed that the
early blockade of BK1R reduced parasite burden in endothelial cells or
cardiomyocytes in a direct proportional to the number of penetrating
parasites. Noteworthy, T. cruzi trypomastigotes infected cell types
overexpressing the inducible BK1R in the absence of ACE inhibitors,
suggesting that carboxypeptidase N/M-dependent generation of
[des-Arg]-kinins (BK1R ligand) is prioritized over ACE-dependent degra-
dation of the intact kinins (BK2R ligand). Another interesting aspect that
emerged from the studies of host–parasite interaction was the evidence of
‘‘crosstalk’’ between BK2R and BK1R (Todorov et al., 2003). As discussed
further below, it is possible that Dm28c T. cruzi may take advantage of the
ubiquitous B1KR pathway to opportunistically invade cardiovascular
cells in the inflamed heart tissues.
5.2.3. Interstitial oedema induced by trypomastigotes: Role of

the kinin system
Todorov et al. (2003) were the first to demonstrate that Dm28c trypomas-
tigotes activate the kinin system in vivo. Using mouse paw oedema as a
readout, studies in BK2R/ or BK1R/ mice infected with trypomasti-
gotes revealed that BK2R mediates the early-phase vascular responses
(2–3 h), whereas the upregulated BK1R pathway accounts for the late
phase (24 h) reaction. Noteworthy, the oedematogenic inflammation in
wild-type mice was consistently mild (in BALB/c mice) or negligible (B6
mice), except for animals purposefully deprived of ACE activity by sys-
temic administration of captopril before parasite inoculation. These
results underscored the importance of ACE/kininase II as a modulator
of inflammatory oedema in mice infected subcutaneously (s.c.) with
Dm28c trypomastigotes.
Given the possibility that blood vessel injury by needle injection could
synergize with parasite products to propel activation of the KKS,
Monteiro et al. (2006) analysed the impact of topical application of
Dm28c trypomastigotes in microcirculatory preparations of the hamster
cheek pouch (HCP). The results from intravital microscopy studies
revealed that the parasites induce a mild BK2R-dependent plasma leakage
response in the HCP, consistent with the mouse oedema studies. In both
models, the vascular reactions were potentiated by captopril and
mitigated by Z11777, a highly specific irreversible inhibitor of cruzipain
(Doyle et al., 2007). These results strongly suggested that the level of
bioactive kinins generated in peripheral sites of T. cruzi infection
(‘‘steady-state’’ conditions) depends on the balance between cruzipain
and ACE.
In a crucial observation, Monteiro et al. (2006) observed that Dm28c
epimastigotes did not elicit significant FITC-dextran leakage in captopril-
treated HCP, despite the fact that these avirulent parasite stages express
high levels of cruzipain. These results suggested that expression of cru-
zipain was necessary but insufficient for trypomastigotes to induce
plasma leakage via the BK2R pathway. Consistent with this hypothesis,
purified cruzipain (enzymatically active) failed to induce plasma leakage
in the captopril-treated HCP superfusate. However, the combination of
cruzipain and purified HK to captopril-HCP led to a full-blown plasma
leakage via the BK2R pathway. Based on these findings, Monteiro and
co-workers proposed that the rate-limiting step governing extent of kinin
release by cruzipain is the level of plasma-borne kininogens available
in peripheral sites of infection. As a corollary, the authors predicted that
(i) in ‘‘steady-state’’ tissues (i.e. in the absence of a pre-established inflam-
mation), the levels of kininogens in interstitial spaces are not sufficiently
high to propitiate appreciable proteolytic release of vasoactive kinins,
either in tissues exposed to avirulent epimastigotes or to purified
cruzipain, and (ii) trypomastigotes might be empowered with proinflam-
matory molecules (absent in epimastigotes) which rapidly induce the
diffusion of plasma-borne proteins (including kininogens) into the inter-
stitial spaces. Efforts to identify this putative molecule converged to the
glycophosphatidyl-linked mucin anchor of trypomastigotes (tGPI),
originally characterized as a potent TLR2 ligand by Almeida and
Gazzinelli (2001). According to these workers, tGPI possesses an unsatu-
rated fatty acid at the sn-2 position (TLR2 agonist) of the alkylacylglycerol
moiety, which is absent in the counterpart GPI anchors of epimastigotes.
Consistent with a role for tGPI, Monteiro and co-workers demonstrated
that Dm28 trypomastigotes failed to elicit appreciable oedema both in
TLR2/ and in neutrophil-depleted mice, irrespective of treatment with
ACE inhibitors. Moreover, assays performed in captopril-treated mice
(wild-type, BK2R/, TLR2/ and neutrophil-depleted) injected with
the combination of purified tGPI (TLR2 ligand) and cruzipain (enzymati-
cally active) demonstrated that tGPI and cruzipain synergistically
induced footpad oedema via the TLR2/neutrophil/BK2R-dependent
pathway, while ACE/kininase II has an anti-inflammatory role, since it
interferes with the transcellular ‘‘crosstalk’’ between TLR2 and BK2R.
It is well established that activated neutrophils are capable of inducing
endothelial barrier disruption through a variety of mechanisms (DiStasi
and Ley, 2009). Intravital microscopy observations in HCP suggested that
neutrophils play a role in the dynamics of oedematogenic inflammation
induced by Dm28c trypomastigotes (Monteiro et al., 2006). After noting

that the peak of plasma leakage was sligthly delayed in relation to leuko-
cyte mobilization, Schmitz et al. (2009) studied the role of innate receptors
as the initiators of T. cruzi-elicited inflammation. First, they demonstrated
that resident macrophages stimulated in vitro by Dm28c trypomastigotes
robustly secreted neutrophil-attracting CXC chemokines (KC/MIP-2) in
TLR2-dependent manner. Next, they verified that repertaxin (CXCR2 antag-
onist) blocked neutrophil-dependent influx of plasma proteins into the
interstitial spaces, thus reducing the initial influx of plasma-borne kinino-
gens (cruzipain substrate) in peripheral sites of infection (Fig. 5.1). Com-
bined, these studies suggested that TLR2/CXCR2/neutrophils control the
rate-limiting step (kininogen diffusion to interstitial spaces) of the microvas-
cular response which is required for over activation of the kinin system in
peripheral sites of T. cruzi infection (Fig. 5.1). Once formed, the vasoactive
kinins amplify oedematogenic inflammation initiated by TLR2/CXCR2/
neutrophils through positive feedback cycles of endothelium BK2R activa-
tion, which can be further prolonged at expense of activation of the inducible
BK1R pathway (Todorov et al., 2003). In summary, the flow of information
between innate immunity (TLR2-driven) and the proteolytic wave
(cruzipain/BK2R-driven) of inflammation is modulated by the kinin-
degrading activity of ACE/kininase II.
5.2.4. ACE is a negative modulator of TH1 induction by kinin

danger signals released in peripheral sites of infection
DCs are a heterogeneous population of professional antigen-presenting
cells (APCs) that are widely but sparsely distributed in peripheral tissues
and lymphoid organs (Shortman and Naik, 2007). Strategically positioned
in T cell-rich areas of secondary lymphoid tissues, the resident DCs are
specialized in antigen presentation to CD4þ and CD8þ T cells. In
steady-state conditions, immature DCs contribute to the maintenance of
peripheral tolerance because these APCs display MHC-restricted antigen
peptides to virgin T cells in the absence of co-stimulatory molecules.
However, during infection, immature DCs develop the competence to
initiate adaptive immunity after sensing the presence of inflammatory
cues (‘‘danger’’ signals) generated in peripheral sites of infection and/or
in the lymphoid tissue environment (Sansonetti, 2006). Once drained by
lymphatics, microbial antigens and proinflammatory molecules (includ-
ing kinins) are transported to the DC-rich cortical areas of the lymph
node. After internalizing antigens via specialized scavenger receptors,
the lymphoid-resident DCs may spread their antigen cargo to lym-
phoid-resident DCs via release of exosomes and/or apoptotic body
uptake (Sansonetti, 2006). While the antigens are processed and presented
in MHC-restricted manner in the surface of these specialized APCs,
FIGURE 5.1 Mechanistic model depicting how the proinflammatory activities of kinins
and endothelins may converge to aggravate myocardial pathology in Chagas heart
disease. Lower side of panel, sparsely distributed the heart of chronically infected
patients, the heart cells containing pseudocysts sooner or later disrupt, releasing
numerous trypomastigotes to the interstitial spaces. Acting as typical microbial PAMPS,
tGPI-mucin (TLR2 ligands) shed by the TCTs are sensed by TLR2 constitutively expressed
by resident macrophages (left side of panel). Next, the activated macrophages secrete
neutrophil-attracting CXC chemokines (KC/MIP-2), which in turn bind to CXCR2
expressed by neutrophils/endothelium (upper left). Neutrophils activated by CXC
chemokines secrete vascular permeability factors which then disrupt the integrity of the
endothelium barrier. This allows for incipient leakage of plasma proteins, including
kininogens and ET-1 (present at high levels in the blood of patients with cardiac disease)
into peripheral sites of infection (upper side of panel). T. cruzi trypomastigotes process
kininogens associated to GAGs, liberating kinins via cruzipain (CZP). The biological
activity of the short-lived kinins (BK2R agonist) is mitigated by the kinin-degrading
activity of ACE/kininase II. The vigour of the inflammation steered by the TLR2/CXCR2/
neutrophil pathway may eventually overcome the regulatory constraints imparted by
ACE/kininase II. The build-up in the extravascular levels of vasoactive kinins leads to
overt activation of the kinin system, due to feedback loops of activation of endothelium
BK2R/BK1R. Further downstream, T. cruzi may then take advantage of the odedemato-
genic inflammation to invade cardiovascular cells through the cooperative activation of
the antigen-bearing DCs concomitantly sense the presence of microbe-

derived ‘‘danger’’ motifs through distinct pattern-recognition receptors
(PRRs), such as TLRs or intracellular NOD2-like receptors (NLR; Kumar
et al., 2011). In addition, DCs may sense the threat to tissue integrity via
receptors for endogenous proinflammatory mediators, such as ATP, uric
acid (Sansonetti, 2006) and BK (Aliberti et al., 2003; Monteiro et al., 2007).
Stabilized by cognate interactions with co-stimulatory molecules (CD80/
86, CD40 and MHC), the prolonged encounters between antigen-bearing
DCs and naı̈ve T cells are essential for TCR activation. During the course
of DC/T cell interaction, the ‘‘mature’’ APCs deliver polarizing cytokines.
For example, IL-12p-70 is critically required for TH1 development.
In 2003, our group reported that exogenous lysyl-BK (LBK) potently
induces the maturation (upregulation of IL-12 and co-stimulatory mole-
cules) on wild-type CD11cþ DCs while failing to elicit such responses in
BK2R/ DCs (Aliberti et al., 2003). In keeping with these in vitro observa-
tions, studies in ovalbumin-immunized mice confirmed that exogenous
LBK induced TH1 polarization via the BK2R/IL-12-dependent innate path-
way. Subsequently, Monteiro et al. (2006) suggested that kinins released in
peripheral sites of T. cruzi infection upregulated IL-12 production by
CD11cþ DCs in the draining lymph node and steered TH1 development
via the BK2R pathway. Noteworthy, these effects were only observed in
infected mice pretreated with captopril, thus implying that ACE/kininase
II offsets the linkage between innate immunity (TLR2 dependent) and the
downstream proteolytic pathways that guide TH1 development via the
BK2R/IL-12-dependent pathway (Monterio et al., 2006; reviewed by
Scharfstein et al., 2008). Analysis of T cell recall responses to parasite
antigens by lymphocytes isolated from draining lymph nodes revealed
that TH1 induction was compromised in TLR2/ or neutrophil-depleted
mice. Importantly, the deficient TH1 responses of TLR2/ or neutrophil-
depleted mice were fully restored by mixing purified HK to the sus-
pension of living trypomastigotes shortly before footpad injection.
In both cases, the HK-dependent rescuing of TH1 responses was nullified
BKRs and ETRs (Andrade et al., 2011). The interstitial oedema driven by kinins is further
intensified (top, right), increasing the levels of ET-1 in the interstitial spaces. Sustained
inflammation may also lead to upregulated expression of B1KR in the myocardium,
offering a window of opportunity for parasite invasion of cardiovascular cells. The
increase in intracellular parasite load translates as upregulated expression of endothe-
lins, which may then aggravate infection-associated vasculopathy and myocardial
fibrosis via ETRs. In addition, the upregulated expression of BK1R in the endothelium
lining may favour the recruitment of circulating anti-parasite IFN-g/TNF-a-producing
CD4þ T effector and CD8þ T effectors to the heart parenchyma. For the sake of
simplicity, the panel does not illustrate the impact of TLR2/B2R activation on DC
maturation and on TH1 development, at early stages of T. cruzi infection (Monteiro et al.,
2006, 2007).
by HOE-140 or by mixing purified HK with trypomastigotes pretreated

with K11777 (irreversible cruzipain inhibitor). Collectively, these experi-
ments supported the concept that plasma-borne kininogens diffusing in
interstitial spaces undergo proteolytic processing by cruzipain, liberating
endogenous signals (kinins) that subsequently convert BK2Rþ/þ CD11cþ
DCs into inducers of TH1 polarization (Scharfstein et al., 2007). Further
indications that the TLR2/BK2R axis bridges inflammation to innate/
adaptive immunity emerged from studies in a mouse model of mucosal
inflammation induced by the periodonto-bacterium P. gingivalis (Monteiro
et al., 2009). Acting cooperatively, P. gingivalis LPS (TLR2 ligand) and gingi-
pains (kinin-releasing proteases) induce mucosal inflammation and stimu-
late antibacterial (fimbriae antigens) TH1/TH17 responses via the previously
described trans-cellular TLR2/BK2R ‘‘crosstalk’’. Notably, in contrast to the
T. cruzi infection model, ACE inhibitors did not interfere with B2R-driven
stimulation of antibacterial TH1/TH17 responses in the P. gingivalis infection
model. Although not addressed experimentally, it is likely that the require-
ment for ACE blockade was superfluous in the model of P. gingivalis-elicited
mucosal inflammation because gingipains are not sensitive to inhibition by
the cystatin-like domains of soluble kininogens.
5.2.5. DCs activated by kinins induce immunoprotective type-1

effector T cells in mice systemically infected by
Trypanosoma cruzi
Although the subcutaneous model of T. cruzi served as paradigm to
investigate the role of KKS in mechanisms linking inflammation to
immunity, the impact on host resistance could not be determined because
these mice resisted acute challenge with Dm28c T. cruzi. Seeking for an
alternative model, Monteiro et al. (2007) compared the phenotypes of
BK2Rþ/þ mice and BK2R/ mice in the classical intraperitoneal model
of acute infection. Strikingly, the BK2R/ mice displayed a highly sus-
ceptible phenotype, succumbing to acute T. cruzi challenge within
30 days. Efforts to characterize the mechanisms underlying the immune
dysfucntion of BK2R/ mice failed to reveal profound defects in the
intralymphoid (spleen) at early stages of infection: the frequencies of
antigen-specific IFN-g-producing CD8þ T cells and CD4þ T cells were
fairly similar in wild-type and BK2R-deficient mice. However, there was a
significant drop in the frequency of intracardiac type-1 effector T cells in
BK2R-deficient mice. Further, as the acute infection progressed in BK2R/
mice, the immune deficiency was intensified, involving both the extra-
lymphoid and lymphoid compartment. Intriguingly, the decayed TH1
response of BK2R/ was accompanied by a corresponding rise in IL-17-
producing T cells (TH17). The premise that the deficient adaptive response
of BK2R/ mice was a secondary manifestation resulting from impaired
BK2R/ DC maturation was confirmed by systemically injecting
wild-type BK2Rþ/þ DCs (i.v.) into the susceptible BK2R/ mice before
injecting the pathogen. Remarkably, this DC transfer manoeuvre rendered
the recipient BK2R/ mice resistant to acute T. cruzi challenge and
restored their capability to generate protective IFN-g-producing CD4þ
CD44þ and CD8þ CD44þ effector T cells, while conversely suppressing
the potentially detrimental TH17 (CD4þ subset) anti-parasite responses.
Using expression of IL-12 and co-stimulatory molecules (CD86, CD80,
CD40) as readout for DC maturation in vitro, Monteiro et al. (2007) further
demonstrated that Dm28c trypomastigotes potently activate BK2Rþ/þ
CD11cþ DCs (splenic origin) but not BK2R/ DCs. Moreover, the authors
showed that trypomastigotes pretreated with the irreversible cruzipain
inhibitor (Z11777) failed to robustly activate wild-type DCs, thus suggest-
ing that the BK2R agonist (DC maturation signal) is indeed released by
cruzipain. Dm28c trypomastigotes induced the maturation of splenic
CD11cþ DCs derived from TLR2/ and TLR4 mutant (C3H/HeJ) via
BK2R, thus precluding cooperative signalling between this GPCRs and
either PRRs. While not excluding the contribution of TLR9 (Bafica et al.,
2006) or NOD2 (Silva et al., 2010) as potential sensors of T. cruzi, these
results were consistent with the concept that kinin ‘‘danger’’ signals pro-
teolytically released by trypomastigotes activate BK2Rþ/þ DCs, converting
these APCs into inducers of type-1 immunity (Monteiro et al., 2007;
Scharfstein et al., 2007). Since the spleen is continuously exposed to plasma
proteins, it is conceivable that Dm28 trypomastigotes might be faced with
abundant levels of blood-borne kininogens bound to their docking sites (e.
g., sulfate proteoglycans) present on cell surfaces and/or extracellular
matrixes. As a corollary, we may predict that the levels of kinin ‘‘danger’’
signals proteolytically generated in the parasitized/inflamed splenic
stroma may suffice to convert conventional CD11cþ DCs into TH1 indu-
cers. As part of an initial effort to determine if some of these mechanistic
principles are extended to the settings of human infection, Coelho dos
Santos et al. (2010) have recently reported that ACE inhibitors convert
human monocytes into drivers of TH17-type responses against T. cruzi.
5.3. FUTURE DIRECTIONS
Focusing on the molecular pathways that govern host-parasite interac-

tions at the interface between the microcirculation and immunity, in this
chapter we have reviewed experimental findings indicating that infec-
tion-associated vasculopathy may be linked to the proinflammatory activ-
ities of a limited group of T. cruzi molecules. Special attention was given
to discuss progress made in endothelin research, which culminated in
the discovery that myocardial fibrosis is aggravated as result of ET-1
upregulation by T. cruzi-infected cardiomyocytes (Tanowitz et al., 2005).
Adding further complexity to this picture, Andrade et al. (2011) have

recently reported that T. cruzi trypomastigotes (Dm28c strain) evoke
edematogenic inflammation and invade cardiovascular cells through
mechanisms involving interdependent signaling of ETAR, ETBR and
BK2R (Andrade et al., 2011). Based on these findings, the authors hypothe-
sized that the trypomastigotes may take advantage of the accumulation of
plasma borne-proteins (including kininogens) and endothelins (ET-1) in
extravascular tissues to infect cardiovascular cells more efficiently
through the cooperative activation of ETRs/BKRs (Fig. 5.1). Future stud-
ies may clarify if T. cruzi trypomastigotes may also exploit the inducible
BK1R to persist in the inflamed myocardium. This possibility comes to
mind, in light of evidences emerging from research in diabetes and
hypertension, showing that prooxidative signals generated by ET-1 and
angiotensin II are able to upregulate B1KR expression in vascular smooth
muscle cells (Morand-Contant et al., 2010). In view of this interesting
precedent, we may predict that ET-1-driven induction of BK1R expression
in cardiovascular cells may offer a window of opportunity for parasite
invasion of cardiovascular cells via the inducible kinin pathway (Fig. 5.1).
Furthermore, considering that patients with chronic Chagas disease dis-
play elevated levels of ET-1 in the bloodstream (Salomone et al., 2001), it is
also possible that trypomastigotes may induce the diffusion of blood
borne ET-1, along with kininogens and other plasma proteins (Fig. 5.1),
following the sequential activation of TLR2/CXCR2>BKR/ETRs
(Andrade et al., 2011). Admittedly, however, rather than exclusively
serving as an ubiquitous gateway for parasite invasion of cardiovascular
cells, BK1R engagement may also stimulate host defense by driving endo-
thelium trans-migration of immunoprotective type-1 effector T cells into
the parasitized heart (Fig. 5.1). Ongoing studies should clarify if the BK1R
engagement may reciprocally intensify ETR signaling, thus forging a
feedback loop that might further aggravate myocardial fibrosis during
the chronic stage of infection.
The discovery that tGPI and cruzipain act cooperatively to activate the
kinin system via the TLR2/CXCR2/neutrophil-dependent pathway
(Monteiro et al., 2006) offered a paradigm to investigate the molecular
basis of the variable proinflammatory phenotypes of T. cruzi strains.
Accordingly, parasite strains expressing low levels of TLR2 may not be
able to efficiently induce the diffusion of plasma proteins (including
kininogens) in peripheral sites of infection. If true, we may predict that
these parasite strains may not be capable of generating high-levels of
kinins in peripheral sites of infection, irrespective of the expression levels
of cruzipain (kinin-releasing protease). It is also possible that the proin-
flammatory phenotypes of T. cruzi isolates may vary due to differences in
the efficiency of shedding of lipid vesicles bearing tGPI-linked mucins
(Trocoli-Torrecilhas et al., 2009). Considering that T. cruzi is able to
activate innate sentinel cells through alternative PRRs, e.g., TLR4

(Oliveira et al., 2010), TLR9 (Bafica et al., 2006) or NOD1 (Silva et al.,
2010), additional studies are required to determine if parasite-induced
activation of TLR4 and/or TLR9 may also conduce to plasma leakage,
perhaps favoring activation of the kinin system in TLR2-independent
manner. Another mechanism that may underlie the variable phenotype
of T. cruzi strains is the expression profiles of cruzipain isoforms (Lima
et al. (2001). For example, it is well established that cruzipain 2 (Dm28c
strain) has narrow substrate specificity as compared to the major cruzi-
pain isoform, i.e., the parasite kininogenase (Scharfstein et al., 2010).
Predictably, strain-dependent variability in the ratio of expression
between these two cruzipain isoforms may have impact on T. cruzi ability
to invade host cells expressing BKRs (influence on tissue tropism) as well
on its capacity to induce interstitial edema and TH1 responses via the
kinin pathway. For similar reasons, variations in the expression levels of
chagasin, a tight-binding endogenous inhibitor of papain-like cysteine
proteases- originally described in T. cruzi (Monteiro et al., 2001), may
also influence the phenotype of T. cruzi strains. This possibility is sup-
ported by evidences (Aparicio et al., 2004) indicating that TCTs of the G
strain, which are poorly infective, display increased chagasin/cruzipain
ratios as compared to Dm28c. Importantly, the infectivity of the G strain
was enhanced upon addition of cruzipain-rich culture supernatants from
Dm28 TCTs. In the same study, the authors pointed out that that vesicles
shed by TCTs might serve as cruzipain substrates, presumably generating
hitherto uncharacterized infection-promoting signals (Scharfstein, Lima,
2008). Hence, strain-dependent differences in the expression levels of tGPI
and cruzipain isoforms may influence host/parasite balance because,
these factors act cooperatively, enhancing parasite infectivity while at the
same time integrating innate immunity to the proinflammatory proteo-
lytic cascades that upregulate generation of TH1-type effector cells.
ACKNOWLEDGEMENTS
This research was supported by funds from the Instituto Nacional de Biologia Estrutural
e Bio-Imagem do CNPq; PRONEX (26/110.562/2010), FAPERJ; CNPq; financed in part by
NIH Grant AI-076248 (HBT). D. A. was supported in part by a Fogarty International Center–
NIH Training Grant (D43-TW007129). The authors acknowledge the help of Rafaela Serra in
the preparation of the illustration (Fig. 5.1).
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CHAPTER 6
Autoimmunity
Edecio Cunha-Neto,*,†,‡ Priscila Camillo Teixeira,*,‡
Luciana Gabriel Nogueira,*,‡ and Jorge Kalil*,†,‡

6.2. Natural History of Chagas Disease 134
6.3. Heart-Specific Inflammatory Lesions in CCC: Parasite
Antigen-Driven Immunopathology? 134
6.4. Immunopathogenesis of CCC 135
6.5. Autoimmunity in Chagas Disease 137
6.5.1. Autoantibodies 138
6.5.2. Autoreactive T cells 139
6.6. Molecular Mimicry 140
6.7. Conclusion 142
References 144
Abstract The scarcity of Trypanosoma cruzi in inflammatory lesions of chronic

Chagas disease led early investigators to suggest that tissue damage
had an autoimmune nature. In spite of parasite persistence in chronic
Chagas disease, several reports indicate that inflammatory tissue
damage may not be correlated to the local presence of T. cruzi. A
significant number of reports have described autoantibodies and self-
reactive T cells, often cross-reactive with T. cruzi antigens, both in
patients and in animal models. Evidence for a direct pathogenetic role
of autoimmunity was suggested by the development of lesions after
* Laboratório de Imunologia, Instituto do Coração, Hospital das Clı́nicas, Faculdade de Medicina,

Universidade de São Paulo, São Paulo, SP, Brazil
{
Disciplina de Imunologia Clı́nica e Alergia, Faculdade de Medicina, Universidade de São Paulo, São Paulo,
SP, Brazil
{
Instituto de Investigação em Imunologia—INCT, São Paulo, SP, Brazil

129
130 Edecio Cunha-Neto et al.
immunization with T. cruzi antigens or passive transfer of lymphocytes

from infected animals, and the amelioration of chronic myocarditis in
animals made tolerant to myocardial antigens. Autoimmune and
T. cruzi-specific innate or adaptative responses are not incompatible
or mutually exclusive, and it is likely that a combination of both is
involved in the pathogenesis of chronic Chagas disease cardiomyop-
athy. The association between persistent infection and autoimmune
diseases—such as multiple sclerosis or diabetes mellitus—suggests
that post-infectious autoimmunity may be a frequent finding. Here,
we critically review evidence for autoimmune phenomena and their
possible pathogenetic role in human Chagas disease and animal
models, with a focus on chronic Chagas disease cardiomyopathy.
6.1. INTRODUCTION
Chronic Chagas disease Cardiomyopathy (CCC) is one of the few well-

defined examples of human post-infectious autoimmunity, where an
infectious episode with an established pathogen—the protozoan parasite
Trypanosoma cruzi—triggers multiple autoimmune phenomena, most
related to documented molecular mimicry, and organ-specific damage.
Indeed, several bona fide autoimmune diseases, like multiple sclerosis
(Ablashi et al., 1998) and insulin-dependent diabetes mellitus (el-Zayadi
et al., 1998), have been associated with persistent infections, which sug-
gest that post-infectious autoimmune diseases are more frequent than
previously thought.
The timescale dissociation between primary infection with high tissue
and blood parasitism and tissue pathology, allied to the scarcity of T. cruzi
in CCC heart lesions, prompted investigators as early as 80 years ago to
suggest that the mononuclear cell infiltrate should directly damage the
heart, perhaps in an autoimmune fashion (Torres, 1929). A significant
number of reports have described autoantibodies and self-reactive T
cells, many times cross-reactive with T. cruzi antigens, both in patients
and in animal models (summarized in Table 6.1). Evidence for a direct
pathogenetic role of autoimmunity was suggested by the development of
lesions or functional damage after immunization with T. cruzi antigens or
passive transfer of lymphocytes and autoantibodies from infected ani-
mals (summarized in Table 6.2). The amelioration of chronic myocarditis
in animals made tolerant to myocardial antigens also suggested a
pathogenic role for autoimmunity in Chagas disease. In this chapter, we
review the evidence for the role of pathological autoimmunity in the
pathogenesis of Chagas disease.
TABLE 6.1 Molecular mimicry after T. cruzi infection
Molecular
Host component T. cruzi antigen Host definition Reference
Neurons, liver, kidney, unknown M, R Mab Snary et al. (1983)

testis
Neurons unknown R Mab Wood et al. (1982)
Neurons Sulphated glycolipids H Mab Petry et al. (1987a,b) and Petry and Eisen
(1988, 1989)
Heart tissue unknown M Serum IgG McCormick and Rowland (1989)
Heart and skeletal muscle Microsomal fraction H Mab Laucella et al. (1996a,b)
Human cardiac myosin B13 protein H rDNA, Ab, Cunha-Neto et al. (1995, 1996) and
heavy chain T cell clones Abel et al. (1997)
Human cardiac myosin Cruzipain M Ab Giordanengo et al. (2002)
heavy chain
95 kDa myosin tail T. cruzi cytoskeleton M Mab Oliveira et al. (2001)
Skeletal muscle calcium- SRA Rb, H AS Acosta et al. (1983) and Santos-Buch et al.
dependent SRA (1985)
Smooth and striated 150 kDa protein H, M Serum IgG Zwirner et al. (1994)
muscle
Glycosphingolipids glycosphingolipids H, M Serum IgG Vermelho et al. (1997)
MAP (brain) MAP H, M rDNA, AS Kerner et al. (1991)
Myelin basic protein T. cruzi soluble extract M Serum IgG, T Al-Sabbagh et al. (1998)
cells
28 kDa lymphocyte 55 kDa membrane H, M Mab Hernandez-Munain et al. (1992)
membrane protein protein
(continued)
TABLE 6.1 (continued)
Molecular
Host component T. cruzi antigen Host definition Reference
47 kDa neuron protein FL 160 H rDNA, AS Van Voorhis and Eisen (1989) and
Van Voorhis et al. (1991, 1993)
23 kDa ribosomal protein 23 kDa ribosomal H Ab Bonfa et al. (1993)
protein
Ribosomal P protein Ribosomal P protein H rDNA, Ab, SP Levitus et al. (1991)
b1 adrenoreceptor M2 Ribosomal P0 and P2b H rDNA, Ab, SP Ferrari et al. (1995), Kaplan et al. (1997),
cholinergic receptor proteins Lopez Bergami et al. (1997, 2001),
Masuda et al. (1998) and Mahler et al.
(2001)
b1-adrenoreceptor M2 150 kDa protein H, M Mab Cremaschi et al. (1995)
cholinergic receptor
M2 cholinergic receptor unknown H Ab Motran et al. (1998)
38-kDa heart antigen R13 peptide from M IgG1, IgG2 Hernandez et al. (2003)
ribosomal protein
P1, P2
Cha antigen SAPA, 36 kDa M Ab, T cell Girones et al. (2001b)
TENU2845
Calreticulin Calreticulin H,M Ab Ribeiro et al. (2009)
M, mouse; H, human; Rb, rabbit; R, rat; AS, antiserum; Ab, patient antibody; Mab, monoclonal antibody; rDNA, recombinant DNA; SP, synthetic peptides.
TABLE 6.2 Evidence for pathological autoimmunity in Chagas disease
T. cruzi antigen Host Effect References
Effects of immunization with T. cruzi antigens

T. cruzi microsomal fraction Rb Myocarditis Teixeira and
Santos-Buch
(1975)
T. cruzi SRA M Myocarditis Acosta and Santos-
Buch (1985)
T. cruzi microsomal and M Myocarditis Ruiz et al. (1985)
cytoplasmic fractions
Recombinant ribosomal M ECG Lopez Bergami et al.
protein P2b alteration (1997)
R13 peptide from ribosomal M ECG Motran et al. (1998)
protein P0 alteration
Immunological effectors Host Effect References
Effects of passive transfer of antibodies or T cells from chronically

T. cruzi-infected hosts
Splenocytes M Focal myocarditis Laguens et al. (1981)
CD4þ T-cell lines M Demyelination Hontebeyrie-Joskowicz
et al. (1987)
CD4þ T splenocytes M Focal myocarditis dos Santos et al. (1992)
and Silva-Barbosa
et al. (1997)
Anti-T. cruzi MAb M cAMP synthesis, Zwirner et al. (1994)
Increased cardiac and Cremaschi et al.
contractility (1995)
Mouse anti-receptor M Modulation of Mijares et al. (1996)
Ab calcium channels
Anti-M2 muscarinic H Conduction defect de Oliveira et al. (1997)
Ab from arrythmic in rabbit hearts and Masuda et al.
patients (1998)
Anti-M2 receptor O2 H Decreased Goin et al. (1991, 1994)
loop Ab from contractility of rat and Leiros et al.
Chagasic patients atria (1997)
Ab against T. cruzi P H Accelerate beating Ferrari et al. (1995) and
protein/b1 on rat Kaplan et al. (1997)
adrenoreceptor cardiomyocytes
Antigen Host Effect References
Effects of immunological tolerance induction with heart antigens

Myosin-enriched heart M Modulation of chronic Pontes-de-Carvalho
homogenate myocarditis and et al. (2002)
fibrosis
Myosin M Acute myocarditis was Leon et al. (2001,
not modulated 2003)
M, mouse; Rb, rabbit; H, human; Ab, antibody; Mab, monoclonal antibody.

6.2. NATURAL HISTORY OF CHAGAS DISEASE
The high parasite load typical of the acute infection ensues a strong
innate and adaptative immune response against T. cruzi, leading to the
control—but not the complete elimination—of tissue and blood parasit-
ism, establishing a low-grade persistent infection regardless of the clinical
progression of the disease (Martin et al., 1987).
Chagas disease cardiomyopathy, the most clinically significant conse-
quence of T. cruzi infection, is an inflammatory cardiomyopathy that
occurs in 25–30% of patients, 5–30 years after infection. About a third of
patients developing CCC present a particularly lethal form of dilated
cardiomyopathy, with shorter survival than idiopathic dilated cardiomy-
opathy, often presenting with severe arrhythmia and heart block (Bocchi
and Fiorelli, 2001; Mady et al., 1994). Five to 10 percentage of infected
patients develop denervation of parietal smooth muscle in the oesopha-
gus and colon, with clinical obstructive disease (Koberle, 1968). Cardiac or
digestive ‘syndromes’ of chronic Chagas disease may also present in
isolated or overlapping forms. Sixty to 70 percentage chronically
T. cruzi-infected individuals remain devoid of both cardiac and digestive
manifestations and are otherwise asymptomatic (also called ‘indetermi-
nate’ patients). Functional damage of the autonomic nervous system is
also observed, affecting a subgroup of patients presenting the cardiac,
digestive or asymptomatic forms of chronic Chagas disease (Amorim and
Marin Neto, 1995).
6.3. HEART-SPECIFIC INFLAMMATORY LESIONS IN CCC:

PARASITE ANTIGEN-DRIVEN IMMUNOPATHOLOGY?
The major histopathological feature attending dilated cardiomyopathy in

CCC is the presence of a diffuse myocarditis, with intense heart fibre
damage and significant fibrosis, in the presence of very scarce T. cruzi
forms (Higuchi et al., 1987; Higuchi Mde et al., 1993). Our group demon-
strated a significant correlation between myocarditis and fibrosis and
ventricular dilation in the Syrian hamster model of CCC (Bilate et al.,
2003). Since it is known that T. cruzi establishes a lifelong, low-grade
infection, the possibility that chronic myocardial inflammation and tissue
damage in CCC are a consequence of recognition of parasite antigen on
target tissue must be entertained (Higuchi et al., 1997; Kalil and Cunha-
Neto, 1996). A direct role for heart parasitism has been proposed after the
identification of T. cruzi antigen and DNA in CCC hearts by immunohis-
tochemical and PCR techniques (Higuchi Mde et al., 1993; Jones et al.,
1993). In addition, T. cruzi-specific CD8þ T cells have been isolated from
Autoimmunity 135
endomyocardial biopsies of a CCC patient (Fonseca et al., 2005),

providing evidence for the recruitment and expansion of T. cruzi-specific
T cells in the myocardium. In experimental T. cruzi infection, a higher
inoculum or parasite load has been associated to more aggressive chronic
heart inflammation or disease (Bilate et al., 2003; Marinho et al., 1999).
Several findings, however, fail to lend support to local recognition of
T. cruzi as the major trigger of heart tissue damage at the chronic phase of
Chagas disease. Low-grade parasite persistence is universal in CCC and
asymptomatic patients (Riarte et al., 1999; Sartori et al., 1998) and not
linked to the development of CCC (Britto et al., 1995; Pereira et al., 1992).
Other evidence against it include: (i) T. cruzi DNA has been detected in
hearts of both asymptomatic patients, just as frequently as among CCC
patients (Anez et al., 1999; Olivares-Villagomez et al., 1998); (ii) CD4þ T-
cell clones obtained from the heart tissue of a CCC patient failed to
recognize several recombinant and crude T. cruzi antigens (Cunha-Neto
et al., 1996); (iii) low-grade parasitism associated to focal inflammatory
foci in the absence of any organ functional damage is widespread in
several organs apart from the heart (Barbosa and Andrade, 1984;
Vazquez et al., 1993, 1996); (iv) immunohistochemistry and in situ hybri-
dization failed to disclose an association between inflammatory lesions
and the presence of T. cruzi antigen or DNA in hearts from Chagas disease
patients (Elias et al., 2003; Palomino et al., 2000). Taken together, evidence
suggests that the local presence of parasites may not be sufficient—or
even necessary—for inducing inflammatory tissue damage.
6.4. IMMUNOPATHOGENESIS OF CCC
Susceptibility factors leading 30% of T. cruzi-infected patients to develop

CCC are largely unknown. Since the bulk of evidence indicates that the
inflammatory infiltrate is a significant effector of heart tissue damage, we
will review the effect of cytokines and chemokines in the pathogenesis of
CCC.
Chronic infection with T. cruzi induces a systemic shift in the periph-
eral blood mononuclear cell (PBMC) cytokine profile towards Th1 cyto-
kines, with suppression of Th2 cytokines (Abel et al., 2001; Cunha-Neto
et al., 1998b; Gomes et al., 2003; Ribeirao et al., 2000). PBMC from CCC
patients displays an increased production of IFN-g by T cells (Abel et al.,
2001; Gomes et al., 2003) or CCR5þ CXCR3þ CD4þ and CD8þ T cells, as
compared to asymptomatic patients (Gomes et al., 2005). This has been
linked to decreased production of IL-10 (Gomes et al., 2003). In addition,
PBMC from CCC patients displays a reduced number of IL-10-producing
CD4þCD25high regulatory T cells and CD4þCD25highFoxP3þ regu-
latory T cells, as well as increased numbers of CD4þCD25þCTLA4þ
regulatory T cells (Araujo et al., 2007) as compared to PBMC from patients

in the asymptomatic form of Chagas disease. This is in line with the
finding of increased numbers of FoxP3þ mononuclear cells in myocardial
sections from asymptomatic as compared to CCC patients (de Araujo
et al., 2011). All chronically T. cruzi-infected patients, even from the
asymptomatic form, display increased plasma levels of TNF-a as com-
pared to seronegative individuals. Further, patients displaying severe
CCC present significantly higher plasma levels of TNF-a and CCL2
(Ferreira et al., 2003; Talvani et al., 2004). The proinflammatory and Th1-
type cytokine profile described above among chronically T. cruzi-infected
patients may be related to the ability of molecules from persisting T. cruzi
parasites to stimulate strong innate immunity and continuously induce
the production of IL-12 and other proinflammatory cytokines (Camargo
et al., 1997). It has recently been reported that later stages of CD4þ T-cell
differentiation are associated with more severe stages of Chagas disease,
suggesting that chronic T. cruzi infection might exhaust long-lived mem-
ory T cells (Albareda et al., 2009).
The inflammatory infiltrate of CCC heart lesions is composed by
macrophages (50%), T cells (40%; 2:1 predominance of CD8þ over
CD4þ T cells) and B cells (10%) (Higuchi Mde et al., 1993; Milei et al.,
1992). CD8þ T cells in CCC heart tissue were found to express Granzyme A
(Reis et al., 1993a). The demonstration of restricted heterogeneity of T-cell
receptor Va transcripts in heart biopsies from CCC patients (Cunha-Neto
et al., 1994) is in line with similar findings in established auto-
immune diseases (Heber-Katz and Acha-Orbea, 1989).
Heart-infiltrating mononuclear cells predominantly produce IFN-g
and TNF-a, consistent with the peripheral cytokine profile (Abel et al.,
2001; Reis et al., 1993a, 1997); expression of the cytokines IL-4, IL-6, IL-7
and IL-15 has also been described (Fonseca et al., 2007; Higuchi Mde et al.,
1993; Reis et al., 1993a, 1997). Accordingly, CCC heart tissue also displays
increased expression of adhesion molecules, HLA class I and class II
molecules (Reis et al., 1993b). Recent studies have shown that FoxP3þ
cells are significantly more abundant in myocardial sections from asymp-
tomatic than in CCC patients or in infected individuals, suggesting that
regulatory T cells are less abundant in CCC than in asymptomatic hearts
(de Araujo et al., 2011). In addition, increased expression of mRNA for
chemokines CCL2/MCP-1, CXCL10/IP-10 and CXCL9/MIG as well as
their receptors CCR2 and CXCR3 was observed in CCC heart tissue,
(Cunha-Neto et al., 2005), consistent with chemokine-driven migration
of monocytic and Th1 T cells to the CCC heart. Gene expression profiling
of CCC myocardial tissue showed that 15% of genes known to be selec-
tively upregulated in CCC are IFN-g inducible (Cunha-Neto et al., 2005).
Moreover, exposure of neonatal murine cardiomyocytes to IFN-g upre-
gulates expression of atrial natriuretic factor (Cunha-Neto et al., 2005), a
Autoimmunity 137
marker of cardiomyocyte hypertrophy and heart failure. Together, these

observations suggest that IFN-g-mediated chronic myocardial inflamma-
tion could contribute to the pathogenesis of CCC, both by eliciting direct
inflammatory damage and by modulation of cardiac cell gene expression.
Mechanisms underlying differential progression to CCC by only 30%
of chronically T. cruzi-infected patients are still incompletely understood.
Familial aggregation of CCC cases has been described (Zicker et al., 1990),
suggesting the existence of a genetic component in susceptibility. Associ-
ation of polymorphic markers of innate immunity genes such as TNF-a,
lymphotoxin-a, MAL/TIRAP (an adaptor protein involved in the TLR2-
and TLR4-signalling pathway), BAT1 (an inhibitor of inflammatory cyto-
kines), NFKBIL1 (potential inhibitor of NFKB) and CCL2 with CCC has
been reported (Ramasawmy et al., 2006a,b, 2008, 2009, reviewed in
Cunha-Neto et al., 2009). Further, we have shown that severe CCC
patients carrying the high TNF-a expresser genotype have shorter
survival (Drigo et al., 2006). Identification of key genes and potent
genetic combinations coupled with environmental factors may lead to
the identification of T. cruzi-infected individuals that will progress
to CCC.
6.5. AUTOIMMUNITY IN CHAGAS DISEASE
The observation that most tissue pathology occurs many years after acute
infection, when parasites were very scarce in tissue, led investigators as
early as 80 years ago (Torres, 1929) to suggest that the mononuclear cell
infiltrate should directly damage the heart, perhaps in an autoimmune
fashion. Early studies were characterized by the lack of molecular defini-
tion of the antigen systems employed; most used tissue or T. cruzi homo-
genates. Peripheral T cells from experimentally infected mice and CCC
patients displayed responses against cardiac tissue homogenate (de la
Vega et al., 1976; Gattass et al., 1988). Non-infected cardiomyocytes
were targets of cytotoxicity by PBMC from chronically infected rabbits
(Santos-Buch and Teixeira, 1974) and CCC patients (Teixeira et al., 1978).
Repeated injection of T. cruzi subcellular fractions induced myocardial
inflammatory lesions in mice and rabbits (Acosta and Santos-Buch, 1985;
Teixeira and Santos-Buch, 1975).
Cossio et al. (1974) described antibodies binding to vascular endothe-
lium and interstitium in mice in the serum of CCC patients, that could be
absorbed with T. cruzi epimastigotes (Table 6.1), but these were found to
be antibodies against a-galactosyl moieties, structures present in rodent,
but not in human tissue (Khoury et al., 1983). Experimentally infected
mice frequently developed T. cruzi-heart muscle cross-reactive antibodies
(Laucella et al., 1996b; McCormick and Rowland, 1989). Conversely,
mice with experimental autoimmune myocarditis induced by immuniza-

tion with heart homogenate developed anti-T. cruzi antibodies (Chambo
et al., 1990).
Several mechanisms have been suggested to play a role in the trigger-
ing of autoimmunity after infection. The three mechanisms described
below have been demonstrated in Chagas disease patients or murine
models and could generate experienced, effector autoreative T or B cells
capable of inducing tissue damage.
(i) Antigen exposure. T. cruzi infection promotes tissue damage and a
consequent exposure of intracellular proteins, along with activation of
innate immunity and myocardial inflammatory response during the acute
and chronic phases of infection, with upregulation of MHC class I and
class II proteins (Reis et al., 1993b). Self-epitopes may be presented by
tissue dendritic cells in the context of MHC and upregulated costimula-
tory molecules (Smith and Allen, 1992). T-cell sensitization to cardiac
myosin has been shown to occur during acute T. cruzi infection (Leon
et al., 2001).
(ii) Molecular mimicry. T and B cells recognize parasite antigens that
present molecular mimicry with antigenically similar epitopes in host
antigens, generating cross-reactive autoimmune responses.
(iii) Polyclonal activation. Acute murine T. cruzi infection induces
antibody production that lacks a T. cruzi specificity and includes
self-antigens, suggesting polyclonal B cell activation (Minoprio et al.,
1988). The T. cruzi-secreted protein TcPA45 has been described as a
T cell-independent B cell mitogen in mice (Minoprio, 2001).
6.5.1. Autoantibodies
During T. cruzi infection, mice can display antibodies specific for various
autoantigens contained in target tissues. Chronically T. cruzi-infected
mice display anti-tubulin IgG antibodies (Ternynck et al., 1990). Sera
from acutely or chronically infected mice recognized cardiac myosin,
desmin and actin (Leon et al., 2001; Tibbetts et al., 1994). In human Chagas
disease, there is a net loss of neurons from the autonomic system along the
hollow viscerae and the heart (Koberle, 1968), and sera from over 80% of
Chagas disease patients contained anti-neuron autoantibodies (Ribeiro
dos Santos et al., 1979). Antibodies against sciatic nerve homogenate
have been found in sera from Chagas disease patients (Gea et al., 1993).
Antibodies against ribonucleoproteins (Bach-Elias et al., 1998) have been
detected during T. cruzi infection. Autoantibodies against galectin-1 are
correlated with the severity of cardiac damage in CCC (Giordanengo
et al., 2001). The Cha human autoantigen and its major B cell epitope
Cha are recognized by sera from Chagas disease patients (Girones et al.,
2001a,b).
Autoimmunity 139
Agonistic antibodies against adrenergic G-protein-coupled receptors

and the second loop of muscarinic (M2) cholinergic receptors have been
described (Borda et al., 1984; Goin et al., 1991, 1994, 1997; Sterin-Borda
et al., 1991). Sera from CCC patients interfere with electric and mechanical
activities of embryonic myocardial cells in vivo (Costa et al., 2000; Kaplan
et al., 1997), and sera from CCC patients induce arrhythmia in rabbit
hearts (de Oliveira et al., 1997). Anti-muscarinic receptor antibodies and
abnormal vagal modulation occur early in Chagas disease patients, inde-
pendently of the presence of left ventricular dysfunction (Ribeiro et al.,
2007). Chagas disease patients showing colonic denervation syndrome
display agonistic anti-M2 muscarinic cholinergic receptors (Sterin-Borda
et al., 2001). Differential patterns of autoantibodies towards cardiovascu-
lar receptors have been associated to CCC, asymptomatic and megacolon
Chagas patients (Wallukat et al., 2011). The presence of such agonistic
anti-receptor antibodies does not correlate with heart symptomatology
but rather with dysfunction of the autonomic nervous system (Goin et al.,
1997). However, the pathogenic potential of non-functional (i.e. non-ago-
nistic) autoantibodies is still a matter of debate. Complement C5–C9
membrane attack complexes were found in membranes of cardiomyocyte
from CCC heart tissue (Aiello et al., 2002), suggesting that complement
activation—perhaps induced by autoantibodies—could play a role in
heart tissue damage.
6.5.2. Autoreactive T cells

The first evidence for the T-cell recognition of a defined heart-specific
autoantigen was provided by Rizzo et al. (1989), who showed that CD4þ
T cells from chronically T. cruzi-infected mice proliferated in vitro in the
presence of syngeneic cardiac myosin. Acutely T. cruzi-infected mice
developed delayed-type hypersensitivity response against cardiac myo-
sin and displayed intense myocarditis (Leon et al., 2001); anti-myosin
autoimmunity was found not to be essential for acute T. cruzi myocarditis
(Leon et al., 2003). Induction of tolerance with a myosin-enriched cardiac
homogenate plus anti-CD4 antibody prior to T. cruzi infection resulted in
reduction of chronic myocarditis and fibrosis when compared to non-
tolerized infected mice (Pontes-de-Carvalho et al., 2002). Myosin is the
most abundant heart protein, making up to 50% of muscle protein by
weight (Harrington and Rodgers, 1984). It is a major antigen in several
instances of heart-specific autoimmunity (Caforio et al., 1992;
Cunningham et al., 1997; Neu et al., 1987b; Vashishtha and Fischetti,
1993); moreover, immunization with cardiac myosin in complete
Freund’s adjuvant induces severe T-cell-dependent myocarditis in genet-
ically susceptible mice (Liao et al., 1993; Neu et al., 1987a, 1990; Smith and
Allen, 1991). A recent study has shown that T cells from patients with the
gastrointestinal form of Chagas disease recognize an epitope in myelin

basic protein, suggesting this could be the target of autoimmunity leading
to denervation characteristic of the megacolon/oesophagus (Oliveira
et al., 2009).
Passive transfer of lymphoid cells can validate the pathogenic role of
autoimmune T cells. The transfer of T-cell populations from chronically
infected mice to nerve sheaths of naı̈ve syngeneic recipients induced nerve
inflammatory lesions (Hontebeyrie-Joskowicz et al., 1987). Moreover,
injection of CD4þ T cells from BALB/c mice chronically infected with T.
cruzi adjacent to newborn syngeneic hearts that had been grafted into
naı̈ve BALB/c recipients resulted in complete rejection of the transplanted
heart (dos Santos et al., 1992), in the absence of T. cruzi DNA (Mengel and
Ribeiro-dos-Santos, 1998). Using a similar system, but employing different
T. cruzi-mouse strain combinations, another group reported that inflam-
mation could only be found in the presence of T. cruzi (Tarleton et al.,
1997). A heart-specific CD4þ T-cell line from a chronically T. cruzi-infected
mouse induced death of embryonic cardiac cells in vitro (Ribeiro-Dos-
Santos et al., 2001). Transfer of this T-cell line to BALB/c nude mice
simultaneously immunized with syngeneic heart homogenates resulted
in intense myocarditis (Ribeiro-Dos-Santos et al., 2001). Adoptive transfer
of splenic T cells from chronically infected mice to naı̈ve
recipients induced myocarditis in the latter and triggered antibody
response against the Cha autoantigen (Girones et al., 2001b). Passive
transfer and tolerance induction experiments are summarized in Table 6.2.
6.6. MOLECULAR MIMICRY

There have been several reports of immunological cross-reactivity/anti-
genic mimicry between more or less defined T. cruzi and host self-anti-
gens (summarized in Table 6.1). To determine whether exposure to
T. cruzi antigens alone in the absence of active infection is sufficient to
induce autoimmunity, Bonney et al. immunized mice with heat-killed
T. cruzi (HKTC). This immunization was capable of inducing acute car-
diac damage, associated with the generation of polyantigenic humoral
and cell-mediated autoimmunity with similar antigen specificity to that
induced by infection with T. cruzi (Bonney et al., 2011). Antibodies recog-
nizing calcium-dependent ATPase from the heart muscle sarcoplasmic
reticulum membranes (SRA—sarcoplasmic reticulum antigen) cross-reac-
tively recognized microsomal membranes from T. cruzi (Acosta et al.,
1983). Immunization with T. cruzi calreticulin induces antibodies that
recognize human and murine heart calreticulin and induces focal inflam-
matory heart infiltrates (Ribeiro et al., 2009).
Cross-reactive neuron-T. cruzi antibodies have been frequently
described, as displayed in Table 6.1 (Petry et al., 1987a; Snary et al.,
Autoimmunity 141
1983; Wood et al., 1982). Sulphated glycolipids and neutral glycosphingo-

lipids found in T. cruzi are essentially the same as found in mammalian
hosts and are cross-reactively recognized by antibodies formed along
infection (Petry and Eisen, 1989; Vermelho et al., 1997). Administration
of monoclonal antibodies cross-reactively recognizing sulphated glycoli-
pids in T. cruzi and neurons induced immediate paralysis and death
by respiratory insufficiency (Petry and Eisen, 1989; Petry et al., 1988).
A cross-reactive epitope was identified between T. cruzi FL-160, and a
neuronal 47 kDa protein (Van Voorhis et al., 1991, 1993), but the autoanti-
body failed to correlate with any clinical form of Chagas disease (Cetron
et al., 1992). Cross-reactivity between T. cruzi and myelin basic protein
was observed at the level both of antibodies and T cells in experimentally
infected mice (Al-Sabbagh et al., 1998) (Table 6.1). Sera from T. cruzi-
infected mice and Chagas disease patients contained cross-reactive anti-
bodies recognizing microtubule-associated proteins from T. cruzi and
fibroblasts (Kerner et al., 1991). Sera from CCC patients possessed anti-
bodies against a C-terminal epitope of T. cruzi ribosomal P2b protein
which is conserved in mammalian ribosomal P protein (Levin et al.,
1989; Levitus et al., 1991). Agonistic anti-b1-adrenergic and M2 musca-
rinic receptors cross-reactive with different T. cruzi antigens were
reported (Cremaschi et al., 1995; Ferrari et al., 1995; Kaplan et al., 1997;
Masuda et al., 1998). Immunization of mice with T. cruzi ribosomal pro-
tein P2b (Lopez Bergami et al., 1997) and the R13 peptide from ribosomal
P protein (Motran et al., 1998) induced electrocardiographic alterations, in
the absence of myocardial inflammation (summarized in Table 6.2).
Cardiac myosin is a target of T cell and antibody recognition by acute
and chronically T. cruzi-infected mice (Iwai et al., 2001; Rizzo et al., 1989;
Tibbetts et al., 1994). Myosin-specific delayed-type hypersensitivity
response could be induced in mice by immunization with protein extract
of T. cruzi, in the absence of detectable cardiac damage, suggestive of
cross-reactivity between cardiac myosin and T. cruzi antigens (Leon et al.,
2004). Mice immunized with cruzipain, a major cystein protease from
T. cruzi, devoid of enzymatic activity developed cross-reactive anti-car-
diac myosin heavy chain autoantibodies, electrocardiographic conduc-
tion disturbances and myositis (Giordanengo et al., 2000a,b).
Cunha-Neto et al. (1995) detected anti-human ventricular cardiac
myosin heavy chain IgG antibodies in similar levels among sera from
individuals in the CCC, asymptomatic and healthy soronegative subjects
(Cunha-Neto et al., 1995). Affinity-selected anti-human ventricular car-
diac myosin heavy chain antibodies from Chagas disease patients sera
specifically recognized a defined T. cruzi antigen (Cunha-Neto et al.,
1995), the recombinant tandemly repetitive protein B13 (Gruber and
Zingales, 1993) (Table 6.1). Cardiac myosin-B13 cross-reactive antibodies
were predominantly found in sera from CCC rather than asymptomatic
patients (Cunha-Neto et al., 1995). CD4þ T-cell clones expanded
from heart tissue of a CCC patient in the absence of exogenous antigen
cross-reactively recognized cardiac (but not skeletal) myosin heavy chain

and T. cruzi protein B13 (Table 6.1; Cunha-Neto et al., 1996). However,
in vitro sensitization of lymphocytes from a T. cruzi seronegative individ-
ual with T. cruzi B13 protein or its peptides elicited B13-cardiac myosin-
cross-reactive T-cell clones (Abel et al., 1997; Cunha-Neto et al., 1998a; Iwai
et al., 2005). The T-cell response to B13 protein was restricted to HLA-DR1,
HLA-DR2 and HLA-DQ7, and B13 peptides were able to bind to these HLA
molecules (Abel et al., 2005). A B13 peptide-specific T-cell clone was estab-
lished from an HLA-DQ7 individual, that cross-reactively recognized
cardiac myosin b chain peptide (5–19). Although only 5 of 15 amino acids
residues were homologous between two peptides, amino acid scanning
analysis and molecular modeling of HLA-DQ7:peptide complexes indi-
cated that TCR-exposed side chains in the cardiac myosin and B13 peptide
were almost identical (Abel et al., 2005; Iwai et al., 2005). In addition, we
identified multiple very low homology cross-reactive epitopes between B13
protein and human cardiac myosin (Iwai et al., 2005). The recognition of
multiple low-homology, cross-reactive epitopes in a single autoantigenic
protein indicates intramolecular degenerate recognition which may poten-
tially increase the magnitude and frequency of occurrence of the T-cell-
driven autoimmune response in CCC and other autoimmune diseases.
This leads to the hypothesis that in vivo sensitization with B13 antigen
along T. cruzi infection could break immunological tolerance towards
cardiac myosin and elicit cardiac myosin-responsive T cells in vivo.
6.7. CONCLUSION
Chagas disease is a conundrum of several clinical syndromes triggered by

T. cruzi infection in a group of susceptible individuals. Expression of
clinical syndromes can be non-overlapping. It is therefore not surprising
that several different systems of molecular mimicry have been identified.
Inasmuch as several of the cross-reactive immune responses may be
simply secondary to sequence conservation or degeneracy in immune
recognition (Mason, 1998), and thus being inconsequential to pathogene-
sis, it is likely that some instances of cross-reactive recognition may play
an important pathogenetic role. Several reports displayed in Table 6.2 that
match criteria for pathologic autoimmunity (Rose and Bona, 1993) have
been identified as follows: (i) the identification of T-cell cross-reactive
antigens, with reproduction of pathobiological changes by passive trans-
fer in murine models in the absence of T. cruzi parasites; (ii) the ameliora-
tion of inflammation as a consequence of tolerance induction to
myocardial antigens; and (iii) the induction of cross-reactive autoimmu-
nity and end-organ dysfunction after immunization with T. cruzi or heart
antigens. The isolation of T. cruzi-heart antigen cross-reactive T cells from
Autoimmunity 143
myocardial tissue of CCC patients is considered important indirect evi-

dence for pathological autoimmunity. It is likely that the persistence of a
parasite which induces strong innate immunity and proinflammatory
cytokines may continuously boost the production of potentially patho-
genic Th1 T cells cross-reactively recognizing T. cruzi and heart-specific
epitopes. Such Th1 T cells may migrate to heart tissue in response to
locally expressed CXCR3 ligand ckemokines. Once they reach myocardial
tissue, cross-reactive T cells could be activated by cardiac antigen even in
the absence of T. cruzi antigens. This would elicit local production of Th1
cytokines. Local production of Th1 cytokines could exert their pathophys-
iological role by causing direct inflammatory damage, as well as modu-
lating cardiac cell gene expression. Functional agonistic autoantibodies
directed against adrenergic or cholinergic receptors may also have an
important role on autonomic system disorders and play a role in heart
conduction disorders and arrhythmias. Genetic polymorphisms of
immune response genes may affect recognition, migration and effector
characteristics of autoreactive T cells and autoantibodies. Finally, it must
be stressed that autoimmune and T. cruzi-specific innate or adaptative
responses are not incompatible or mutually exclusive, and it is likely that
a combination of both is involved in the pathogenesis of CCC (Fig. 6.1).
T. cruzi
Acute infection
Antigen exposure Molecular mimicry Polyclonal activation
Loss of tolerance to heart antigen
Genetic susceptibility T. cruzi persistence

(SNPs)
Functional Pathogenic Innate

autoantibodies autoreactive T cells immunity
Chronic chagas disease cardiomyopathy
FIGURE 6.1 Potential role of autoimmunity in the pathogenesis of chronic Chagas

disease cardiomyopathy.
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CHAPTER 7
ROS Signalling of Inflammatory
Cytokines During Trypanosoma
cruzi Infection
Shivali Gupta,* Monisha Dhiman,* Jian-jun Wen,*
and Nisha Jain Garg*,†,‡
Contents 7.1. Reactive Oxygen Species and Source 154

7.2. Inflammatory Cytokines During Trypanosoma cruzi
Infection and Chagas Disease 156
7.3. ROS Signalling of Cytokine Responses 158
7.4. The Impact of Oxidative Stress and Cytokine Mediators
and Cardiac Dysfunction 160
7.5. Conclusions and Future Directions 162
References 164
Abstract Inflammation is a host defence activated by exogenous (e.g. patho-

gen-derived, pollutants) or endogenous (e.g. reactive oxygen spe-
cies—ROS) danger signals. Mostly, endogenous molecules (or their
derivatives) have well-defined intracellular function but become
danger signal when released or exposed following stress or injury.
In this review, we discuss the potential role of ROS in chronic
evolution of inflammatory cardiovascular diseases, using our
experiences working on chagasic cardiomyopathy as a focus-point.
* Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA
{
Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA
{
Faculty of the Center for Tropical Diseases, Sealy Center for Vaccine Development, and the Institute for
Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, USA

153
154 Shivali Gupta et al.
7.1. REACTIVE OXYGEN SPECIES AND SOURCE

Broadly defined, reactive oxygen species (ROS; e.g. O2, OH, and H2O2)
are derivatives of molecular oxygen. The site and extent of ROS produc-
tion has important consequences and determines the ultimate cell/tissue
fate. ROS can be formed in the heart, vascular tissue, splenocytes, and
blood leukocytes through the action of specific oxidases and oxygenases
(e.g. xanthine oxidase, NADPH oxidase—NOX), peroxidases (e.g. myelo-
peroxidase), the Fenton reaction, and as by-products of the electron trans-
port chain of mitochondria (Turrens, 2003). Further, cyclooxygenase,
lipooxygenase, and cytochrome P-450 enzymes produce ROS as a by-
product during arachidonic acid metabolism (Cohen, 1994). Nitric oxide
(NO) is produced by the enzymatic activity of nitric oxide synthases
(NOS), which oxidize L-arginine, transferring electrons from NADPH.
Different NOS isoforms have been identified for example, inducible
NOS (iNOS) in phagocytic cells, mtNOS in mitochondria, (eNOS) in
endothelial cells, and neuronal nNOS (Andrew and Mayer, 1999). Read-
ers are referred to recently published review articles for further details on
ROS biochemistry (D’Autreaux and Toledano, 2007) and the role of iNOS
and NO in Chagas disease (Gupta et al., 2009b).
We focus on two major ROS producers relevant in Chagas disease
here. The prototypic NOX (gp91phox), renamed as NOX2, was first identi-
fied in phagocytes (neutrophils, macrophages). When activated, NOX
catalyses a rapid ROS production by the one-electron reduction of O2,
referred as respiratory burst that serves as the first line of host defence
against microbes. Presently, seven mammalian NOX homologs have been
identified, namely NOX1–NOX5, dual oxidase 1 and 2 (DUOX1 and
DUOX2). In cardiovascular system, NOX1, NOX2, NOX4, and NOX5
have been identified. NOX1 is expressed mainly in vascular smooth
muscle cells (VSMCs). NOX2 and NOX4 are expressed in endothelial
cells, cardiomyocytes, fibroblasts, and VSMCs (Weintraub, 2002). NOX5
has been reported in human endothelial cells and smooth muscle cells but
is not found in rodents (Belaiba et al., 2007).
The earliest studies have reported cytochemical detection of NOX at
plasma membrane of peritoneal mouse macrophages during interaction
with Trypanosoma cruzi (Cardoni et al., 1997). Others have used in vitro
assay systems or animal models and demonstrated that T. cruzi-mediated
macrophage activation results in increased levels of O2 formation, likely
by NOX-dependent oxidative burst (Alvarez et al., 2004; Melo et al., 2003;
Munoz-Fernandez et al., 1992). We have extended these observations and
shown that splenocytes of infected mice and in vitro cultured macro-
phages respond to T. cruzi infection by activation of NOX2 and a substan-
tial increase in ROS production (Dhiman and Garg, 2011). A robust
ROS Signalling of Inflammatory Cytokines During Trypanosoma cruzi Infection 155
response of splenocytes of infected mice to T. cruzi antigenic lysate sug-

gested that parasite factors (and not active invasion) are sufficient to
activate NOX and ROS generation. Indeed, T. cruzi-derived components
are recognized by toll-like receptors (GPI-anchor and Tc52: TLR2; GIPL:
TLR4; and TcDNA: TLR9) and NOD-like receptors, implicated in NOX
activation (Kayama and Takeda, 2010; Lipinski et al., 2009; Silva et al.,
2010). Yet, further studies are required to identify the T. cruzi-generated
stimuli that activate TLR- and NOD-signalling mechanisms and initiate
translocation of cytosolic components (p47phox, p67phox, and G-protein)
and NOX assembly during Chagas disease.
In the heart, in response to T. cruzi infection, infiltrating activated
neutrophils and macrophages produce NOX- and myeloperoxidase-
dependent ROS and are a major source of oxidative stress during the
acute stage (Garg, unpublished data). Endothelial activation of xanthine
oxidase in response to T. cruzi infection and resultant increase in O2
production is also shown (Berry and Hare, 2004; Hernandez et al., 2009).
Other NOX isoforms, and other forms of ROS producers that may be
expressed by various cell types in the heart and induced by hormones,
haemodynamic forces, or local metabolic changes in the heart, remain to
be identified in experimental models of T. cruzi and human patients.
Recent studies provide evidence for mitochondrial release of O2 as a
major source of ROS and oxidative stress in chagasic myocardium. The
heart is highly dependent on mitochondria for the energy required for its
contractile and other metabolic activities. Mitochondria represent 30% of
the total volume of cardiomyocytes and provide 90% of the cellular ATP
energy through oxidative phosphorylation. Though mitochondrial degen-
erative changes, that is, swelling, irregular membranes, and loss of cristae,
were recognized by electron microscopic analysis of heart biopsies from
chagasic patients and experimental animals (Carrasco Guerra et al., 1987;
Garg et al., 2003; Palacios-Pru et al., 1989; Parada et al., 1997), the signifi-
cance of these observations have been explored in the last decade only.
It is now documented that chagasic hearts sustain mitochondrial dysfunc-
tion at gene expression, protein, and biochemical activity levels. Global
microarray profiling of gene expression has identified alterations in sev-
eral of the mitochondrial function related transcripts in the myocardial
biopsies of infected humans (Cunha-Neto et al., 2005) and experimental
animals (Garg et al., 2004; Mukherjee et al., 2003). Further studies
documented a decline in the activities of respiratory complexes, NADH-
ubiquinone reductase (CI) and ubiquinol-cytochrome c reductase (CIII;
Vyatkina et al., 2004) and ATP synthase (CV) complex (Uyemura et al.,
1996), in chagasic murine hearts. The functional effect of these perturba-
tions was shown by decreased mitochondrial respiration (Uyemura et al.,
1995), and reduction in myocardial and mitochondrial ATP level
(Nian et al., 2004; Wen et al., 2006) in chagasic experimental models.
Mitochondrial dysfunction also contributed to increased ROS produc-

tion. The rate of electron leakage and O2 formation in mitochondria is
closely related to the coupling efficiency between the respiratory chain
and oxidative phosphorylation. The CI and CIII complexes are the main
sites for electron leakage to O2 and O2 generation in mitochondria
(Chen et al., 2003; Ide et al., 1999), and their compromised activity in
chagasic myocardium was associated with excessive electron leakage
and ROS production (Vyatkina et al., 2004). Inhibition studies have
shown that CI was not the main source of increased ROS in chagasic
hearts. Instead, defects of the myxothiazol-binding site in CIII complex
resulted in enhanced electron leakage towards the Qo-centre and contrib-
uted to increased ROS generation in chagasic cardiac mitochondria (Wen
and Garg, 2008). Thus, conditions conducive to oxidative stress are pre-
sented in the chagasic heart.
Cardiomyocytes are a major cell type producing mitochondrial ROS in
chagasic conditions. Utilizing the adult rat primary cardiomyocytes, and
murine and human cardiomyocyte cell lines, we demonstrated that
invading T. cruzi elicit substantial ROS production that was further
enhanced by inflammatory milieu (IFN-g, TNF-a, and IL-1b). Inhibition
studies showed that ROS in cardiomyocytes infected by T. cruzi were not
produced by NOX, xanthine oxidase, and myeloperoxidase. Instead,
mitochondrial electron leakage to O2 was enhanced at the respiratory
chain resulting in O2 formation that coincided with a loss of mitochon-
drial membrane potential (Dcm) and inhibition of CI and CIII complex
activities (Gupta et al., 2009a). Cardiomyocytes lacking mtDNA (rho),
and, therefore, functional electron transport chain, exhibited no increase
in ROS in response to T. cruzi, thus, validating mitochondria as primary
source of ROS in cardiomyocytes (Gupta et al., 2009a).
7.2. INFLAMMATORY CYTOKINES DURING TRYPANOSOMA

CRUZI INFECTION AND CHAGAS DISEASE
Several inflammatory cytokines have been shown to contribute to cardiac
dysfunction under various pathophysiological conditions associated with
heart failure, including I-R injury, myocardial infarction, atherosclerosis,
hypertrophy, and acute viral myocarditis (Aukrust et al., 2005; Hori and
Nishida, 2009; Khaper et al., 2010; Laura et al., 2010; Neumann et al., 1993;
Nian et al., 2004; Tedgui and Mallat, 2006). Cytokines and chemokines
implicated in the progression of heart failure include TNF-a, IL-1, IL-6,
IL-8, IL-13, IL-18, IFN-g, cardiotrophin-1, monocyte chemoattractant pep-
tide-1 (MCP-1), and macrophage inflammatory protein-1 alpha (MIP-1a),
the pro-inflammatory mediators; and transforming growth factor-beta
(TGF-b) and IL-10 that are the anti-inflammatory mediators (Aukrust
et al., 2005; Chandrasekar et al., 2003; Damas et al., 2001; Euler-Taimor

and Heger, 2006; Frangogiannis et al., 2007; Mann, 2002; Stumpf et al.,
2008; Torre-Amione et al., 1996). Of these, TNF-a and IFN-g are of special
interest as the circulating and tissue levels of these cytokines at both the
protein and mRNA levels are elevated in chagasic patients and consid-
ered to reflect the severity of heart failure (Cunha-Neto et al., 1998;
Samudio et al., 1998; Van Voorhis, 1992).
The innate and adaptive immune responses triggered by the parasite
and its derived surface molecules (e.g. GPI anchors) during the acute
phase lead to exacerbated production of inflammatory cytokines (e.g. IL-
12, TNF-a, IFN-g) and chemokines such as CCL3 (MIP-1a), CXCL10 (IP-
10), and CCL5 (RANTES) (Hardison et al., 2006a,b; Machado et al., 2005).
The enhanced expression of cytokines (TNF-a, IL-1b, and IL-6) and che-
mokines (e.g. RANTES, MIP-2) at mRNA and protein levels has also been
noted in the myocardium of T. cruzi-infected experimental models
(Chandrasekar et al., 1998; Machado et al., 2000; Talvani et al., 2000). It is
generally accepted that macrophages and dendritic cells (DCs), upon
phagocytosing parasites, produce cytotoxic NO and ROS to kill the invad-
ing T. cruzi (Gazzinelli et al., 1992; Munoz-Fernandez et al., 1992). The
activated macrophages also express IL-12, TNF-a, and costimulatory
molecules that prime the IFN-g-producing specific T cells that then
migrate to the target organs in response to chemokines produced in
infected tissues. Using the genetic knockout mice or the antibodies for
depletion of specific immune molecules, it is shown that blockage of type 1
cytokines (IFN-g, TNF-a) correlates with increased susceptibility to T. cruzi
infection (Miller et al., 1997; Reed, 1988; Zacks et al., 2005). Contrarily, a
complete absence of Th2 or anti-inflammatory cytokines has severe nega-
tive effects on the infected host. For example, IL-10-deficient mice infected
with T. cruzi develop a syndrome similar to endotoxic shock due to the
enhanced production of TNF-a and IFN-g (Holscher et al., 2000; Reed et al.,
1994). Collectively, these results point to the importance of both inflam-
matory and anti-inflammatory responses during T. cruzi infection and
indicate that IL-4 þ IL-10/TNF-a þ IFN-g ratio may be an important
determinant of desirable outcome. Indeed, we have shown using a vacci-
nation approach that a polarized response with dominance of IFN-g, TNF-
a, and CD8þ T cells in the acute phase and IL-4, IL-10, and CD4þ T cells in
the chronic phase was most efficacious in providing protection from T.
cruzi infection and disease. This was because the protective effects of CD8þ
T cells and type 1 cytokines (IFN-g and TNF-a) were not suppressed by IL-
4 and IL-10 cytokines leading to an effective control of acute infection in
vaccinated mice. Later on, vaccinated mice switched to type 2 dominance
that suppressed the activation and infiltration of IFN-g- and TNF-a-pro-
ducing immune cells that otherwise cause tissue damage and injury in
chagasic heart (Gupta and Garg, 2010).
Similar to experimental studies, the inflammatory infiltrate found in

the heart tissue of chagasic patients contains phagocytes, and CD4þ and
CD8þ T cells. Chagasic patients exhibit a Th1 type (IFN-g) cytokine profile
with suppression of Th2 type cytokines (IL-4, IL-10), and elevated plasma
levels of TNF-a persist in chronic stage (Cunha-Neto and Kalil, 2001;
Cunha-Neto et al., 1998; Higuchi et al., 1997; Higuchi Mde et al., 1993).
Peripheral blood mononuclear cells from chronic chagasic patients pro-
duce more IFN-g and less IL-10 than indeterminate patients (Souza et al.,
2004). Moreover, increased myocardial expression of adhesion molecules,
MCP-1, IP-10, and MIG and their receptors CCR2 and CXCR3, and cyto-
kines IFN-g, TNF-a, IL-4, IL-6, and IL-15 has been reported in chagasic
patients by several researchers (Machado et al., 2000, 2005; Silva et al.,
1995; Teixeira et al., 2002). Gene expression profiling of myocardial tissue
from chagasic experimental animals and human patients showed that
15% of genes known to be selectively upregulated are IFN-g-inducible
(Cunha-Neto et al., 2005). These observations, point to the pathologic
significance IFN-g and TNF-a in chagasic cardiomyopathy.
Besides inflammatory cells, non-immune cells also respond to T. cruzi
infection by cytokine production. Infection of endothelial cells with T. cruzi
caused induction of IL-1b and IL-6 (Tanowitz et al., 1992). A released
surface protein of T. cruzi, trans-sialidase, induced IL-6 production in
isolated endothelial cells (Saavedra et al., 1999). Our finding of increased
TNF-a and IL-1b mRNAs in infected cardiomyocytes suggested that car-
diomyocytes also respond to T. cruzi by inflammatory cytokine production
(Ba et al., 2010). Whether cytokine response by the non-immune cells is a
component of innate immunity or a bystander effect to T. cruzi infection is
not known and remain to be investigated in future studies.
7.3. ROS SIGNALLING OF CYTOKINE RESPONSES
In addition to their capacity to kill microbes, ROS are critical signalling

intermediates linking the innate and adaptive immune systems by trig-
gering the production of pro-inflammatory cytokines (TNF-a, IL-1b) by
macrophages and DCs of the innate immune system. The innate immune-
derived signal, required for maturation of the adaptive immune response,
is dependent on the redox-sensitive signalling pathways (Curtsinger
et al., 1999; Pape et al., 1997; Tse et al., 2004). ROS elicits a wide spectrum
of cellular responses through the activation of intracellular signalling
pathways (Hensley et al., 2000; Tanaka et al., 2001; Thannickal and
Fanburg, 2000; Ueda et al., 2002). Of note is the redox regulation of
MEKK (MAPK/ERK kinase kinase), PKC (protein kinase c), NIK (NF-k
B-inducible kinase) cascades, and transcriptional factors (e.g. NF-kB,
AP-1, Nrf-2) that translate extracellular signals into intracellular
responses (Tanaka et al., 2001) and differentially regulate the expression

of pro-inflammatory mediators (Dong et al., 2002), and protective antiox-
idants such as g-glutamyl cysteine synthetase (gGCS), manganese super-
oxide dismutase (MnSOD), and hemeoxygenases (Rahman, 2003;
Rahman and MacNee, 2000). The Rel/NF-kB transcriptional pathway is
an important intracellular signalling pathway for both innate and
acquired immunity, and it has received most attention from the perspec-
tive of the role of ROS in activation of pro-inflammatory gene transcrip-
tion in human myocardium. The noticeable pro-inflammatory mediators
and chemotactic factors that are regulated by ROS-mediated NF-kB acti-
vation include MCP-1, IL-6, TNF-a, IL-1a, and IL-1b. Cytokines and
chemokines, in turn, also stimulate cascade of events leading to increased
oxidative stress (Aukrust et al., 2001; Dhingra et al., 2007; Guggilam et al.,
2007; Nakamura et al., 1998; Tatsumi et al., 2000). Further, neurohor-
mones, such as catecholamines, angiotensin II, aldosterone, endothelin-
1, are potential contributors to the pro-inflammatory phenotype of heart
failure and known to enhance oxidative stress both directly and indirectly
(Sano et al., 2001; Sun et al., 2006; Wei et al., 2002; Wu et al., 2005). The
currently available literature has not addressed the mechanistic role of
cytokines and neurohormones in elicitation of ROS and oxidative stress in
chagasic conditions. We, therefore, focus on ROS signalling of cytokine
responses that have been examined in recent studies.
Inhibition studies with cultured and primary macrophages showed
that NOX/ROS was a critical regulator of cytokine production in response
to T. cruzi infection. In vivo studies using splenocytes of T. cruzi infected
mice, with or without in vitro stimulation with parasite antigens, vali-
dated the above observations and demonstrated that inhibition of NOX
by apocynin or DPI or use of ROS scavenger substantially inhibited the
activation and proliferation of phagocytes and inflammatory mediators
(IL-1, IL-6, IFN-g, and TNF-a; Garg, unpublished data). Further studies
will be required to delineate if NOX/ROS signal nuclear transport and
activation of transcription factors (e.g. NF-kB and AP-1) and promote
cytokine gene expression, or if NOX/ROS elicit immune cell proliferation
and thereby indirectly alter cytokine profile in infected mice.
Others have shown that T. cruzi trypomastigotes (or Tc-proteins e.g.
trans-sialidase) activate NF-kB in a number of cell types, including epi-
thelial cells, endothelial cells, myocytes, and fibroblasts (Hall et al., 2000;
Huang et al., 1999, 2003). NF-kB activation increased the resistance to
infection in many of these cell types. Except for myocytes, it, however,
remains to be seen whether ROS play a role in cytokine gene expression in
non-phagocytic cells invaded by T. cruzi. In cardiomyocytes infected by
T. cruzi, mtROS elicited cytokine gene expression via multiple mechan-
isms. One, mtROS enhanced nuclear translocation of RelA (p65), thereby
activating NF-kB-dependent gene expression of inflammatory cytokines
(e.g. TNF-a, IFN-g, IL-1b; Ba et al., 2010). Two, ROS caused 8-hydroxygua-
nine (8-oxoG) lesions and DNA fragmentation that signalled polyadenosine
ribose polymerase 1 (PARP-1) activation, evidenced by poly-ADP-ribose
(PAR) modification of PARP-1, and other proteins in infected cardiomyo-
cytes. PARP-1 signals DNA repair via PARylation of histones; however, its
hyperactivation may have pathophysiological effects ranging from catalytic
activation of inflammatory and hypertrophic gene expression, depletion of
NADþ pool, and cell death (Balakumar and Singh, 2006; Pacher and Szabo,
2008). Inhibition of PARP-1 using RNAi or chemical inhibitor (PJ34), or by
removal of ROS using an antioxidant, was beneficial in blocking the mtROS
formation and DNA damage (Ba et al., 2010). Importantly, we found that
PARP-1 inhibition also regulated cytokine gene expression, albeit via a
different mechanism. PARP-1 did not directly interact with p65, and it did
not signal RelA (p65) translocation to nuclei in infected cardiomyocytes.
Instead, PARP-1 contributed to PAR-modification of RelA (p65)-interacting
nuclear proteins and assembly of NF-kB transcription complex. These stud-
ies suggested that ROS-PARP-1-RelA signalling pathway contribute to
inflammatory cytokine production in cardiomyocytes infected by T. cruzi.
It remains to be seen whether mitochondria serve as activator of an innate
defence response by cardiomyocytes upon T. cruzi exposure, or these events
are bystander effects of T. cruzi infection of the host cells.
7.4. THE IMPACT OF OXIDATIVE STRESS AND CYTOKINE

MEDIATORS AND CARDIAC DYSFUNCTION
We believe that both oxidants and pro-inflammatory cytokines affect

cardiac function in chagasic cardiomyopathy over time in a biphasic
manner. As detailed above, studies in experimental models and human
patients demonstrate that infected host sustains oxidative stress due to
T. cruzi-elicited splenic NOX/ROS and enhanced mitochondrial release of
ROS in the myocardium and also produce inflammatory cytokines by
phagocytic and T cells, some of these activities being essential for parasite
control. The host also responds by an increase in antioxidant reserve and
anti-inflammatory responses to control the injuries that can be elicited by
oxidative stress and inflammatory cytokines. Glutathione (GSH), gluta-
thione peroxidase (GPx), and MnSOD have been shown to be most critical
in cardiac antioxidant defences, particularly in protecting the cardiomyo-
cytes from oxidative injury (Marczin et al., 2003). Our studies demon-
strated that host responds to acute T. cruzi infection by upregulating
glutathione antioxidant defence constituted by GPx, glutathione S-reduc-
tase, and GSH. However, after the initial burst, the glutathione defence
was unresponsive to chronic oxidative stress. In chronic phase,
pro-oxidant milieu in the heart was evidenced by (a) increased ROS
levels; (b) decreased activity of MnSOD; (c) insensitivity of glutathione

defence to oxidative stress; and (d) increased GSSG, and lipid (malon-
dialdehydes—MDA) and protein (carbonyl) oxidation products (Wen
et al., 2004). A similar pro-oxidant status in seropositive human patients
is evidenced by (a) increased GSSG and MDA contents; (b) decreased
MnSOD, GPx activity, and GSH contents (de Oliveira et al., 2007; Perez-
Fuentes et al., 2003); and (c) inhibition of CIII activity (Wen et al., 2006).
Treatment of chagasic experimental animals with an antioxidant tipped
the balance in favour of preservation of mitochondrial and cardiac func-
tion. Infected mice and rats, treated with an antioxidant, exhibited a
substantial increase in mitochondrial function evidenced by improved
complex activities and ATP synthesis, and decreased ROS production.
Antioxidant-treated rodents also exhibited a significant decline in the
myocardial accumulation of MDA-, HNE-, and carbonyls-adducts in
chronic phase (Wen et al., 2006). Importantly, preventing the oxidative
injuries in chronic stage preserved the cardiac haemodynamics that oth-
erwise were compromised in chagasic rats (Wen et al., 2010). Others have
shown a decline in oxidative stress in human chagasic patients given
Vitamin A (Macao et al., 2007). All of these observations support the
idea that antioxidant depletion and inefficient scavenging of ROS, result-
ing in sustained oxidative stress, are of pathological importance in human
CCM progression.
A mixed cytokine response to T. cruzi infection, elicited in both
humans and animals (reviewed in Zacks et al., 2005), is a mixed blessing
as it results in control of acute parasitaemia, and physiological hypertro-
phy or minimal to no myocarditis, but fails to eliminate infection. Conse-
quently, ensuing parasite persistence results in a prolonged maladaptive
phase that is clinically asympotomatic but ultimately leads to pathological
state of heart failure associated with pathological hypertrophy, fibrosis,
apoptosis, necrosis, and pro-oxidant and pro-inflammatory responses
(Arnaiz et al., 2002; Chandrasekar et al., 1998; de Oliveira et al., 2007;
Machado et al., 2000; Pereira Barretto et al., 1986; Perez-Fuentes et al.,
2003; Petkova et al., 2001; Rossi et al., 2003; Talvani et al., 2000; Wen et al.,
2004). It appears that a summed up balance between pro-oxidant and
pro-inflammatory mediators with antioxidant and anti-inflammatory
mediators determines the overall response of either protection (physio-
logical) or damage (pathological) during chronic phase of Chagas disease.
The re-expression of foetal genes (ANP, BNP, ask-actin, and b-MHC) is
a hallmark of hypertrophic remodelling, and a considerable body of
evidence shows the redox regulation of various signalling cascades and
remodelling responses in cardiac diseases of various etiologies (Liaudet
et al., 2009). Current evidence supports the involvement of the following
pathways: (i) ERK-1/2 (Xiao et al., 2001, 2002) and the small GTPase Ras
(Kuster et al., 2005) in response to a-adrenergic agonist stimulation and
angiotensin II (Nakagami et al., 2003; Satoh et al., 2006), (ii) MAPKs in

pressure-overload hypertrophy (Li et al., 2002), and (iii) NF-kB and apo-
ptosis-signal-regulated kinase 1 (ASK-1) in response to angiotensin II
infusion (Satoh et al., 2006). ASK-1 is upstream of p38MAPK and JNK in
the MAPK signalling cascade, and both of these have been shown to be
activated by NOX/ROS (Matsuzawa and Ichijo, 2005). Inhibition or scav-
enging of free radicals has been shown to modulate the ERK signalling
and hypertrophic responses in neonatal and adult cardiomyocytes
(Nakamura et al., 1998; Tanaka et al., 2001). In Chagas disease, our
observation of a decline in the expression of hypertrophic markers and
collagen deposition in response to antioxidant treatment suggested that
ROS signals pathological hypertrophic remodelling in chagasic myocar-
dium. The role of ROS of mitochondrial, but not of inflammatory, origin
in signalling hypertrophy in chagasic hearts was evidenced by the obser-
vation that NOX and MPO, the classical mediators of inflammatory ROS,
were equally depressed upon treatment of infected rodents with anti-
parasite drug (benznidazole) and ROS scavenger (phenyl-a-tert-butyl
nitrone—PBN), yet hypertrophic phenotype was depressed in PBN-trea-
ted rodents only (Wen et al., 2010). The specific signalling pathways
regulated by ROS in T. cruzi-induced hypertrophy await further
elucidation.
Besides ROS, experimental studies have shown that the inflammatory
cytokines (e.g. TNF-a, IL-1b, and MCP-1) also promote myocardial hyper-
trophy and contribute to the development and progression of heart failure
(Gullestad and Aukrust, 2005). Further studies are required to identify
whether inflammatory cytokines, noted to be enhanced in chagasic exper-
imental animals and human patients (reviewed in Cunha-Neto et al.,
1998; Zacks et al., 2005), synergistically enhance the ROS-mediated
signalling cascades involved in activation of hypertrophic responses in
chagasic hearts.
7.5. CONCLUSIONS AND FUTURE DIRECTIONS
Host responds to T. cruzi infection by eliciting inflammatory cytokines

(TNF-a, IFN-g) and ROS production. Sustained ROS generation of inflam-
matory and mitochondrial origin, coupled with an inadequate antioxi-
dant response, results in inefficient scavenging of ROS in the heart and
leads to long-term oxidative stress. Thus, while ROS are essential for
activation of inflammatory responses and pathogen control in acute
stage, the persistent oxidative stress denies the control of inflammatory
state. Further, intracellular T. cruzi or Tc-antigens that persist during late
or chronic infection might interact with the immune and non-immune
cells in the myocardium and subsequently activate signalling cascades

(e.g. NF-kB pathway) that trigger the production of inflammatory cyto-
kines (TNF-a, IL-1b), ROS-induced DNA damage, and hypertrophy in
cardiomyocytes. Importantly, inflammatory cytokines and ROS create a
complex feedback mechanism that can positively sustain the stress
responses, and thus, play an important role in cardiac remodelling and
evolution of chronic Chagas disease.
The above discussed literature strongly suggests the important patho-
physiological role of increased oxidative stress and inflammatory
responses in the genesis of heart failure in various cardiomyopathies
including chagasic heart disease. In experimental settings, beneficial
effects of antioxidants (e.g. Vitamin A, C, and E, PBN), especially in the
short term, have been documented both in human patients (Macao et al.,
2007) and in animal models of Chagas disease (Wen et al., 2006, 2010).
However, it must be noted that large-scale randomized trials testing the
efficacy of anti-cytokine (TNF-a; reviewed in Khaper et al., 2010) and
antioxidant (reviewed in Houston, 2010) therapies in humans have not
been successful in preventing the heart disease. Several possible factors
including dosage used, period of treatment, bioavailability of the com-
pounds, and the number of enrolled patients have been put forward to
explain the lack of desired outcome. What may be needed are specific
agents that target defined ROS sources, inflammatory cytokines, and
deleterious redox-dependent signalling pathways. Specific targeting of
ROS-dependent pathways receives support from the beneficial effects of
drugs that are already in use. For example, statins that inhibit the activa-
tion of small GTPases such as Rac, and thereby affect NOX activity, are
widely used to treat heart patients. Several experimental studies have
indicated favourable outcome of statin use in regulating cardiac hyper-
trophy and remodelling after myocardial infarction, and preliminary
studies in small number of patients indicate that the benefits are also
afforded in patients with congestive heart failure (Chen et al., 2004;
Ichihara et al., 2006; Takemoto et al., 2001). We propose that substantial
effort should be made in delineating the complex interrelationships
between the oxidative stress and inflammatory mediators, wherein the
promise of antioxidant and anti-inflammatory therapies in controlling
progressive chagasic cardiomyopathy can be realized.
ACKNOWLEDGEMENTS
The work in NJG’s laboratory has been supported in part by grants from the American Heart
Association, John Sealy Memorial Endowment Fund for Biomedical Research, American
Health Assistance Foundation, and National Institutes of Health. S. G. is an awardee of a
Postdoctoral Fellowship from the Sealy Center of Vaccine Development.
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CHAPTER 8
Inflammation and Chagas
Disease: Some Mechanisms
and Relevance
André Talvani* and Mauro M. Teixeira†,‡
Contents 8.1. The Multiple Roles of Inflammation in

Chagas Disease 172
8.2. Experimental Models for Studying Inflammation and
Immune Mechanisms in Chagas Disease 173
8.3. Mediators of Protection in Experimental
8.4. Mediators of Inflammation and Their Role
in Mediating Tissue Damage and Protection
in Experimental Trypanosoma cruzi Infection 177
8.4.1. Chemokines 181
8.4.2. Lipid mediators 185
8.4.3. Endothelin 185
8.5. Conclusion 186
References 187
Abstract Chagas cardiomyopathy is caused by infection with flagellated

protozoan Trypanosoma cruzi. In patients, there is a fine balance
between control of the replication and the intensity of the inflam-
matory response so that the host is unable to eliminate the parasite
* Laboratório de doença de Chagas, Departamento de Ciências Biológicas & NUPEB, Universidade Federal de
Ouro Preto, Ouro Preto, Minas Gerais, Brazil
{
Laboratório de Imunofarmacologia, Departamento de Bioquı́mica e Imunologia/ICB, Universidade Federal
de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
{
Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

171
172 André Talvani and Mauro M. Teixeira
resulting in the parasite persisting as a lifelong infection in most

individuals. However, the parasite persists in such a way that it
causes no or little disease. This chapter reviews our understanding
of many of the mediators of inflammation and cells which are
involved in the inflammatory response of mammals to T. cruzi
infection. Particular emphasis is given to the role of chemokines,
endothelin and lipid mediators. Understanding the full range of
mediators and cells present and how they interact with each
other in Chagas disease may shed light on how we modulate
disease pathogenesis and define new approaches to treat or
prevent the disease.
8.1. THE MULTIPLE ROLES OF INFLAMMATION IN

CHAGAS DISEASE
Inflammation is a fundamental disease process, side by side in relevance
with neoplastic transformation and cellular degeneration. The major goal
of the inflammatory process is to restore integrity and function of cells
and tissues after injury by harmful agents (biological, chemical or physi-
cal). The inflammatory process can, therefore, be viewed as an adaptive
response that is triggered by noxious stimuli and conditions, including
infection by pathogenic microorganisms. The ancient Greeks were first to
recognize and describe cardinal signs of inflammation (inflammation
derives from the Latin word ‘‘inflammare’’—to set on fire): heat, redness,
pain and swelling. Loss of function is another cardinal sign of inflamma-
tion that was added later. All of them reflect physiological events which
are part of a classical acute inflammatory response (Rocha e Silva, 1978).
The inflammatory response involves the release of a large number of
soluble mediators, including amines, lipid mediators, complement, oxy-
gen derivatives, adhesion molecules, cytokines and chemokines (Garside
and Brewer, 2010; Sorokin, 2010). The most frequent and desirable out-
come after an inflammatory response is full restoration of tissue integrity
and function. However, when the inflammatory process is chronic, not
controlled, excessive, misplaced or insufficient (e.g. after certain infec-
tions), tissue damage may occur (Garcia et al., 2010). Persistence of the
inflammatory process, be it due to persistence of the stimulus or absence
of endogenous regulatory mechanisms, may lead to chronic inflammation
and ensuing tissue injury and fibrosis.
Chagas disease is caused by infection with the protozoan parasite
Trypanosoma cruzi. In the acute setting, there is massive inflammatory
response in the various infected tissues and it is believed that this inflam-
mation is necessary to clear infection. Indeed, absence or decrease of
inflammatory responses will lead to excessive parasite replication and
Inflammation and Chagas Disease: Some Mechanisms and Relevance 173
death of the animal. An excessive inflammatory response, as it occurs in

the absence of control mechanisms, may lead to tissue damage and death
of the host (Aliberti et al., 2001; Golgher and Gazzinelli, 2004; Teixeira
et al., 2002). The host, therefore, needs to strike a fine balance between
control of parasite replication and the intensity of the inflammatory
response. In humans, this fine balance is achieved in such a way that
the host is unable to eliminate the parasite and it persists as a lifelong
infection in most individuals. In the clinical context, this is referred to as
the indeterminate form of Chagas disease. In the chronic setting, con-
trolled immune and inflammatory mechanisms are acting systemically or
in infected tissues in a subtle way to keep infection under control and to
modulate inflammatory and immune responses as to prevent tissue dam-
age. In the absence of inflammatory and immune responses, there can be
enhanced parasite replication and host death in the absence of immune
protective mechanisms, as seen in infected patients taking immunosup-
pressant drugs (Bacal et al., 2010). On the other hand, some 10–30% of
infected individuals will go on to develop heart disease or mega syn-
dromes. In these patients who develop severe chronic disease, there is
evidence that there are deficient regulatory mechanisms (e.g. relative loss
of IL-10 in relation to IFN-g production) that may contribute to disease
pathogenesis (Dutra et al., 2005; Gomes et al., 2003). Therefore, it is clear
that in Chagas disease, and indeed in most infectious diseases, the inflam-
matory and immune response of the host need to act in fine balance to
destroy the parasite, or achieve a level of coexistence compatible with no
or little disease, and restore tissue integrity (Garcia et al., 2010).
Here, we review some of the mediators of inflammation and cells
which are involved in the inflammatory and immune response of mam-
mals against T. cruzi infection. Understanding the full range of mediators
and cells expressed and how they interact with each other in Chagas
disease may shed light on how we understand pathogenesis and define
new approaches to treat or prevent the disease.
8.2. EXPERIMENTAL MODELS FOR STUDYING

INFLAMMATION AND IMMUNE MECHANISMS
IN CHAGAS DISEASE
If we are to examine the relative role of mediators and cells in Chagas

disease, it is necessary to comprehend the limitations of experimental
models that try to reproduce the clinical, pathological and immune para-
meters of Chagas disease. Rodents, especially mice, are the most widely
used species for studying T. cruzi infection in vivo. There are several
reasons which favour the use of these animals, including (i) easy of
obtaining and handling, (ii) low cost, (iii) most genetic lineages are
susceptible to T. cruzi infection and (iv) diversity of genetic background

including isogenic and knockout animals (Costa, 1999; Romanha et al.,
2010). In mice and in other species, the strain and load (number) of
parasites inoculated are essential parameters to define survival and levels
of parasitaemia (Bértoli et al., 2006). For example, time to death varies
according to parasite genetic background, from few days (e.g. Y strain) to
a few weeks (e.g. Colombian strain) in the absence of anti-T. cruzi therapy.
A major limitation of published studies in mice is that most experimental
studies have focused on modelling acute T. cruzi infection. Indeed, a great
deal of experiments in mice has focused on evaluation of parameters such
as parasitaemia and lethality at early time points (days to few weeks) after
infection. This short time course is useful to study chemotherapy,
immune responses and the ability of the host to deal with the parasite,
but is less useful to understand the chronic situation in humans, in whom
the acute phase of disease lasts for 2–4 months and is mostly unnoticed
(Fig. 8.1). In humans, acute infection usually subsides and gives place to a
chronic phase that will last the entire life of the individual or will cause his
death (Rocha et al., 2007). Therefore, acute murine models of T. cruzi
infection are useful to understand the mediators of inflammation and
immunity necessary to deal with acute infection but less so to study the
chronic disease observed in humans. Murine models have also been
elected as ideal to test anti-T. cruzi molecules (Paula-Costa et al., 2010;
Romanha et al., 2010; Waghabi et al., 2009a).
Several newer developments are worth mentioning because of the
move in the direction of chronic murine models in which heart damage,
rather than acute measures such as lethality and parasitaemia, is a major
defining parameter of morbidity. Modelling chronic Chagas in mice
may be more relevant for the human disease and sheds light on
Months Years Decades

Time line
1 2 3 4 1 2 3 4 5 1 2 3 4 5 6 of infection
Mice
Dogs
Human
Acute phase
Chronic phase
FIGURE 8.1 Timeline of Trypanosoma cruzi infection in human and experimental models.
Timeline demonstrated above is the temporal course followed by different hosts after
T. cruzi infection and takes in consideration the approximate time in months, years and
decades described for these models (mice, rats, dogs) in the literature.
immunopathogenetic mechanisms operating in Chagas disease, in spite

of the shorter lifespan of mice. Examples of interesting systems which
have been developed include use of certain parasite strains and lower
inocula of virulent strains. For example, (i) some parasite strains (e.g.
Brazil strain) will cause little parasitaemia and lethality and will induce
significant heart damage as determined by echocardiography or magnetic
resonance imaging (Chandra et al., 2002a,b; Jelicks et al., 2002); (ii) lower
inoculum (50–100 parasites per animal) with the Colombian strain may
also cause longer term infection (4–8 months) that is accompanied by
chronic pathological changes in the heart (Garcia et al., 2005; Soares
et al., 2010; Talvani et al., 2000).
Rats have also been used to study certain aspects of Chagas disease,
especially central nervous system damage, hormonal alterations and
myocardites. Even these animals have been used to investigate chronic
aspects of heart disease; they are highly informative models to evaluate
acute immune and neurological aspects of T. cruzi infection. Indeed,
young animals display more susceptibility to T. cruzi infection, whereas
adult rats revealed to develop a mild heart disease with extremely low
parasitaemia. This phenomenon suggests there exists a close relation
between immune response and disease, in particular, conducted by anti-
bodies that can either induce lysis of parasites or facilitate trypomastigote
removal from the circulation (Pascutti et al., 2003).
Monkeys (Carvalho et al., 2003) and dogs (de Lana et al., 1992; Guedes
et al., 2002) have been used as alternatives for studies of chronic T. cruzi
infection. The putative advantage of dogs in the context of chronic Chagas
disease is the longer life span of these animals (around 15–20 years).
Young dogs are susceptible to infection with T. cruzi and develop acute
and chronic phases, with indeterminate and cardiac forms, in a similar
manner to human infection, including the presence of diffuse inflamma-
tion and fibrosis (Laranja and Andrade, 1980). In addition, these animals
have cellular and humoral immune responses, clinical and pathological
manifestations of congestive heart failure that resemble those observed in
human chagasic cardiomyopathy (de Lana et al., 1992; Diniz et al., 2010;
Guedes et al., 2010; Melo et al., 2011). On the other hand, there are clear
limitations: the expense, and ethical and moral concerns associated with
the use of dogs and the lack of genetic tools to be used in these animals, to
cite the most relevant in our view.
As observed in Fig. 8.1, there is a clear temporal issue when one tries to
study any particular pathogenesis mechanism(s) in experimental models
that try to mimic T. cruzi infection. Chagas disease presents an acute phase
followed by a chronic phase that, particularly in human, the chronic
cardiac disease is more important clinically. Therefore, selection of the
appropriate animal model to the appropriate phase of the disease is crucial
to gain correct insight into disease pathogenesis. There are no reasons to
prevent use of any particular animal species and parasite strain to model
Chagas disease. Any animal model has limitations. The appropriate use of
these tools must be guided by the question to be answered and kinetic
issues related to the course of infection need to be fully taken into account.
8.3. MEDIATORS OF PROTECTION IN EXPERIMENTAL

TRYPANOSOMA CRUZI INFECTION
Experiments in rodents have shown that several mediators of the inflam-
matory and immune responses are essential for the ability of the host to
deal with T. cruzi infection. The first line of defence against T. cruzi occurs
at the site of parasite entry in which resident cells, likely of the monocytic
lineage, interact with parasites. Innate immune cells may interact with
protozoan-derived glycosylphosphatidylinositol (GPI) anchors via pat-
tern recognition receptors (Toll-like receptors ‘‘TLR’’ family) located on
the plasma surface membrane (TLR-1, 2, 4, 5 and 6) or in the cytoplasm
endosomal membrane (TLR-3, 7, 8 and 9) of the cell (Bafica et al., 2006;
Campos and Gazzinelli, 2004; Koga et al., 2006; McGettrick and O’Neill,
2010). Non-TLR receptors, including mannose receptor and cytosolic
receptors of the nucleotide-binding oligomerization domain (NOD)-like
and retinoic acid-inducible gene I (RIG-I)-like receptor families, have also
been recently shown to elicit immune activation events when bound to
T. cruzi (Silva et al., 2010). Binding of parasite molecules to cells of the
monocytic lineage will release several pro-inflammatory mediators,
including cytokines (TNF-a and IL-12), chemokines and nitric oxide—
NO (Camargo et al., 1997; Coelho et al., 2002; Talvani et al., 2009). These
interactions provide the first line of defence, albeit of limited efficacy,
against infection and provide the necessary inflammatory signals for the
adaptive immune response.
IL-12 stimulates natural killer cells to amplify the synthesis of IFN-g
and generation of type I lymphocyte response which stimulates macro-
phages to release more TNF-a, IL-12, NO and chemokines, generating an
amplified positive feedback system (Bastos et al., 2007; Ropert and
Gazzinelli, 2000; Teixeira et al., 2002). Macrophages activated by IFN-g
and TNF-a play an important role in the control of parasite growth in the
initial phases of the infection. It is believed that macrophage-derived NO
is crucial for this process (Silva et al., 1995; Vespa et al., 1994). NO is a free
radical generated from enzymatic deamination of L-arginine by NO
synthases (Clark and Rockett, 1996). During the last decade, in vitro and
in vivo experiments have suggested that other mediators (e.g. eicosanoids,
cytokines and chemokines) also contribute to induce macrophage
activation and promotion of NO synthesis, possibly by regulating iNOS
expression (Coelho et al., 2002; Talvani et al., 2002, 2003, 2009).
The production of chemokines is essential to recruit leukocytes into

infected tissues (dos Santos et al., 2001; Paiva et al., 2009; Yamauchi et al.,
2007). By attracting leukocytes, chemokines are relevant for mediating
protection against infection but also contribute to tissue inflammation and
eventual damage. For instance, in the absence of CCR5 (receptor for
CCL5/RANTES), CD4þ and CD8þ T cells do not migrate into the infected
heart and there is uncontrolled parasite replication (Machado et al., 2005),
which culminates in the death of the animals.
During the acute phase of the experimental infection in mice, CD4þ T
cells are the predominant subset followed by CD8þ T cells. As disease
progresses, CD8þ T cells take over as the predominant inflammatory cell
type. Both CD4þ/CD8 and CD8þ/CD4 T cells in the infected myocar-
dium were characterized as activated cells expressing LFA-1high, VLA-
4high and CD62Llow (dos Santos et al., 2001). Similar to what has been
discussed above and by others, CD4þ and CD8þ T cells are relevant to
control parasite replication (e.g. there is uncontrolled replication and high
parasitism in their absence), but again they may contribute to cause tissue
inflammation and damage as disease progresses (Brener and Gazzinelli,
1997; Gomes et al., 2003; Reis et al., 1993; Silverio et al., 2010).
8.4. MEDIATORS OF INFLAMMATION AND THEIR ROLE

IN MEDIATING TISSUE DAMAGE AND PROTECTION
IN EXPERIMENTAL TRYPANOSOMA CRUZI INFECTION
In addition to the protective immune mediators involved in T. cruzi infection
discussed above, there have been several studies showing the role of other
mediators of the inflammation, including chemokines, platelet-activating
factor (PAF), leukotriene B4 (LTB4), lipoxygenase 5 (5-LO) and transforming
growth factor-b (TGF-b), in the context of experimental Chagas disease
(Table 8.1). Wild-type and genetically modified knockout or transgenic
rodents (especially mice) and cell cultures (e.g. peritoneal macrophages)
stimulated with parasites or their soluble antigens have been the most useful
experimental models used to identify the role of inflammatory proteins/
peptides in the interaction with T. cruzi during acute events. Table 8.1 brings
a list describing studies in which molecules involved with the inflammatory
response have been evaluated and describes briefly the studies performed.
An incomplete picture of the interplay of these various molecules in the
context of acute and chronic Chagas disease is given in Fig. 8.2. Describing in
detail the role of each of the mediators pointed in Table 8.1 is beyond the
scope of this review. Below, we will focus on a few mediators with which
our group has worked more extensively—chemokines, lipid mediators and
endothelin. These will be used to exemplify the multiple roles of inflamma-
tion and its mediators in the context of Chagas disease.
TABLE 8.1 Inflammatory mediators in the pathogenesis of Trypanosoma cruzi infection
Inflammatory mediators Roles during Trypanosoma cruzi infection References
Platelet activator factor (PAF) " Production of CCL5 in vivo and " production of Aliberti et al. (1999),
CCL2 þ LTB4 in vitro; " NO and mice survival Rodrigues et al. (1999),
# trypanocidal activity and parasitaemia peak in vivo; Talvani et al. (2003)
" of parasite ecto-phosphatase activities
Leukotriene B4 (LTB4) " NO and TNF-a synthesis in vitro; " trypomastigotes Talvani et al. (2002), Wirth
killing in vitro and Kierszenbaum
(1985)
5-Lipoxygenase (5-LO) # Heart tissue parasitism in vivo; " erythrocyte oxidative Borges et al. (2009),
stress; " IFN-g, TNF-a, IL-6 and NO in vivo Pavanelli et al. (2010)
Prostaglandin E2 (PGE2) " Inflammatory infiltration, parasite nests and cardiac Abdalla et al. (2008),
fibrosis and its " production is due by activation of Sterin-Borda et al. (1996)
muscarinic receptors in CD8T cells
Nitric oxide (NO) " Killing of parasites by murine macrophages; " apoptosis Chandra et al. (2002a),
of T cells; modulates chemokine production by T. cruzi- Durand et al. (2009),
infected myocytes; " ventricular dilation and systolic Machado et al. (2008),
dysfunction in acute murine chagasic myocarditis Silva et al. (2003)
Brain (BNP) and atrial (ANP) In human: correlated with # LV ejection fraction, Moreira Mda et al. (2008),
natriuretic peptides " LV end-diastolic diameter, " LV premature Ribeiro et al. (2002),
complexes, " NYHA class; good predictors of death or Talvani et al. (2004c)
necessity for heart transplant (p < 0.0001)
Endothelin (ET-1) Triggered by T. cruzi-derived molecules, " NO, Camargos et al. (2004),
inflammation and fibrosis in heart tissues; " levels of Petkova et al. (2000),
ET-1 in patients with CC; " right ventricular internal Rachid et al. (2006),
diameter, " left ventricular end-diastolic, " diameter/ Salomone et al. (2001),
fractional shortening and " wall thickness in mice; Tanowitz et al. (2005)
blockade of ET-1 receptor # parasitaemia, tissue
parasitism and inflammation
Chemokines # Parasite growth and triggers the chemotaxis and Guedes et al. (2010),
(MCP-1/CCL2, RANTES/ morphogenesis of trypomastigote forms; " activation Marino et al. (2005),
CCL5 and MIP-1a/CCL3) and recruitment of heart inflammatory infiltrate; Paiva et al. (2009),
" uptake and killing of intracellular parasites by Talvani et al. (2004a),
inducing NO synthesis and production by Yamauchi et al. (2007)
macrophages and cardiomyocytes; in humans, " in
serum levels and variant in CCL2 -2518AA genotypes
suggest severe CC; " expression and seric levels are
associated with severe CC in patients and dog models
Chemokine receptors (CCR4, Participate in the control of parasite growth and in the Guedes et al. (2010),
CCR5, CCR2, CXCR4) development of a protective immune response during Hardison et al. (2006),
acute infection; " CXCR3 and CCR5 (heart) and # CCR5 Machado et al. (2005),
(PBMC) associated with severe CC; # expression levels Marino et al. (2005),
of CXCR4 in severe patients Talvani et al. (2004b)
Tumour necrosis factor " Leukocytes activation with " production of Bilate et al. (2007), Lula
(TNF-a) and interferon- inflammatory cytokines and chemokines; # parasite et al. (2009), Talvani
gamma (IFN-g) replication in murine macrophages, " (TNF-a) and et al. (2000), Talvani
# (IFN-g) associated with fibrosis; " serum levels in et al. (2004b)
patients with severe CC
Interleukin-10 " Percentage of CD4 and CD8 co-expressing CCR3 and Costa et al. (2009), Gomes
(IL-10) IL-10 in asymptomatic patients, # expression associated et al. (2005), Silva et al.
with worse cardiac function; controls (1992)
Th-1-like immune response and prevents excessive
damage in host inflamed tissue
Transforming growth factor " Invasion of cardiac fibroblasts and myocytes and Araújo-Jorge et al. (2008),
(TGF-b) modulates pro-inflammatory cytokines; " intracellular Silva et al. (1991),
parasite cycle; " fibrosis, disorganize GAP connexin-43 Waghabi et al. (2009a,b)
junctions and " heart remodelling
(continued)
TABLE 8.1 (continued)
Inflammatory mediators Roles during Trypanosoma cruzi infection References
Toll-like receptors (TLR—2, 4, TLR-2 þ NF-kB in response to T. cruzi " cardiomyocyte Bafica et al. (2006), Campos
7, 9) and nucleotide-binding hypertrophy; macrophages activation on innate and Gazzinelli (2004),
oligomerization domain immunity is TLR-dependent; natural host resistance Koga et al. (2006),
(NOD) (# parasitaemia and " surviving) is TLR-4 and NOD- Oliveira et al. (2004),
dependent; TLR# chemokine production; TLR9 has a Petersen et al. (2005),
primary role in the MyD88-dependent induction of Silva et al. (2010)
IL-12/IFN-g, products from NF-kB stimulation in vivo
are NOD-dependent in T. cruzi infection
" CD8 T cells apoptosis; " cardiac infiltration and Fas Guillermo et al. (2007), de
ligand/CD95L associated with myocarditis; # activated Oliveira et al. (2007),
T cells, # NO production and # parasites load Rodrigues et al. (2008)
Matrix metalloproteinases " TNF-a, IFN-g, nitrite and nitrate; " heart inflammation Gutierrez et al. (2008),
(MMPs) and # survival rate in T. cruzi-infected mice Nogueira de Melo et al.
(2010)
Adiponectins # Levels are associated with # inflammation and Nagajyothi et al. (2008,
" cardiovascular disease 2009)
C-reactive protein (CRP) " parasite invasion to cardiac cells through a CRP-like López et al. (2006),
molecule on T. cruzi surface; " levels associated with Aparecida da Silva et al.
worsening human heart function (with or without (2010), Melo Coutinho
T. cruzi infection) et al. (1998)
CC, Chagas cardiomyopathy; PBMC, peripheral blood mononuclear cells; LV, left ventricle; NYHA, New York Heart Association; RANTES, regulated upon activation, normal T
cell expressed and secreted; MIP-1a, macrophage inflammatory protein-alpha; MCP-1, monocyte chemoattractant protein; ", increase; #, decrease.
Acute phase Amastigote

Chronic
Chronic
cardiomyopathy
cardiomyopathy Collagen
T. cruzi
GPI-mucin
Cardiac form
↑ of TNF-a, CCL2, CCL5, TGF-b, ET-1,
NK s MMPs with leukocyte recruitment, necrosis,
p tor
cells ece apoptosis, fibrosis, cardiomyocyte hypertrophy
Dr
s /NO
LR
IF
T
N-
g
Activated Indeterminate form

phagocytic cells
med ator y
(↑ of regulatory cells,
Par
rs
FOXP3 and IL-10)

n
iato
a
vatio
mm
site ctors
fa
Infla
inte
Acti
rac
tive
PMN TNF-a
IFN-g
Mac IL-12 Mediators Control of parasite Tissue parasite
PAF replication and persistence
acting on
T cells LTB4 ↑ of inflammation
CCL2
EC
CCL3
CCL5
Cardiomyocytes NO
Chronic phase
(PMN, polymorphic mononuclear cells; Mac, macrophages; EC, endothelial cells; NO, nitric
oxide; NK, natural killer; GPI, glycophosphatidilinositol; TLR, toll like-receptors; NOD, nucleotide-
binding oligomerization domain; MMPs, matrix metalloproteinases; ET-1, endothelin-1; , increase)
FIGURE 8.2 Interconnectivity of inflammatory mediators in the acute and chronic

phases of Chagas disease. The presence of Trypanosoma cruzi activates pattern recog-
nize receptors (TLR and/or NOD) on phagocytic cells to produce inflammatory med-
iators. These mediators act on various immune cells activating them and promoting the
release of other soluble inflammatory and regulatory mediators, which may play a role
on the control of parasite replication. Parasite persistence in the host is a major
characteristic of Chagas disease. Regulatory cells and factors may contribute to parasite
persistence but also contribute to prevention of tissue damage. In some patients,
fibrogenic cardiac disease develops and is associated with marked inflammation of
infected tissues. Mediators of inflammation found in this chronic disease-prone stage
are similar to those necessary for control of acute infection.
8.4.1. Chemokines
Chemokines are small (8–14 kDa) inducible cytokines that recognize a
large group of seven transmembrane-spanning G-protein-coupled ser-
pentine receptors displayed on the leukocyte surface and are involved
in normal trafficking of leukocytes to lymphoid and nonlymphoid organs

and recruitment of these cells to inflammatory sites (Le et al., 2004;
Murphy, 1994). Initial studies have shown that cardiomyocytes infected
with Colombian strain of T. cruzi were able to express mRNA for a range
of CC chemokines, including CCL5/RANTES, CCL2/MCP-1, CCL3/
MIP-1a and CCL4/MIP-1b (Talvani et al., 2000, 2002). T. cruzi-infected
peritoneal macrophages are also able to produce chemokines and respond
to them by increasing parasite uptake and NO production. Enhanced
uptake and NO production eventually lead to better control of parasite
replication in macrophages (Aliberti et al., 1999). Interestingly, chemo-
kines may cooperate with IFN-g and TNF-a to facilitate NO production
and parasite killing (Aliberti et al., 2001). Another interesting cooperation
between IFN-g and chemokine production occurs in vitro and in vivo.
IFN-g enhanced and modified the in vitro synthesis of chemokines by
macrophages infected with T. cruzi or stimulated with tGPI-mucins
(Aliberti et al., 2001; Coelho et al., 2002). Similarly, in vivo, as IFN-g
production occurs during the onset of infection in the hearts of mice,
there is modification of the chemokine milieu towards production of
chemokines associated with recruitment of Th1 cells and macrophage
activation (Aliberti et al., 2001; Hardison et al., 2006; Talvani et al., 2000;
Teixeira et al., 2002). Indeed, in a murine model, TNF-a, IFN-g and IFN-g-
induced chemokines RANTES/CCL5 (regulated upon activation, normal
T cell expressed and secreted), MIG (monokine induced by IFN-g) and
CRG-2/IP-10/CXCL10 (cytokine response gene 2/interferon g-inducible
protein 10), as well as JE/MCP-1/CCL2 (monocyte chemoattractant pro-
tein-1) and MIP1-a/CCL3 (macrophage inflammatory protein-1a, MIP-
1a) were described as dominant cytokines expressed in situ during
chronic phase of T. cruzi-elicited myocarditis and possible contributing
to the driving recruitment of activated T cells (Talvani et al., 2000).
Expression of inflammatory cytokines and chemokines was correlated
with the presence of inflammatory cells within the heart, including
CD4þ, CD8þ T cells and macrophages, with peak at day 30 and remaining
highly expressed through 120 days post-infection. More recently, a new
study was performed using the same mouse and parasite genetic back-
ground—C57BL6 and Colombian strain, respectively—showing that CXC
chemokine ligand 9 (CXCL9), CXCL10, CC chemokine ligand 2 (CCL2)
and CCL5 were prominently expressed during initial phase, whereas
transcripts for CXCL9, CXCL10, and CCL5 remained elevated during
chronic infection (Hardison et al., 2006). As expected, the peak of chemo-
kine expression levels was coincident with the increased IFN-g expression
and inflammation within the heart, reinforcing an important role for these
molecules in host defence. Therefore, there appears to be a positive
interaction between chemokines and IFN-g that lead to optimal control
of T cruzi infection. As mentioned above, this interaction is not sufficient
to attain total control of infection and parasites persist. In the long term,
however, chemokines/IFN-g interaction may contribute to the heart dam-
age observed in patients (Cunha-Neto et al., 2009). Indeed, experiments
using Met-RANTES, an N-terminally modified human RANTES/CCL5
capable of inhibiting CCR1 and CCR5, showed that treatment with the
drug decreased the infiltration of CD4þ and CD8þ T cells and deposition
of fibronectin in the heart of infected animals, without decreasing or
interfering with parasitism (Marino et al., 2004).
Few studies have attempted to understand the possible role of chemo-
kines in the context of human Chagas cardiomyopathy (Dutra et al., 2005).
Studies have been carried out in patients with established disease in
comparison with those who were infected but without disease and non-
infected subjects. The idea of the studies was to evaluate whether levels of
chemokines or chemokine receptors would increase in Chagas disease
and correlate with disease severity. It was found that high plasma levels
of CCL2 increased in patients with Chagas disease, especially in those
with heart dysfunction, correlated with the degree of heart dysfunction
(Talvani et al., 2004b). When spontaneous production of CCL2 by periph-
eral blood mononuclear cells (PBMC; in vitro assay) was examined, it was
noticed that levels of CCL2 were enhanced in patients with Chagas
disease, irrespective of their clinical condition (Talvani et al., 2004b).
These studies would suggest that CCL2 marks the severity of the disease
and may be important for Chagas disease progression. In support of a
possible role of CCL2 in Chagas disease, it has been shown that patients
presenting a ccl2 promoter polymorphism at position -2518A/G, which is
recognized to increase serum level of the protein by influencing its tran-
scriptional activity, had a fourfold greater risk of developing CC than
those without this genotype (Ramasawmy et al., 2006). However, elevated
levels of chemokines, such as CCL2, and chemokine receptors on leuko-
cytes may alter in patients with cardiomyopathy irrespective of the cause
(Sigusch et al., 2006; Stumpf et al., 2008). Therefore, it is not possible to
conclude for a direct role of CCL2 in the context of Chagas disease and the
elevated levels of the chemokines may simply mark, and not be the cause
of, the alterations observed in chronic heart failure. However, these stud-
ies do suggest that CCL2 may be a marker of heart dysfunction measure
in blood akin to the role of TNF-a and BNP (Lula et al., 2009; Ribeiro et al.,
2002, 2006; Talvani et al., 2004b,c).
Another member of the chemokine family that has received some
interest in the past few years is the chemokine receptor CCR5—a receptor
for CCL3, CCL5, CCL8 and CCL14. Studies in patients demonstrated that
circulating CD3þCD8þ T cells expressing high levels of CCR5 associated
with mild cardiomyopathy form when compared with uninfected indivi-
duals or those patients presenting severe form of the disease (Talvani
et al., 2004a). These data supported two previous studies showing that
CCR5 promoter point mutation (CCR5-59029G), associated with CCR5low

expression was significantly increased in asymptomatic patients (Calzada
et al., 2001; Fernandez-Mestre et al., 2004). It is not easy to interpret
human PBMC data, but it is possible to hypothesize that the decrease of
CCR5þ cells in blood of patients with severe disease may be reflecting the
migration of CCR5þ cells into tissues. If the latter tenet is true, blockade of
entry of CCR5þ cells into tissue may prevent disease progression. Indeed,
we have previously shown that chronic blockade of CCR5 with Met-
RANTES decreased migration of T cells into the heart and decreased
disease severity in models of T. cruzi infection in mice (Marino et al.,
2004). Alternatively, CCR5 may mark a population of regulatory cells
which are known to be elevated in experimental infection (Mariano
et al., 2008). There is now compelling evidence that CD4þCD25þ
T cells—called regulatory T cells or simply Treg—are able to maintain
immune tolerance and homeostasis, preventing autoimmunity and mini-
mize harmful inflammatory responses to mammalian hosts (Hori and
Sakaguchi, 2004). Circulating naturally arising CD4þCD25high regulatory
T cells, which express a family of transcription factor Foxp3, were higher
in patients with asymptomatic than in those with cardiac form of Chagas
disease (Araujo et al., 2007). In a study in rats, blockade of CCL4 was
associated with increased heart inflammation and fibrosis, suggesting a
role for the migration of CCR5þ regulatory cells in the control of tissue
damage in that species (Roffê et al., 2006). The role of CCR5 for the
recruitment of FOXP3þ cells in Chagas remains to be determined in detail.
The chemokine CCL3 (also known as MIP-1a) has been shown to be
important for the dyskinesis in the left ventricle wall observed in the
hearts of mice infected with the Brazil strain of T. cruzi (Durand et al.,
2006). The role of CCL3/CCL5 and their receptors CCR1/CCR5 was also
investigated in rats by DNA vaccination encoding both chemokines and
through Met-RANTES treatment, respectively. Simultaneous treatment
with vaccines encoding for both chemokines or treatment with Met-
RANTES increased heart parasitism, inflammation, fibrosis and
decreased local IFN-g production (Roffê et al., 2010), clearly contrasting
with available data in murine models of T. cruzi infection (Marino et al.,
2004). These data in rats reinforce the important role of chemokines
during T. cruzi infection but suggest that caution must be taken when
expanding the therapeutic modulation of the chemokine system in mice
to the human infection. In conclusion, it is clear that chemokines play a
central role in the context of Chagas disease. Most studies have focused on
a few chemokines that drive tissue inflammation and are necessary to
deal with infection. However, there are chemokines that are important to
drive regulatory cells. As for the role of inflammation in the context of
Chagas, different set of chemokines appear to drive immune response or
disease. Defining these chemokines will not be an easy task especially in
light of results discussed above showing that different species may pres-
ent different outcomes when faced with the same infectious challenge.
Moreover, as discussed above, studies in human are not simple as one
usually compares patients with disease with those without disease. There
is not a long-term follow-up but cross-sectional studies performed in
individuals who have developed disease. As discussed, levels of chemo-
kines (and of other mediators of inflammation) may reflect the present
condition of the patient (e.g. heart failure) and not the cause that led to
that condition.
8.4.2. Lipid mediators

We have previously demonstrated that PAF and another lipid mediator
LTB4 were able to induce NO and TNF-a production in cultured T. cruzi-
infected peritoneal macrophages and killed parasites in a NO-dependent
manner. These data were reinforced in vivo, when blockade of PAF and
LTB4 receptors was associated with higher parasitaemia and lower sur-
vival in infected mice (Aliberti et al., 1999; Talvani et al., 2002, 2003).
Biosynthesis of LTB4 occurs mainly in granulocytes, monocytes/macro-
phages and mast cells and it is dependent on the enzyme 5-LO. Consis-
tently with a role for LTB4 in the context of T cruzi-associated heart
inflammation, deletion or blockade of 5-LO showed that 5-LO derived
was associated with reduction in inflammatory indices, in collagen depo-
sition, in migration of CD4þ and CD8þ and IFN-g producer cells into the
myocardium (Pavanelli et al., 2010). Despite the decreased inflammatory
response, control of infection was eventually attained in all animals
suggesting that the inflammatory response triggered by 5-LO is not cru-
cial for control of parasitism (Borges et al., 2009; Panis et al., 2011;
Pavanelli et al., 2010). Altogether, these studies demonstrate that lipid-
derived mediators, especially PAF and LTB4, appear to be important in
the initial phases of the infection. Two major functions can be ascribed to
these molecules. They facilitate parasite uptake and iNOS expression by
mononuclear cells and consequently play a role in controlling parasite
replication initially. Subsequently, these molecules may be relevant for
the migration of leukocyte subsets to the myocardium and contribute to
local damage. It is not known whether these mediators will be relevant in
the context of chronic Chagas disease.
8.4.3. Endothelin
Endothelin 1 (ET-1), a potent vasoconstrictor, is another important exam-
ple of mediator released by endothelial cell and myocardium whose
involvement in chronic events of T. cruzi infection was proposed since
the beginning of the 1990s. Experimental studies involving rodents
infected with a cardiotropic strain of T. cruzi have shown the presence of

intense vasculitis in accordance with high plasma levels of ET-1 and an
increased expression of mRNAs for the precursor molecule preproET-1
and also ET-1 in the myocardium (Petkova et al., 2000). Another study
reinforced the contribution of ET-1 to the pathogenesis of murine chagasic
cardiomyopathy using mice with ET-1 genes deleted in cardiomyocytes.
In these mice, there was reduction in inflammatory infiltration and fibro-
sis in the heart and reduction in infection-associated changes in right
ventricular internal diameter and in left ventricular end-diastolic diame-
ter, in fractional shortening, and in the relative wall thickness by echocar-
diography (Tanowitz et al., 2005). In addition to having contractile effects,
it is clear that ET-1 may have proliferative effects on endothelial cells and
may also induce the release of mediators of inflammation from leukocytes
(Abraham and Distler, 2007). In rats, ET-1 appears to be important for
initial control of parasite replication. Blockade of ET-1 receptors decreases
inflammation associated with infection in some organs, but there was no
major role of the molecule in driving tissue inflammation in rats
(Camargos et al., 2004; Rachid et al., 2006, 2010). The effects of ET-1 on
the course of infection appeared to be secondary to the ability of ET to act
on seven transmembrane-spanning G-protein-linked receptors, ET(A)
and ET(B), and interfere with the production of NO. Therefore, despite
its well-known pro-inflammatory effects in various situation and, the
observation that ET-1 is released in the course of T. cruzi infection, this
mediator appears to play no central role in driving tissue inflammation
but it is important in driving vascular dysfunction.
8.5. CONCLUSION
Chronic Chagas disease is characterized by sparse inflammatory infil-

trate, minimal parasitaemia and constant low-grade tissue parasitism
(Benvenuti et al., 2008; Ben Younès-Chennoufi et al., 1988; Marin-Neto
et al., 2007). These low-grade inflammation and immune response are
sufficient to keep infection under control, as demonstrated by activation
of disease in patients taking immunosuppressant drugs, and causes little
damage in the great majority of infected patients. Indeed, the indetermi-
nate form of the disease or minimal heart damage is the most frequent
outcome of infected patients. In some individuals, heart disease, and
occasionally oesophageal and colonic disease occurs, and disease is
thought to result from the combined effect of persistent parasitism and
parasite-driven tissue inflammation. Herein, we reviewed some of the
molecules that drive tissue inflammation and showed that not all media-
tors of inflammation are necessary to drive the recruitment of cells
involved in control of parasite replication. Indeed, some mediators have
mostly a detrimental effect and appear to drive tissue damage. In this

respect, blocking some mediators of the inflammatory response may aid
in the control of progression of disease, for example, blockade of CCR1/5
with Met-RANTES (Marino et al., 2004). However, most studies described
to date have been performed in mice and have not really addressed the
chronic disease in humans. In contrast, due to its very chronic nature,
studies in chagasic patients have not been adequate to determine whether
presence of certain inflammatory mediators in plasma actually showed
their importance for pathogenesis or simply represented the clinical state
of the patient. For example, levels of CCL2 in plasma of chagasic patients
correlated with the degree of heart failure (Talvani et al., 2004b). It is clear,
therefore, that it will not be simple to translate findings in experimental
situations to humans and to decide which will be ideal molecule to
evaluate whether anti-inflammatory treatment will provide additional
benefit for patients with Chagas disease.
ACKNOWLEDGEMENTS
We recognize the financial support of Conselho Nacional de Desenvolvimento Cientı́fico
e Tecnológico (CNPq), Coordenação de Aperfeiçoamento Pessoal de Ensino Superior
(CAPES), Fundação de Amparo a Pesquisas do Estado de Minas Gerais (FAPEMIG), Inter-
national Society for Infectious Disease (ISID/EUA) and Drugs for Neglected Disease initia-
tive (DNDi). M. M. T. and A. T. are recipients of productivity awards from CNPq.
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CHAPTER 9
Neurodegeneration and
Neuroregeneration in
Chagas Disease
Marina V. Chuenkova and Mercio PereiraPerrin

9.2. Involvement of the Autonomic Nervous System
in CD Pathogenesis 197
9.2.1. Pathophysiological evidence of
neurodegeneration in the heart 198
9.2.2. GI neurodegeneration 200
9.3. Mechanism of Neuronal Damage 202
9.3.1. Parasitism 202
9.3.2. Acute inflammation 202
9.3.3. Autoimmunity 205
9.4. Neuroregeneration 206
9.5. Trans-Sialidase/Parasite-Derived Neurotrophic Factor 209
9.5.1. Trans-Sialidase 209
9.5.2. Parasite-derived neurotrophic factor 211
9.5.3. PDNF and Trypanosoma cruzi receptor-
independent intracellular signalling 218
9.6. Conclusions 220
References 221
Abstract Autonomic dysfunction plays a significant role in the development

of chronic Chagas disease (CD). Destruction of cardiac parasympa-
thetic ganglia can underlie arrhythmia and heart failure, while
lesions of enteric neurons in the intestinal plexuses are a direct
Department of Pathology and Sackler School of Graduate Students, Tufts University School of Medicine,
Boston, Massachusetts, USA

195
196 Marina V. Chuenkova and Mercio PereiraPerrin
cause of aperistalsis and megasyndromes. Neuropathology is gen-

erated by acute infection when the parasite, though not directly
damaging to neuronal cells, elicits immune reactions that can
become cytotoxic, inducing oxidative stress and neurodegenera-
tion. Anti-neuronal autoimmunity may further contribute to neu-
ropathology. Much less clear is the mechanism of subsequent
neuronal regeneration in patients that survive acute infection.
Morphological and functional recovery of the peripheral neurons
in these patients correlates with the absence of CD clinical symp-
toms, while persistent neuronal deficiency is observed for the
symptomatic group. The discovery that Trypanosoma cruzi trans-
sialidase can moonlight as a parasite-derived neurotrophic factor
(PDNF) suggests that the parasite might influence the balance
between neuronal degeneration and regeneration. PDNF function-
ally mimics mammalian neurotrophic factors in that it binds and
activates neurotrophin Trk tyrosine kinase receptors, a mechanism
which prevents neurodegeneration. PDNF binding to Trk receptors
triggers PI3K/Akt/GSK-3b and MAPK/Erk/CREB signalling cascades
which in neurons translates into resistance to oxidative and nutri-
tional stress, and inhibition of apoptosis, whereas in the cytoplasm
of infected cells, PDNF represents a substrate-activator of the host
Akt kinase, enhancing host-cell survival until completion of the
intracellular cycle of the parasite. Such dual activity of PDNF
provides sustained activation of survival mechanisms which, while
prolonging parasite persistence in host tissues, can underlie distinct
outcomes of CD.
9.1. INTRODUCTION
Chronic Chagas disease (CD) is the most lethal endemic infection in the
Western hemisphere and regardless of the recent progress on vector
control remains a significant public health issue in Latin America
(Coura, 2007; Rassi et al., 2000). Despite nearly one century of research,
the pathogenesis of CD is not completely understood and many questions
regarding disease progression and treatment are still unsolved, mainly
due to extremely complex nature of the parasite interaction with the
vertebrate host.
During the acute phase of Trypanosoma cruzi infection, the parasite has
the ability to infect a wide variety of cells in most tissues; however, the
clinical picture of CD is dominated by cardiologic and gastrointestinal
(GI) manifestations. Chagasic cardiomyopathy is the most important
clinical presentation of CD and comprises a wide range of symptoms,
including congestive heart failure, arrhythmias, heart blocks, sudden
death, thromboembolism, and stroke.
Neurodegeneration and Neuroregeneration in Chagas Disease 197
Despite its obvious clinical importance, the exact mechanism whereby

parasitism causes cardiac tissue damage in the chronic phase is still
largely unknown. Several theories have been proposed to explain the
development of chronic CD heart disease: microvascular disturbances,
direct parasite damage to myocardium, immune-mediated myocardial
injury and destruction of the cardiac autonomic nervous system (ANS),
which is commonly referred to as the neurogenic theory (Davila et al.,
2004; Leon and Engman, 2003; Tarleton, 2001).
9.2. INVOLVEMENT OF THE AUTONOMIC NERVOUS

SYSTEM IN CD PATHOGENESIS
The foundation to neurogenic theory was laid by the pioneering work of

Köberle in the 1950s, but neuropathology in CD has been recognized
almost from its original discovery by Carlos Chagas, when he and the
cardiologist E. Vilella described in the early 1920s abnormal heart rate in
response to atropine in chagasic patients (Punukollu et al., 2007). Köberle
employed the technique of neuronal counts to analyze pathological altera-
tions in the heart and GI of CD patients and found neuronal depopulation
for most of them so striking that he postulated it should constitute the
main pathological mechanism in CD (Koberle, 1970). The ganglionic
damage and absolute reduction in subepicardial intramural neuronal
countings were confirmed by other investigators (Marin-Neto et al.,
2007; Oliveira, 1985; Punukollu et al., 2007; Ribeiro et al., 2001; Simoes
et al., 2000) and corroborated by studies in animals experimentally
infected with the T. cruzi, which demonstrated that cardiac neuronal
parasitism was associated with periganglionitis and degeneration of ner-
vous fibres and Schwann cells (de Souza et al., 1996; Machado et al., 1998;
Rodrigues et al., 2002). Because the intramural cardiac ganglia are mostly
parasympathetic, the neurogenic theory of CD pathogenesis suggests that
Chagas cardiomyopathy is underlined by ‘‘parasympathetic reduction’’
in the affected organs. Selective destruction of post-ganglionic vagal
neurons of the intra-cardiac plexus during the acute phase of CD
(Davila et al., 1991; Machado et al., 1998; Ribeiro et al., 2001, 2005;
Sterin-Borda et al., 1997; Vasconcelos and Junqueira, 2009) leaves sympa-
thetic nervous system unopposed, and the result of such sympatho-vagal
imbalance would eventually lead to a catecholamine-induced cardiotoxi-
city, cardiomyopathy and heart failure (Davila et al., 2004; Koberle, 1968).
Indeed, evidence links sympathetic hyper-activation to idiopathic heart
failure, one of the proposed mechanisms being apoptotic death of cardi-
omyocytes caused by overstimulation of their adrenergic receptors
(Olshansky et al., 2008).
9.2.1. Pathophysiological evidence of neurodegeneration in

the heart
9.2.1.1. Parasympathetic dysautonomia
Consistent with this theory, anatomic parasympathetic denervation in CD
patients shown by morphological studies resulted in impaired heart rate
regulation detected by pharmacological and physiological tests. Overall,
these studies showed that patients with CD are usually deprived of the
tonic inhibitory action normally exerted by the parasympathetic system
on the sinus node and lack the vagus nerve-mediated mechanism to react
to transient changes in blood pressure with appropriate changes in heart
rate (Amorim and Marin Neto, 1995; Marin-Neto et al., 2007). In these
cases, ECG often reveals premature atrial or ventricular heart beats,
ventricular tachycardia, atria/ventricular fibrillation or various degrees
of heart block (Punukollu et al., 2007).
Also according to the neurogenic theory, the rarefaction of the para-
sympathetic nerve terminals and the reduction in vagal ganglionic trans-
mission should result, similar to non-chagasic HF patients (Olshansky
et al., 2008), in adaptive up-regulation of post-synaptic muscarinic ACh
receptors (mAChRs) in the cardiac muscle. Indeed in rats and mice
infected with T. cruzi increased density of M2-muscarinic receptors was
restricted to the ventricle with selective degeneration of vagal fibres
(Peraza-Cruces et al., 2008; Rocha et al., 2006). Such increase could further
contribute to myocardial damage through reaction with anti-M2-mAChRs
auto-antibodies with agonist-like activity detected in both chagasic and
non-chagasic patients with heart failure (Olshansky et al., 2008; Sterin-
Borda and Borda, 2000), and may explain the bradycardia and the atria–
ventricular conduction blockage, frequently associated with chronic car-
diopathy in CD (Hernandez et al., 2003).
The cardiac parasympathetic dysautonomia is an early phenomenon
in the course of T. cruzi infection. It was shown to precede left ventricular
systolic dysfunction (Ribeiro et al., 2001), which is a major pathological
feature of CD cardiomyopathy and the main predictor of the death risk,
suggesting that vagal nerve damage should have a unique role in the
pathogenesis of the heart CD (Ribeiro et al., 2005).
Indeed, early vagal impairment in the hearts of CD patients could be a
mechanism that triggers sudden death due to malignant ventricular
tachyarrhythmias and fibrillation which are major causes of sudden
death among patients with cardiac CD. This possibility is supported by
autopsy reports of highly denervated hearts in CD patients who died
suddenly (Baroldi et al., 1997; Lo and Hsia, 2008; Marin-Neto et al., 2007).
The other consequence of vagal nerve damage in the heart can be
dilatation of the left ventricle and apical aneurism—a distinct abnormality
described especially often in association with CD (Marin-Neto et al., 2007;
Punukollu et al., 2007; Tanowitz et al., 2009). The parasympathetically

denervated heart lacks the physiological mechanism of rate self-regula-
tion and has to respond to blood flow demands, such as physical exercise,
with greater increase in stroke volume which can lead to increased
ventricular afterload, chamber dilatation and aneurism (Marin-Neto
et al., 2007).
Finally, damage of the parasympathetic ganglia and vagal nerve term-
inals could also have a negative impact on the cholinergic anti-inflamma-
tory pathway. An inflammatory reflex of the vagus nerve can inhibit
release of pro-inflammatory cytokines (TNF-a, IL-18, IL-6 and IL-1) and
thereby prevent tissue injury and cell death (Tracey, 2007). Consequently,
diminished vagal anti-inflammatory signals promote persistent inflam-
mation, which plays pathogenic role in the progression of left ventricular
dysfunction and heart failure (Aukrust et al., 2005).
Although parasympathetic dysautonomia is not specific that for CD—
parasympathetic neuronal depopulation and decreased vagal tone
characterize heart failure of other etiologies (Olshansky et al., 2008)—
because absolute reduction in the parasympathetic neuronal countings
is more prominent in chagasic patients (Biolo et al., 2010). Compared with
HF from other causes, CD heart failure is usually associated with poorer
prognosis and higher mortality, which can reach above 50% in a 5-year
period (Bestetti and Muccillo, 1997; Mady et al., 1994).
9.2.1.2. Sympathetic dysfunction

As T. cruzi infection progresses, it also causes lesions of the sympathetic
nervous system, which almost eliminates any neural influence upon the
left ventricle in CD patients (Cunha et al., 2003; Iosa et al., 1989; James
et al., 2005).
Contrary to parasympathetic denervation, the sympathetic denerva-
tion is independent of neuronal death or damage in cervical and stellate
ganglia, and target the cardiac post-ganglionic nerve terminals in the heart
(Machado et al., 1994). However, in the adrenal medulla—the principal
site of catecholamine production in the body—the acute T. cruzi infection
induced a clear rarefaction of the preganglionic sympathetic nerve fibres
(Camargos et al., 1996). Overall, such reduced sympathetic activity results
in lower plasma levels of norepinephrine (NE), as demonstrated for
patients with CD heart disease who have class III and IV heart failure in
contrast to sympathetic hyperactivity and elevated plasma NE in the same
class heart failure patients without CD (Iosa et al., 1989). The limited
adrenergic input together with the decrease in the cardiac adrenergic
receptor density and affinity to catecholamines (Camargos et al., 2000;
Lo Presti et al., 2009) explain why symptomatic patients with advanced
CD should not benefit from adrenergic blockers (Davila et al., 1998;
Marin-Neto et al., 2007) and have a clinical course worse than that
of patients with non-chagasic dilated cardiomyopathy (Bestetti and
Muccillo, 1997).
The noradrenergic innervation of atrial blood vessels was usually
more resistant to the infection than the myocardial one, and in experimen-
tal T. cruzi infection, animals with moderate to severe myocardial dener-
vation demonstrated well-preserved vascular sympathetic innervation
(Camargos et al., 2000).
Although it is likely that the origin of chagasic heart disease is more
complex, and other factors such as inflammation and autoimmune reac-
tions contribute to the damage of the myocardium, the striking neuro-
genic disturbances in the hearts of many T. cruzi-infected patients should
have a major pathophysiological impact in the severity of chagasic
cardiomyopathy.
9.2.2. GI neurodegeneration
Approximately 10% of chronic chagasic patients (one-third in certain
regions) can develop GI motor disorders, mostly of oesophagus and
colon, such as aperistalsis, achalasia of the cardia and disturbances of
gastric emptying, which lead to organ obstruction and stagnated food
passage followed by megasyndromes, malnutrition and severe weight
loss (Herbella et al., 2008; Matsuda et al., 2009; Meneghelli, 2004).
Whereas the pathogenic impact of autonomic dysfunction in the
development of cardiac CD is still a matter of debate, it is universally
accepted that denervation in GI is the primary, pathogenic mechanism
in the chronic GI form of CD (Adad et al., 2001; da Silveira et al., 2007a;
de Oliveira et al., 1995; Meneghelli, 2004). In his fundamental study of
200 autopsied cases, Köberle initially showed that the dilatation
and hypertrophy of the oesophagus and colon in CD patients corre-
sponded to 50–90% reduction in ganglion cells in enteric plexuses
(Koeberle, 1963).
Later clinical studies invariably confirmed dramatic (up to 95%) neu-
ronal depopulation in patients with megaesophagus and megacolon and
in T. cruzi-infected animals, and demonstrated that the primary target of
injury by infection in GI are the neuronal ganglia in both extrinsic (para-
sympathetic) and intrinsic (myenteric and submucous plexuses) nervous
systems (Caetano et al., 2006; da Silveira et al., 2008; Iantorno et al., 2007;
Machado et al., 2001; Matsuda et al., 2009; Meneghelli, 2004; Nascimento
et al., 2010).
Basic functions of the gut, such as peristalsis, secretion and blood flow,
are primarily regulated by the intrinsic network of GI ganglia, the enteric
nervous system (ENS), which interacts with the sympathetic and para-
sympathetic neurons but constitutes an independent part of the ANS.
Enteric neurons are organized into myenteric and submucous plexuses,

embedded in the wall of the digestive tract, and consist of about 20
distinct subtypes of neurons which can be classified as intrinsic primary
afferent neurons (monitor the state of the lumen and the gut wall), inter-
connecting neurons and motor neurons (target the muscle layers)
(Laranjeira and Pachnis, 2009). The motor neurons are the most damaged
by T. cruzi infection, which explains why the prevalent disturbance of
intestinal function in CD is a progressive loss of muscular coordination
and motor activity, followed by hypertrophy of the intestinal muscle wall
(Campos and Tafuri, 1973; da Silveira et al., 2007b; de Oliveira et al., 1995;
Matsuda et al., 2009).
Among the motor neurons, inhibitory innervation (vasoactive intesti-
nal peptide (VIP) and nitric oxide synthase (NOS)-positive) is preferen-
tially destroyed by T. cruzi infection (da Silveira et al., 2007a; Ribeiro et al.,
1998; more references), and many patients with chagasic achalasia posi-
tively respond to NO-donor drugs which decrease esophageal sphincter
pressure and increase megaesophagus clearance (Matsuda et al., 1995).
T. cruzi infection has certain effects on excitatory transmission as well.
In contrast to idiopathic achalasia and megacolon, where the excitatory
innervation is preserved, in CD patients with similar symptoms choliner-
gic excitatory (ChATþ) motor neurons are damaged (Adad et al., 2001;
Dantas et al., 1990; Iantorno et al., 2007; Machado et al., 1987), and
substance P (SP, tachykinin), a peptide co-transmitter of excitatory neu-
rons, is decreased (Long et al., 1980; Maifrino et al., 1999a). Another
study, however, demonstrated significant increase in the proportion of
SP-positive excitatory neurons in both enteric plexuses of chagasic mega-
colon (da Silveira et al., 2007a). Disbalance of SP excitatory transmission
associated with several intestinal disorders (Koon and Pothoulakis, 2006;
Margolis and Gershon, 2009) might contribute to the intensity of the
inflammation in CD patients with megacolon (da Silveira et al., 2007b),
because tachykinins (SP), through NK1 receptors on the muscle, induce
secretion of pro-inflammatory IL-1, IL-6, IL-8 and TNF (da Silveira et al.,
2007a). However, these cytokines also inhibit the expression of NK1
tachykinin receptor (Simeonidis et al., 2003), and it is decreased in CD
megacolon (da Silveira et al., 2008), which is likely a feedback mechanism
to restrict inflammation magnitude in the infected colon.
In addition to dramatic loss of enteric neurons in the gut plexuses and
disturbances in peptidergic transmission, there are also preganglionic
lesions and a reduction in the number of dorsal cells of the motor nucleus
of the vagus thought to happen due to retrograde destruction resulting
from loss of enteric neurons (Koberle, 1970).
Along with the severe effects on neurons, infection also damages other
components of GI nervous system, such as interstitial cells of Cajal (ICC)
and enteric glial cells (EGCs). ICC are pacemaker cells responsible for
coordinating peristalsis and mediating nerve impulses in the gut (Hagger

et al., 2000), and EGC provide structural and metabolic support to enteric
ganglia, control intestinal inflammation and produce neurotrophic factors
such as NGF, NT-3 and GDNF involved in preservation of enteric neu-
rons (von Boyen et al., 2006). Reduction in ICC and EGC populations
observed in T. cruzi infection (de Lima et al., 2008; Nascimento et al., 2010;
Ribeiro et al., 1998) would affect mechanical and homeostatic support and
decrease neurotransmission in ENS (Bassotti et al., 2007; Ruhl, 2006)
further promoting neurodegeneration and smooth muscle pathology in
GI of CD patients.
9.3. MECHANISM OF NEURONAL DAMAGE
Though the role of the parasite in the CD dysautonomia is still debated,

most studies have demonstrated the presence of T. cruzi in the nervous
system of CD patients and the close association between the parasite
persistence and the rate of neuronal destruction (da Silveira et al., 2005;
Davila et al., 2004; Marin-Neto et al., 2007; Silva et al., 2003; Zhang and
Tarleton, 1999).
The mechanism of neuronal loss in CD is not completely clear and is
thought to occur as a result of direct parasitism of neurons and support-
ing cells, acute periganglionic inflammation and anti-neuronal
autoimmune reactions (Fig. 9.1).
9.3.1. Parasitism
The peripheral neurons are predominantly damaged during the acute
phase, when parasitemia and tissue parasitism are high (da Silveira et al.,
2009; Rodrigues et al., 2002; Soares and Santos, 1999). At that time, trypo-
mastigotes are often found in myenteric and submucosal plexuses and in
the interstitium of sympathetic and parasympathetic ganglia, where they
target glial and other supporting cells for intracellular parasite proliferation
(Lenzi et al., 1996; Meyer et al., 1982; Tanowitz et al., 1982). Infected cells are
ruptured at the end of the parasite intracellular cycle to release newly
produced trypomastigotes and parasite-derived products. It is possible
that certain T. cruzi molecules may damage neuronal cells, similar to
LYT1, a parasite factor with hemolytic activity (Manning-Cela et al., 2001),
although to-date, no parasite-produced neurotoxins have been described.
9.3.2. Acute inflammation

Accumulating data indicate that the degeneration in ANS is largely
caused by T. cruzi-triggered host immune responses. T. cruzi is a potent
stimulator of the host innate immune system (Aliberti et al., 1996),
T. cruzi
Inflammation
IL-12
TNFα Auto-immunity
Intracellular IFNγ
parasitism
Macrophages
iNOS
Auto-antibody
Apoptosis
NO•
?
Peripheral neurons
Glial cells
Heart Digestive tract
Sudden Heart Aperistalsis

death failure Megasyndromes
FIGURE 9.1 Mechanism of neurodegeneration in Chagas disease. Parasite invasion of

the heart and GI tissues can damage cardiac and enteric peripheral neurons and glial cells
through intracellular parasitism, generation of acute inflammatory response and auto-
immune reactivity. Parasite invasion triggers host innate immune defence reactions
characterized by production of pro-inflammatory mediators TNF-a, IL-12 and IFN-g,
which in concert activate macrophages, stimulate expression of inducible NO synthase
(iNOS), production of NO and derived nitrogen and oxygen radical products, which kill
intracellular parasites, but also damage uninfected cells. TNF-a can directly induce
neuronal apoptosis via death-domain-containing TNF receptor. Anti-neuronal and anti-
myelin autoimmune response further promotes PNS destruction, while intracellular
parasitism of neuronal and glial cells is likely a minor factor.
and local inflammatory responses triggered by T. cruzi invasion of the

heart and GI, while eliminating the infected cells, can also damage
surrounding uninfected neighbours. Strong association has been
demonstrated between denervation in cardiac and intestinal neuronal

plexuses and immune cytotoxic mechanisms, which depend on parasite
residence (da Silveira et al., 2005; Melo and Machado, 2001). Immune cells
with cytolytic potential such as macrophages, NK cells and cytotoxic
lymphocytes were observed in enteric plexuses of CD patients, and the
presence of these cells inversely correlated with the number of surviving
neurons (Adad et al., 2001; Campos and Tafuri, 1973). Inflammation
persists through chronic phase of the infection, and in the GI tract,
especially in dilated portion of chagasic megacolon is associated with
the increased denervation (Adad et al., 2001; da Silveira et al., 2007b),
suggesting correlation between anti-parasite immune response, denerva-
tion and CD symptoms.
Similar findings were made in experimental CD, when immunosup-
pression with cyclophosphamide or cortisone stopped neuronal loss in
the myenteric plexus of infected mice and reduced damage to cardiac
noradrenergic nerve terminals preventing the acute myocarditis despite
increased parasitemia and tissue parasitism (Adad et al., 2001; Caetano
et al., 2008; Guerra et al., 2001).
Further insights into the role of immune cytotoxic responses in auto-
nomic damage in CD were provided by demonstration that depletion of
the complement or radio-sensitive leucocytes failed to prevent early
denervation in the heart (Machado et al., 1994; Melo and Machado,
1998). Gamma-irradiation, which eliminates leucocytes, but excepts
macrophages, rather enhanced damage to adrenergic nerves in the heart
of infected animals, which corresponded to the presence of numerous
macrophages with ultrastructural features of activation (Melo and
Machado, 1998, 2001).
Resistance to T. cruzi infection and effectiveness of parasite elimina-
tion largely depend on the production of pro-inflammatory cytokines
IL-12, TNF-a and IFN-g which synergistically activate macrophages to
produce NO, a free radical effector molecule that controls intracellular
T. cruzi multiplication (Aliberti et al., 2001; Gazzinelli et al., 1992).
The other side of excessive NO production is its general cytotoxicity
associated with neuropathology, as was demonstrated for some neurode-
generative disorders (Knott and Bossy-Wetzel, 2009).
NO can have diverse roles in the NS. Produced by the constitutive
neuronal nitric oxide synthase (nNOS), it functions as a second messen-
ger regulating synaptic plasticity, neuroprotection and inhibitory neuro-
transmission, whereas the inducible NOS (iNOS) generates NO in
response to inflammatory stimuli (Gutierrez et al., 2009b; Knott and
Bossy-Wetzel, 2009). NO produced by iNOS in copious amounts and
for prolonged periods of time is cytotoxic to neuronal cells and can cause
massive necrotic cell death and axonal degeneration (Gu et al., 2010).
NO-releasing macrophages can adhere to intact nerve stripping off
myelin sheath, so NO has been also considered a key effector of demye-

lination in the PNS (Conti et al., 2004), a process also observed for CD
neuropathy (Fazan and Lachat, 1997; Losavio et al., 1993; Molina et al.,
1987). NO production is significantly higher in CD patients and in infected
animals compared to uninfected controls (Garcia et al., 1999; Perez-
Fuentes et al., 1998; Pinto et al., 2002), and several studies have linked
expression of iNOS and overproduction of NO to neurotoxic effects in CD.
Thus, it was demonstrated that iNOS is increased in the denervated
hearts of infected mice, and that IFN-g-induced iNOS activation in the
inflammatory foci along the intestinal wall correlates with damage to
intrinsic intestinal nerves (Arantes et al., 2004; Garcia et al., 1999). At
the same time, infected animals with decreased production of NO due to
immunosuppression, inhibition of iNOS, or knockout of IFN-g or iNOS
genes, presented no reduction in neuron numbers in the heart and myen-
teric plexuses (Arantes et al., 2004; Caetano et al., 2008; Garcia et al.,
1999). Macrophages isolated from iNOS/ mice did not release NO in
response to IFN-g and, co-cultured with neurons, did not affect their
survival rate, while activation of wt-macrophages by IFN-g led to 60%
neuronal death (Almeida-Leite et al., 2007), analogous to myenteric neu-
ronal loss described in experimental models and human studies (Adad
et al., 2001; Koberle, 1970). Interestingly enough, the neuronal survival
rate was not altered in pure neuronal cultures exposed to T. cruzi
(Almeida-Leite et al., 2007) in keeping with similar observations in
immunosuppressed experimental animals (Caetano et al., 2008). The role
of IFN-g in the nervous system is not limited to indirect neuronal damage
via promotion of NO release by macrophages/microglia. In addition,
IFN-g was reported to inhibit dendritic growth and synapse formation
and to induce neurotoxicity through a neuron-specific complex of IFN-g
receptor with a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA)
receptor, thus directly causing neuronal dysfunction (Mizuno et al., 2008).
Finally, damaged vagus nerve, in a negative feedback manner, would
decrease its anti-inflammatory reflex and further the inflammation in
infected tissues by promoting release of pro-inflammatory cytokines,
including TNF-a and IFN-g and extra amounts of NO (Tracey, 2007).
Overall, these data identify NO production by the IFN-g-activated
macrophages as a specific cause of neuronal damage in the acute phase
of T. cruzi infection (Fig. 9.1), demonstrating how the mechanism for
parasite clearance could damage the NS.
9.3.3. Autoimmunity
Sera from CD patients contain auto-antibodies specific for various host
molecules in cardiac, nervous and other tissues (Marin-Neto et al., 2007).
Autoimmune reactions develop as a result of molecular mimicry between
host and parasite proteins, and/or presentation of self-antigens due to

breakdown in self-tolerance and extensive polyclonal immune activation
(Gao et al., 2002, 2003; Soares and Santos, 1999).
The immune self-reactivity to nervous system is demonstrated in
patients with a severe GI form of CD by detection of antibodies and
reactive T lymphocytes to myelin basic protein (MBP), a component of
peripheral myelin (Oliveira et al., 2009). This observation is supported by
earlier studies in infected animals, which showed an antibody activity
against sulfatide, a specific constituent of both T. cruzi parasites and myelin
sheaths of the peripheral nerves (Feldman et al., 1999). As demyelination is
part of neuropathology symptoms observed in the PNS in the course of
T. cruzi infection (Losavio et al., 1993; Molina et al., 1987), it suggests that
myelin of peripheral nerves in CD patients can be targeted by autoimmune
reactions. Antibodies that recognize self-antigens in myenteric plexus,
sciatic nerve, spinal cord and brain, and cross-reacte with parasite surface
epitopes were detected in experimental infection (Gea et al., 1993; Tekiel
et al., 1997; Van Voorhis et al., 1991). Some of these antibodies displayed
pathogenic capacity by causing changes in the sciatic nerve action potential
after epineural injection in uninfected animals (Tekiel et al., 2001).
Functional activity of a different kind was demonstrated for anti-neuro-
nal auto-antibodies, isolated from chronic asymptomatic CD individuals.
Passive immunization with these auto-antibodies, that target neuronal
receptors TrkA, TrkB and TrkC, decreased parasitemia and increased sur-
vival of T. cruzi-infected mice (Lu et al., 2008a,b). In addition, anti-TrkB and
-TrkC antibodies were shown to promote peripheral nerve regeneration
after a nerve crush, and clinical improvement in the animal model of neuro-
degenerative Charcot–Marie–Tooth disease (Sahenk et al., 2010). Given
symptom-free status of the Chagasic auto-antibody donors (Lu et al.,
2008b), presence of these antibodies might even signify a protective effect.
As it has been mentioned, autoimmune reactions are generated in the
late chronic stages of CD, whereas neuronal destruction preferentially
occurs during the acute phase and precedes anti-neuronal autoimmunity
but closely correlates with the anti-parasite cytotoxic immune response.
Such dynamics points to a parasite-directed immune cytotoxicity, rather
than autoimmunity, as the primary cause of neuronal damage in CD.
However, late-stage anti-neuronal auto-reactivity can contribute to and/
or sustain acute neuronal dysfunction (Fig. 9.1) in these 30% of infected
patients who eventually develop symptoms of CD.
9.4. NEUROREGENERATION
The reason why 70% of T. cruzi-infected individuals escape deadly pathol-

ogy of CD, despite retaining, similar to symptomatic patients, pathogenic
parasites, is not known. Equally enigmatic is why an unusually long latent
period of 20–30 years separates initial acute infection and the onset of
chronic disease. The parasite/host factors generally speculated to define
the rate of CD progression and different clinical outcomes are the hetero-
geneity in virulence and tissue tropism among the parasite population,
and the efficiency of host defensive and regenerative responses to
infection (Adesse et al., 2010; Camargos et al., 2000; da Silveira et al.,
2007b; Dutra and Gollob, 2008; Haolla et al., 2009). One of such responses
is the capacity of peripheral neurons to regenerate and compensate
for functional deficits caused by nerve injuries with reinnervation
of target organs. Neuronal plasticity in the peripheral nervous system
(PNS) would allow regrowth and reconnection of the damaged axons,
and sprouting by the intact adjacent axons of new branches into dener-
vated areas (Navarro et al., 2007), thus restoring the PNS functional
integrity lost as the result of neuronal lesions.
That neuronal regeneration is taking place in CD and can be important
for the clinical status of infected patients is indicated by the comparative
physiological and pathological studies which revealed substantial differ-
ences in the extent of organ innervations between asymptomatic and
chronic CD patients. While neuronal deficiency in the symptomatic
group correlated with poor functional characteristics, asymptomatic
patients with better neuronal counts showed normal electrocardiogram
and X-rays of the heart, oesophagus and colon, and were not much
different to seronegative controls (Correia et al., 2007; da Silveira et al.,
2005, 2009; Junqueira and Soares, 2002; Koberle, 1970; Marin-Neto, 1998;
Nascimento et al., 2010; Villar et al., 2004).
The direct demonstration of autonomic recovery after the acute phase
of T. cruzi infection was further provided by experimental CD models.
Functional improvement in the heart and colon of infected animals was
associated with reinnervation of muscle fibres, collateral sproutings of the
damaged nerves and axonal regrowth, remyelination of vagus nerve
fibres and axons of intramuscular nerves (Fazan and Lachat, 1997;
Losavio et al., 1989; Machado et al., 1998; Machado and Ribeiro, 1989;
Maifrino et al., 1999b; Molina et al., 1987; Rodrigues et al., 2002). At 5–6-
month post-infection, that roughly corresponds to chronic stage in
humans, infected animals that survived the acute phase generally demon-
strated normal pattern of both vagal and sympathetic innervation in
myocardium and vasculature (Alves and Machado, 1984; Camargos
et al., 2000; Fazan and Lachat, 1997).
Another indication of active neuronal regenerative processes in
later stages of T. cruzi infection is an increase in the number and func-
tional activity of glial population in the heart and GI tract in both humans
and experimental animals (da Silveira et al., 2008; Fazan and Lachat, 1997;
Nascimento et al., 2010). Schwann and EGCs provide local environment
supportive for regrowth of damaged nerve fibres by secreting the
extracellular matrix constituents and neurotrophic factors, required

for neuronal regeneration (Chernousov and Carey, 2000; von Boyen
et al., 2006). Neurotrophins (NTs) NGF and GDNF, shown to increase
in the hearts of T. cruzi-infected rats (Martinelli et al., 2006), act
directly on neuronal receptors and would promote neurite extension
and formation of new connections, while inducing general resistance
of neuronal ganglia to various neurotoxic conditions (Navarro et al.,
2007).
That the latter can actually be the case in CD, was again first prompted
by studies of Koberle, who demonstrated that surviving neuronal ganglia
in CD patients with chronic infection were less sensitive to the age-related
degeneration compared to the non-infected age-matched individuals
(Fig. 9.2; Koberle, 1970).
It is established that by triggering immune cytotoxic mechanisms,
T. cruzi is responsible for the onset of the denervation process, though
directly the parasites, even in high numbers, are not damaging to neuro-
nal cells (Almeida-Leite et al., 2007; Melo and Machado, 2001; Meyer et al.,
1982). Another indication of neuronal tolerance to T. cruzi burden is the
paucity of symptoms in the immune-privileged CNS. Trypomastigotes
are frequently found in the cerebrospinal fluid, brain and spinal cord in
the absence of neurological symptoms (Hoff et al., 1978; Pittella, 1993),
unless patients are severely immunocompromised (Diazgranados
et al., 2009). In experimental CD, even in younger animals, which
are usually very susceptible to T. cruzi infection, central neurons are
largely preserved regardless of high parasite load and presence of the
proliferating parasites inside astrocytes (Camargos et al., 2000;
Caradonna and Pereiraperrin, 2009; Machado and Ribeiro, 1989). This
ability of T. cruzi to incite little, if any, neuronal death in the brain
certainly deserves future investigation, especially considering severe
meningoencephalitis caused by the brain infection with T. brucei, an
agent of sleeping sickness and a close relative of T. cruzi (Chimelli and
Scaravilli, 1997).
Recent data suggest that T. cruzi parasites may be not only neutral
by-standers but also might contribute to neuronal and other cells regenera-
tion and resistance against external toxic insults (Akpan et al., 2008;
Aoki et al., 2004; Aoki Mdel et al., 2006; Chuenkova and Pereira, 2000;
Chuenkova et al., 2001; da Silveira et al., 2008). It has been demonstrated
that T. cruzi releases a factor that can functionally mimic mammalian NTs
and promote survival of neuronal cells. This parasite-derived neurotrophic
factor (PDNF) activates Trk (tropomyosin-related kinase) receptors and
induces neurite outgrowth and resistance of neurons and glial cells to
apoptosis, and thus may affect the PNS regeneration during T. cruzi
infection.
Heart
Normal
6000
Number of neurons
Chagas
4000
2000
0
0 25 50 75
Years
Esophagus
1500
Normal
Number of neurons
Chagas
1000
500
0
25 50 75
Years
Indeterminate/Megaesophagus
Number of neurons, log 10
Indeterminate
Megaesophagus
100
10
1
30 45 60
Years
FIGURE 9.2 Age-related degeneration of ganglion cells in the heart and oesophagus of
normal and Chagasic individuals. Adapted from Koberle (1968) and Burkauskiene et al.
(2006).
9.5. TRANS-SIALIDASE/PARASITE-DERIVED
NEUROTROPHIC FACTOR
9.5.1. Trans-Sialidase
Trans-Sialidase (TS), also known as PDNF, belongs to one of the largest
T. cruzi protein families (Atwood et al., 2005). It was originally discovered
as a neuraminidase that cleaved monosaccharide sialic (N-acetyl-
neuraminic) acid from glycoconjugates (Pereira, 1983), and later was also
described as TS that can simultaneously catalyze covalent binding of
sialic acid to b-galactosyl residues on the parasite surface or on the host
cells (Parodi et al., 1992; Schenkman et al., 1992).
Expression of TS enzymatic activity is developmentally regulated and
restricted to infective bloodstream trypomastigotes (Cavallesco and
Pereira, 1988), each of them literally covered with more than 10 million
molecules of TS/PDNF, attached to the parasite surface by a GPI anchor.
Upon GPI cleavage, trypomastigotes copiously release TS into the extra-
cellular space and bloodstream at the rate of about 106 mol/min (Agusti
et al., 1997; Rubin-de-Celis et al., 2006; Scudder et al., 1993). Thus during
the acute infection TS/PDNF is present in blood and tissues not only
surface-linked to circulating trypomastigotes, but also as a soluble factor,
mediating a variety of the host–parasite interactions.
By sialylating mucins on the parasite surface, TS/PDNF protects
bloodstream trypomastigotes from complement lysis (Beucher and
Norris, 2008; Buscaglia et al., 2006), while by binding to the host sialil
and b-Gal residues, it promotes parasite attachment to and invasion of,
the host cells (Ming et al., 1993; Todeschini et al., 2004). As a soluble factor,
TS/PDNF was shown to remodel surface of immune cells and augment
T. cruzi immunosuppression in the acute phase of CD by inhibiting
CD8þ lymphocytes cytotoxicity and promoting apoptosis of T cells
(Freire-de-Lima et al., 2010; Mucci et al., 2006).
Typical structure of enzymatically active TS/PDNF represents a
70-kDa globular core that accommodates the catalytic site (Buschiazzo
et al., 2002), followed with a variable number of 12 amino acid repeats in
tandem (long tandem repeat—LTR), also called SAPA (shed acute phase
antigen; Frasch, 2000). LTR/SAPA is not required for sialic acid transfer
but is highly immunogenic (Alvarez et al., 2001) and contributes to para-
site immune evasion by inducing abnormal polyclonal B cell activation
and production of non-specific Igs, characteristic for the acute phase of
CD (Gao and Pereira, 2001; Gao et al., 2002). LTR/SAPA was also shown
to up-regulate secretion of IL-6 (Saavedra et al., 1999), which mediates
anti-parasite protective immune responses (Gao and Pereira, 2002).
The TS catalytic activity, however, is expressed by a relatively small
subset of TS/PDNF molecules. Recent data on sequencing of the parasite
genome and characterization of the proteome demonstrate that T. cruzi
encodes many more species of TS and TS-like molecules as was initially
thought (Atwood et al., 2005; El-Sayed et al., 2005). TS gene family com-
prises 1400 members, of which 220 are expressed as proteins, ranging in
size from 60 to more than 200 kDa. Only 15 of them, produced exclusively
by the invasive trypomastigotes, can function as TSs (Atwood et al., 2005).
Trypomastigotes also express TS/PDNF molecules without catalytic
activity, due to a single mutation of Tyr342 to His, and several other
species of TS family (Frasch, 2000). Intracellular amastigote forms appear

to produce unique subsets of TS molecules without catalytic activity and
LTR/SAPA repeats, while no expression of TS family members was
detected for the insect stage epimastigotes (Atwood et al., 2005).
Such stage-specific restrictions indicate a particular importance of TS/
PDNF proteins for T. cruzi adaptation to a mammalian host, where struc-
turally diverse TS/PDNF family members acquired certain activities
unrelated to the sialic acid transfer (Alves and Colli, 2008; Ratier et al.,
2008).
9.5.2. Parasite-derived neurotrophic factor

Findings over the recent years revealed a new and important function for
TS/PDNF as a neurotrophic factor which activates neuronal Trk receptors
and elicits complex of intracellular responses that promote neuronal
survival, differentiation and resistance to stress.
9.5.2.1. Trk receptors and neuronal regeneration

The interaction of mammalian neurotrophic factors (NTs) and Trk recep-
tors is of the utmost importance for development and maintenance of
both the CNS and PNS. Receptor tyrosine kinases TrkA, TrkB and TrkC
are expressed by neurons, glia and certain non-neuronal cell populations,
and their activation by respective NT ligands NGF, BDNF, and NT-3
control multiple processes in NS, including axonal and dendritic out-
growth, synaptic plasticity and neurotransmission, protection from
endogenous toxic events and neural repair and regrowth (Huang and
Reichardt, 2001). All NTs also bind pan-NT receptor p75NTR which
enhances their selectivity and affinity to respective Trks.
Trk receptors are cell surface transmembrane proteins that possess an
intrinsic, ligand-sensitive tyrosine kinase activity. TrkA, TrkB and TrkC
share a common organization at both extracellular and intracellular levels.
Their extracellular domain consists of two cysteine clusters flanking three
leucine-rich motifs, and two consecutive immunoglobulin-like domains
(Fig. 9.3; Sofroniew et al., 2001), which constitute the major interface for
NT binding and selectivity, controlling TrkA, TrkB and TrkC responsive-
ness to, respectively, NGF, BDNF and NT-3 (Arevalo and Wu, 2006;
O’Connell et al., 2000). Similar to other receptor tyrosine kinases, Trk
cytoplasmic part has a tyrosine kinase domain and several tyrosine-con-
taining motifs. NT binding induces Trk receptor dimerization to which the
intracellular tyrosine kinase domain responds by rapid phosphorylation of
the cytoplasmic tyrosines and generation of phosphorylation-dependent
recruitment sites for adaptor molecules and enzymes that mediate initia-
tion of intracellular signalling (Segal, 2003; Yuan and Yankner, 2000).
This process couples Trks activation to phosphorylation cascades of the
BDNF NT-3
NGF PDNF
Trk receptor
Ig 1
Ig 2
Ras
P Y490 Shc
Raf
P
PI3K P
P MEK
P
GSK3 Akt
P
Erk
Bcl2
P
CREB
Transcription
ROS
Survival Differentiation
– PDNF – PDNF
Neurotransmission
FIGURE 9.3 Trk receptor-mediated signalling by mammalian neurotrophins (NGF, BDNF

and NT-3) and the parasite-derived neurotrophic factor (PDNF)/trans-sialidase. Binding
of neurotrophic factors to Trks causes receptor dimerization, phosphorylation of tyro-
sine residues in the cytoplasmic domain and generation of the docking sites for adaptor
molecules containing PTB or SH2 domains, such as Shc. This results in activation of a
small GTPase Ras, which promotes sequential activation of three kinases in a MAP kinase
cascade: Raf, Mek and Erk. Phosphorylation of Erk leads to its nuclear translocation,
where it activates transcription factors such as CREB, important for axonal elongation
and sprouting. PI3K/Akt pathway is activated by recruitment of PI3K, which enhances
lipid kinase activity leading to phosphorylation of PIP2 lipids to PIP3. Alternatively, PI3
kinase can be activated directly downstream of Ras G-proteins. PIP3 bind to pleckstrin
homology (PH) domains on a variety of proteins, including protein kinases PDK1 and Akt.
Lipid-bound PDK1 phosphorylates and activates Akt, which is critical for the ability of Trk
receptors to mediate neuronal survival.
PI3K/Akt and the Ras/Raf/MAPK/Erk, central in the NT/Trk-promoted

neurotrophic effects (Fig. 9.3). In neurons, these signalling cascades result
in the suppression of a default apoptosic program while enhancing regen-
erative capacity and resistance to stress.
In particular, signalling via PI3K/Akt results in phosphorylation and
inactivation of GSK-3b, leading to microtubule assembly that promotes
axonal growth and increases axon calibre and branching (Zhou et al.,
2004), while Akt phosphorylation of Bad prevents neuronal apoptosis
(Datta et al., 1997). Similarly, the activity of forkhead transcription factor,
FKHRL1, which regulates the expression of several pro-apoptotic proteins,
is inhibited by Akt in response to NTs (Zheng et al., 2002). Moreover, the
NF-kB pro-survival pathway is activated via Akt phosphorylation of IkB
inhibitor, targeting it for degradation (Arevalo and Wu, 2006).
Signalling via MAPK/Erk leads to activation of several downstream
targets that mediate gene transcription, such as the cAMP response ele-
ment-binding (CREB) protein, which controls expression of genes
essential for the survival and differentiation of neurons and axonal regen-
eration (Gao et al., 2004; Ginty et al., 1994).
Among growth factors, NTs are unique in their ability to act as guid-
ance molecules for neuronal growth cones (Tucker et al., 2001; Zheng and
Kuffler, 2000) and as such undoubtedly contribute to successful regener-
ation in the PNS. All NTs promote neurite extension after axotomy, and as
expression of NTs is dramatically up-regulated in the lesioned peripheral
nerves ( Johnson et al., 2008; Meyer et al., 1992), localized sources of NTs
through Trk signalling provide trophic support and serve as pathfinding
cues for regenerating axons (Boyd and Gordon, 2003), inducing turning of
growth cones and sprouting of axons into degenerating stump of the
nerve (Twiss et al., 2006; Vo and Tomlinson, 1999). Increased synthesis
and release of NTs at the site of damage also promote Schwann cell
migration, which precedes and induces axonal elongation into repair
sites (Madison and Archibald, 1994).
9.5.2.2. PDNF interaction with Trk receptors

The discovery that PDNF binds to Trk receptors and functionally mimics
mammalian neurotrophic factors pointed to a new direction in T. cruzi
research, suggesting a possible mechanisms for the parasite involvement
in regeneration of the mammalian PNS during CD.
The remarkable functional similarity between PDNF and NTs starts
with affinity to the receptor (analogous to NGF, PDNF binds TrkA with
an equilibrium binding constant Kd of 1.1 109 Roux and Barker, 2002)
and continues with a series of intracellular events, characteristic for Trk–
ligand interaction. These include receptor dimerization and transpho-
sphorylation of activation loop tyrosine Tyr490 that provides recruitment
site for scaffolding adaptors and activation signals to PI3K/Akt and
Ras/MAP/Erk pathways (Fig. 9.3; Chuenkova and Pereira, 2000, 2001;

Chuenkova and PereiraPerrin, 2004).
9.5.2.2.1. Activation of PI3K/Akt and survival PI3 kinase is central to the

survival-promoting effects via Trk receptors. Trk-dependent activation of
PI3K generates PIP3 phosphoinositides that in collaboration with phos-
phoinositide-dependent kinases activate the protein kinase Akt, which
through its direct and indirect actions on multiple downstream targets
plays the leading role in neuronal survival (Brunet et al., 2001; Yuan and
Yankner, 2000; Yuan et al., 2002). PDNF-induced activation of Akt and
phosphorylation of Akt substrate GSK-3b kinase result in inhibition of
pro-apoptotic GSK-3b and up-regulation of the mitochondrial anti-apo-
ptotic Bcl-2, followed by a reduction in reactive oxygen species (ROS)
formation, inhibition of oxidative stress and caspase-mediated apoptosis
(Fig. 9.3; Chuenkova and Pereira, 2000; Letai, 2006; Maurer et al., 2006;
Woronowicz et al., 2007). Consequently, PDNF treatment supported
peripheral and central neurons, such as DRG, hippocampal and cerebellar
neurons, and neuronal cells through trophic deficiency and neurotoxic
insults—pathological conditions that otherwise would lead to oxidative
stress and neurodegeneration, such as observed in the acute CD
(Chuenkova et al., 2001; Chuenkova and Pereira, 2000, 2003; Chuenkova
and PereiraPerrin, 2004; Weinkauf and Pereiraperrin, 2009; Woronowicz
et al., 2004).
9.5.2.2.2. Activation of MAPK/Erk and differentiation Resistance to neu-

rodegeneration in the PDNF-exposed neuronal cells is also linked to
another Trk-dependent pathway of Ras/MAPK/Erk1/2, implicated in
survival of many neuronal subpopulations (Cheung and Slack, 2004).
PDNF, by inducing MAPK/Erk signalling, prevented activation of cas-
pase-3 and cleavage of the caspase-3 substrate PARP (poly-ADP-ribose
polymerase), a DNA repair enzyme (Cole and Perez-Polo, 2002), and
protected dopaminergic cells against neurotoxin MPTP that causes oxi-
dative stress-related symptoms and pathology in nigrostriatal neurons
analogous to Parkinson disease (Chuenkova and Pereira, 2003).
Sustained activation of MAPK/Erk via PDNF engagement of TrkA,
TrkB and TrkC receptors correlated with neurite outgrowth in the pri-
mary cultures of DRG neurons and differentiation of neuronal PC12 cells
to sympathetic phenotype (Chuenkova and Pereira, 2001; Weinkauf and
Pereiraperrin, 2009; Woronowicz et al., 2007). The molecular events con-
necting PDNF activation of Erk to differentiation include translocation of
phosphorylated Erk to the nucleus, and activation of CREB and CRE-
dependent transcription (Fig. 9.3; Chuenkova and PereiraPerrin, 2005,
2006). CREB activates many genes, including other transcription factors
such as c-fos (Ha and Redmond, 2008), one of the immediate early genes
necessary for initiation and maintenance of neuronal differentiation

(Bonni et al., 1995; Vyas et al., 2002). CREB is also targeted by Akt in the
regulation of neuron survival (Sakamoto and Frank, 2009; Yuan and
Yankner, 2000), and thus can be the point that converges signals from
two pathways activated by PDNF via binding to Trks.
9.5.2.2.3. Synergy with neuropoietic cytokines Similar to classic NTs,

PDNF can act in synergy with neuropoietic cytokines of the IL-6 family.
These cytokines are strongly up-regulated after nerve injury and promote
neuronal regeneration amplifying the effects of conventional NTs (Lai
et al., 1996; Vawter et al., 1996; Wong et al., 1997). Such pattern was
demonstrated when IL-6 family members CNTF and LIF potentiated
PDNF induction of Akt, transcription of Bcl-2 and survival of neuronal
cells (Chuenkova and Pereira, 2000). PDNF and neuropoietic cytokines
activate distinct types of neuronal receptors, Trks and gp130, respectively,
both coupled to PI3K/Akt and MAPK/Erk pathways (Akpan et al., 2008;
Chuenkova and Pereira, 2001; Howlett et al., 2009; Sheu et al., 2000),
where receptor-mediated signals can be integrated to enhance neuronal
regeneration. Such collaborative synergistic effects of cytokines and a
parasite factor at the site of injury might contribute significantly towards
recovery of target neurons especially when the availability of these factors
is limited.
9.5.2.2.4. Induction of cholinergic and adrenergic phenotypes PDNF acti-

vation of Trk receptors and the Cre-dependent transcription can underlie
another important aspect of T. cruzi interaction with the PNS such as
release of neurotransmitters. In acute CD host immune response to infec-
tion damages both catecholaminergic and cholinergic innervations
(Machado and Ribeiro, 1989; Machado et al., 1979) and reduces the NE
and ACh levels in the heart and plasma of CD patients (Davila et al., 2004;
Lo Presti et al., 2009). Such decrease could relate not only to degeneration
of sympathetic and parasympathetic innervation (Rodrigues et al., 2002;
Tafuri and Maria Tde, 1970), but also to reduced activity of neurotrans-
mitter-synthesizing enzymes in the infected and neighbouring cells as
demonstrated for cholinergic gene expression in T. cruzi-infected cardio-
myocytes, fibroblasts and neuronal cells (Akpan et al., 2008; Imai et al.,
2005; Mukherjee et al., 2003). While intracellular T. cruzi down-regulated,
extracellular parasites and PDNF increased expression of cholinergic
locus genes, acetyltransferase (ChAT) and vesicular ACh transporter
(VAChT) (Akpan et al., 2008). Up-regulation of ChAT and VAChT—two
key components of the ACh synthetic cascade that define cholinergic
phenotype in neurons—depended on PDNF engaging TrkA receptor
and activating Akt and Erk1/2 signalling (Akpan et al., 2008).
PDNF also stimulated acquisition of the adrenergic phenotype in

ventral mesencephalic neurons and neuronal PC12 cells (Chuenkova
and Pereiraperrin, 2006), mimicking NGF and other neurotrophic factors,
which can maintain both cholinergic and adrenergic neuronal popula-
tions in the NS (Luther and Birren, 2009; Vaillant et al., 2002). PDNF, via
MAPK/Erk, induced activation of tyrosine hydroxylase (TH)—the rate-
limiting enzyme in the biosynthesis of catecholamines—at transcriptional
level and via post-translational phosphorylation, increasing TH-catalyzed
conversion of tyrosine to dopamine precursor L-DOPA (Chuenkova and
Pereiraperrin, 2006).
Such PDNF activity could be instrumental in restoring Chat expres-
sion, Ach and catecholamine levels in later stages of T. cruzi infection
(Bestetti, 1996; Davila et al., 1995; Machado et al., 1979, 1987), and regen-
eration of peripheral neurons in CD patients (Koberle, 1968).
9.5.2.2.5. Mapping of neurotrophic activity Contrary to the bona fide Trk–

ligands of NT family, T. cruzi PDNF does not bind the pan-NT receptor
p75NTR, and thus is a selective Trk ligand (Chuenkova and PereiraPerrin,
2004). Structural basis of PDNF affinity to Trks is not understood. There is
data suggesting that TS activity of PDNF is necessary for receptor activa-
tion (Woronowicz et al., 2004, 2007). At the same time, other findings
demonstrate that PDNF mutant lacking both sialidase and sialyl-transfer-
ase activity can still elicit full-scale TrkA-dependent signalling. In addi-
tion, NGF binding to TrkA, which does not seem to involve glycosyl
residues (Urfer et al., 1995; Wiesmann et al., 1999), can be competitively
inhibited by PDNF indicating a mechanism distinct from the hydrolysis of
sialil residues (Chuenkova and Pereira, 2000; Chuenkova and
PereiraPerrin, 2005; Chuenkova et al., 1999).
PDNF and NTs have seemingly distinct architecture—for PDNF that
of a six-bladed b-propeller connected via an a-helix to a lectin-like domain
(Buschiazzo et al., 2002)—and NTs are formed by twisted b-sheets
(Nagata, 2010). However, the presence in PDNF sequence of motifs con-
served among NTs (Chuenkova and PereiraPerrin, 2004; Wiesmann et al.,
1999) suggests possible similarity in the molecular basis of binding to Trk
receptors. One of the motifs is located within a segment of 425–445 amino
acids (Fig. 9.4, blue), which, if deleted, eliminates neurotrophic activity of
PDNF (Chuenkova and Pereira, 2000; Chuenkova and PereiraPerrin,
2004; Chuenkova et al., 1999), whereas a synthetic peptide modelled on
this sequence reproduced such activity by activation of TrkA and down-
stream signalling via PI3K/Akt and MAPK/Erk/CREB. Furthermore, the
peptide competes with PDNF for binding to TrkA (Chuenkova and
PereiraPerrin, 2005), which identifies this region as critical for PDNF
interaction with Trks.
1 632 1200
425 445
FIGURE 9.4 TS/PDNF molecule with a TrkA receptor interactive motif (blue) and
substrate sites for Akt kinase (red). Above: primary structure of TS/PDNF with the
N-terminus (grey, residues 1–632) and C-terminal tandem repeats (black ovals, residues
633–1200); below: 3D visualization of the N-terminal surface residues created using
Polyview-3D (based on Amaya et al., 2004).
9.5.2.3. T. cruzi invasion and Trk receptors

Interaction of PDNF with TrkA induces endocytosis of PDNF-receptor
complexes (Woronowicz et al., 2004), and T. cruzi is shown to successfully
exploit this mechanism for cell invasion. Trypomastigotes enter neuronal,
Schwann and some other cells in a Trk-dependable manner, which in
addition requires receptor-mediated signal transduction, as inhibition of
Trk tyrosine kinase activity abrogated parasite invasion, reduced parasite
load and tissue inflammation, and attenuated experimental CD (de Melo-
Jorge and PereiraPerrin, 2007).
The dependence of T. cruzi invasion on the intact Trk-mediated sig-
nalling is in line with the other data describing signalling pathways
downstream of Trk receptors as critical checkpoints in the invasion
process. These include activation of the MAPK and PKC pathways
that enhanced infection of macrophages, endothelial and vascular
smooth muscle cells (Mukherjee et al., 2004; Villalta et al., 1999), and
PI3K-mediated responses, such as accumulation of membrane PIP3 and
mobilization of intracellular Ca2þ stores, important for formation of para-
sitophorous vacuole (Andrade and Andrews, 2005). Thus, it appears that
T. cruzi activation of Trk receptors prior to using them as a vehicle for cell
entry is an essential mechanism for efficient cell invasion.
The importance of Trk receptors in T. cruzi invasion is further under-
lined by the discovery that anti-Trk auto-antibodies, isolated from chagasic
patients, block T. cruzi cell entry, and reduce parasitemia and inflammation
in the heart of infected mice (Lu et al., 2008a,b). Given that these anti-Trk
antibody entry also demonstrated therapeutic agonist-like activity and
improve pathology in various animal models of neurodegeneration
(Sahenk et al., 2010), induction of such autoimmune response might func-
tion as a defence mechanism to control T. cruzi invasion of the PNS.
9.5.3. PDNF and Trypanosoma cruzi receptor-independent

intracellular signalling
T. cruzi recognition of cell surface receptors initiates parasite entrance, via
the parasitophorous vacuole, into the cell cytoplasm, where trypomasti-
gotes differentiate into amastigotes. The latter replicate by binary fission for
several days which can produce hundreds of new parasites per cell. Such
robust intracellular growth requires nutrients and space, and naturally puts
tremendous stress on a host cell. Nevertheless, the T. cruzi-bearing cells not
only stay alive but also demonstrate overwhelming resistance to both
intrinsic and death receptor-provoked apoptosis. Often during infection,
the TH1 cytokine response that induces apoptosis in uninfected cells does
not have this effect on the infected ones suggesting a strong selective
pressure to block apoptosis (Gazzinelli and Denkers, 2006).
Multiple data demonstrate that such T. cruzi strategy to prevent cell
death until completion of the parasite intracellular cycle is based on a
parasite interference with cell signalling network and transcriptional
machinery.
For example, in infected cardiomyocytes, T. cruzi blocked apoptosis
triggered by TNF-a and serum reduction by activation of NF-kB and
inhibition of caspase 3 (Petersen et al., 2006). In infected Schwann and
EGCs, resistance to oxidative stress and death receptor-mediated apopto-
sis correlated with the induction of Akt activation (Chuenkova and
PereiraPerrin, 2009; Chuenkova et al., 2001), and in fibroblasts, T. cruzi
down-regulated expression of the host apoptotic genes while up-regulat-
ing transcription of the anti-apoptotic Bcl-2 (Murata et al., 2008). Accord-
ingly in the hearts of infected mice, Bcl-2 and Bcl-2-interacting protein
were over-expressed together with apoptosis suppressor proteins 1–5 and
FLICE (cFLIP), an inhibitor of death receptor signalling (Hashimoto et al.,
2005; Mukherjee et al., 2008).
In blocking host apoptosis T. cruzi is not unique. Biologically diverse
intracellular protozoan parasites such as Cryptosporidium parvum, Leish-
mania spp., Theileria spp., Toxoplasma gondii and Plasmodium spp. have all
been shown to inhibit the apoptotic response of their host cell by regulat-
ing host pro-survival signalling (Carmen and Sinai, 2007). However,
though such parasite strategies are extensively described, the molecular
basis underlying parasites crosstalk with the host intracellular signalling
molecules remains largely unexplored.
Although T. cruzi, via PDNF, interaction with Trks induces cell resis-
tance to death stimuli, such receptor-mediated effects are time-limited
and cannot solely account for the protection against cell damaging events
resulting from the long-lasting intracellular parasitism.
It was shown that PDNF has a role in the parasite intracellular devel-
opment, because trypomastigotes with elevated expression of PDNF were
released to cell cytosol earlier and proliferate faster (Rubin-de-Celis et al.,
2006). Both trypomastigotes and intracellular amastigotes express GPI-
anchored proteins of TS/PDNF family (Garg et al., 1997; Silveira et al.,
2008) which, surface-bound or shed into the cell cytoplasm (Frevert et al.,
1992), are able to directly interact with the cell signalling effector mole-
cules responsible for regulation of cell fate.
This hypothesis got an experimental support when it was demon-
strated that in infected Schwann cells PDNF, expressed by the intracellu-
lar parasites, integrated into the host PI3K/Akt signalling cascade as a
substrate-activator of Akt (Chuenkova and PereiraPerrin, 2009).
PDNF contains several motifs that correspond to the consensus R/K-
x-R/K-x-x-pS/T required for an Akt substrate (Fig. 9.4; Manning and
Cantley, 2007), and was recognized as such by the host Akt in the
T. cruzi-infected cells (Chuenkova and PereiraPerrin, 2009). Moreover,
accumulation of phosphorylated PDNF in the cytoplasm of infected
cells corresponded to an increase in Akt activation and cell resistance to
apoptosis (Chuenkova and PereiraPerrin, 2009; Chuenkova et al., 2001).
Ectopically expressed in Schwann cells, PDNF reproduced anti-apoptotic
effects of the parasite residence and induced Akt activation and transcrip-
tion of Akt and PI3 kinases, while down-regulating expression of the pro-
apoptotic genes: caspase-9 and caspase recruitment domains (CARD),
transcription factor FOXO, the mitochondrial Bax and NF-B inhibitor
IkB (Chuenkova and PereiraPerrin, 2009). In addition, PDNF induced
expression of glial fibrillary acidic protein (GFAP; MVC, unpublished
observations), implicated in survival and regenerative capacities of glial
cells in enteric CD (Nascimento et al., 2010).
Combination of PI3K/Akt activation and down-regulation of the pro-
apoptotic effectors protected PDNF-expressing cells against apoptotic
death induced by oxidative stress and cytokines TNF-a and TGF-b
(Chuenkova and PereiraPerrin, 2009). In addition to stimulation of NO
production by activated macrophages, TNF-a promotes apoptosis via
‘‘death’’ domain-containing TNFR1 (Shen and Pervaiz, 2006), and is pres-
ent, together with TGF-b, in the inflammatory lesions in the PNS, where
both are shown to cause degeneration of Schwann cells (Skoff et al., 1998).
Anti-apoptotic activity of the intracellular PDNF in conditions similar to
immune response during T. cruzi infection (Gutierrez et al., 2009a; Silva
et al., 2003; Wen et al., 2006) characterizes PDNF as a T. cruzi factor that
defines, at least in part, the mechanism of infected cells resistance to
cytotoxic environment.
How the intracellular PDNF can activate host Akt is not clear. Phos-
phorylation of Ser- and Thr-containing PDNF motifs by Akt can create
sites for interaction with phospho-Ser/Thr-binding intracellular signal-
ling molecules (Seeburg et al., 2005; Yaffe and Elia, 2001) and thus recruit
PDNF into protein–protein signalling complexes. One such example is
presented by 14-3-3 proteins, implicated in multiple signalling pathways,
including that of PI3K/Akt (Barry et al., 2009), and up-regulated by
intracellular T. cruzi (MVC and MPP, unpublished observation). Some
of Akt-phosphorylated PDNF sites overlap with the sequences recog-
nized by 14-3-3 proteins, and in the cytoplasm of the T. cruzi-infected
Schwann cells, PDNF was shown to associate with 14-3-3 (Chuenkova
and PereiraPerrin, unpublished observation). Such interactions can
underlie PDNF integration, as a scaffolding adaptor, in the assembly of
PI3K/Akt signalosome, Akt activation and pro-survival transcriptional
remodelling.
9.6. CONCLUSIONS
As one of the most successful parasitic protozoans, T. cruzi has evolved
active strategies to maintain persistent infection in the vertebrate host. A
spontaneous cure is uncommon, suggesting a balanced interaction between
the parasite and the host, which is clearly beneficial to T. cruzi, while
excessive tissue pathology and host lethality would interrupt the infectious
cycle and undermine the parasite chances for successful transmission. To
prevent such an outcome T. cruzi developed potent adaptive mechanisms
which underlie the remarkable symbiosis inherent in the course of the
chronic asymptomatic CD when the majority of infected population, while
still retaining the pathogen, is clinically normal. The data discussed above
illustrate the natural complexity of T. cruzi residence in the mammalianPNS.
Parasite modulation of the host pro-survival responses ranges from its
complete inhibition to promotion and became an important component of
the T. cruzi pathogenic profile. Destruction of the nervous tissue in the acute
phase is counterbalanced by regeneration in the indeterminate stage of the
infection which leads to preservation of the host as the parasite habitat and
allows maintaining infection at a stable level.
While pathogenic effects of the T. cruzi infection have been extensively
studied, the parasite role in the host regeneration has just began to
be appreciated. However, these processes might contribute to the clinical

manifestations of CD shifting the balance between symptomatic and
asymptomatic outcome. By releasing a molecule with neurotrophic
activity, the parasite can potentially promote formation of the local micro-
environment permissive for survival and regrowth of regenerating nerves
and facilitate functional recovery of the autonomic innervation in the
heart and GI observed in asymptomatic infection. Further studies in this
direction will undoubtedly increase our understanding of the molecular
pathogenesis underlying CD, and may suggest future therapeutic
opportunities to control progression of the disease.
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CHAPTER 10
Adipose Tissue, Diabetes and
Chagas Disease
Herbert B. Tanowitz,*,†,k Linda A. Jelicks,‡
Fabiana S. Machado,# Lisia Esper,#
Xiaohua Qi,} Mahalia S. Desruisseaux,*,†
Streamson C. Chua,†,§,k Philipp E. Scherer,**,††,‡‡ and
Fnu Nagajyothi*

10.2. Adiponectin 237
10.3. Adipose Tissue and Infection 240
10.4. Chagas Disease and Adipose Tissue 241
10.5. Chagas Disease and Glycaemia 245
10.6. Conclusions 246
References 246
* Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA
{
{
Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, New York, USA
}
Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA
}
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA
k
The Diabetes Research Center, Albert Einstein College of Medicine, Bronx, New York, USA
#
Department of Biochemistry and Immunology, Institute of Biological Science, Federal University of Minas
Gerais, Belo Horizonte, Brazil
** Touchstone Diabetes Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA
{{
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
{{
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

235
236 Herbert B. Tanowitz et al.
Abstract Adipose tissue is the largest endocrine organ in the body and is
composed primarily of adipocytes (fat cells) but also contains fibro-
blasts, endothelial cells, smooth muscle cells, macrophages and
lymphocytes. Adipose tissue and the adipocyte are important in
the regulation of energy metabolism and of the immune response.
Adipocytes also synthesize adipokines such as adiponectin which is
important in the regulation of insulin sensitivity and inflammation.
Infection of mice with Trypanosoma cruzi results in an upregulation
of inflammation in adipose tissue that begins during the acute phase
of infection and persists into the chronic phase. The adipocyte is
both a target of infection and a reservoir for the parasite during the
chronic phase from which recrudescence of the infection may occur
during periods of immunosuppression.
10.1. INTRODUCTION
Chagas disease, caused by Trypanosoma cruzi, remains an important cause

of morbidity and mortality in endemic areas of Latin America and among
immigrant populations in non-endemic areas (Tanowitz et al., 2009).
There has been an increase in obesity and type 2 diabetes in the tropical
world including those in which Chagas disease is endemic. For example,
the clinical–nutritional profile of individuals with chronic disease in one
study, evaluated at the Tropical Diseases Nutrition Out-Patient Clinic of
the Botucatu School of Medicine, São Paulo State University, Brazil,
revealed that 94% of patients with Chagas disease were overweight or
obese (Geraix et al., 2007). The relationship between this parasite and
adipose tissue and the adipocyte (fat cell) has not been fully evaluated.
Depending on the individual, adipose tissue may account for 10–50% of
body composition. The adipocyte is the major component of adipose tissue,
and it is well established that it contributes to the pathogenesis of diabetes,
obesity and the metabolic syndrome (Asterholm et al., 2007; Attie and
Scherer, 2009; Horrillo et al., 2010; Nawrocki and Scherer, 2005; Rajala
and Scherer, 2003), and its secretory products have been implicated in
other processes (Attie and Scherer 2009; Nawrocki and Scherer 2005).
Although the adipocyte was once considered to be a static storage com-
partment for triglycerides, it is now appreciated that adipocytes are active
endocrine cells playing a critical role in various metabolic and immune
responses (Halberg et al., 2008; Kaminski and Randall, 2010; Yang et al.,
2010; Zuniga et al., 2010). Adipocytes contribute to these functions by
influencing systemic lipid homeostasis and also through the production
and release of a host of adipocyte-specific and adipocyte-enriched hor-
monal factors and inflammatory mediators, including adipokines. Until
recently, there has been little attention given to the role of adipose tissue
and adipocytes in infectious disease (Desruisseaux et al., 2007).
Adipose Tissue, Diabetes and Chagas Disease 237
Adipose tissue is a heterogeneous tissue composed not only of adipo-

cytes but also of other cell types including fibroblasts, endothelial and
smooth muscle cells and especially in the setting of infection and morbid
obesity, macrophages and leukocytes (Anderson et al., 2010; Weisberg
et al., 2003). It is important to note that in experimental T. cruzi infection,
there is a similar infiltration of macrophages into adipose tissue, which
raises the possibility that similar signalling pathways could be involved.
The mechanisms for macrophage recruitment have included cell dam-
age/death by apoptosis/necrosis, tissue hypoxia and, more recently,
lipolysis (Kosteli et al., 2010).
Different adipose tissue depots display distinct gene expression pat-
terns and vary widely in their size and proximity to neighbouring organs.
As noted, adipose tissue stores lipid in the form of triglycerides as well as
non-esterified cholesterol on the surface of lipid droplets that act as
specialized organelles inside the adipocyte. Since the lipid droplet is
such a large component of the adipocyte, changes in the amount of lipid
stored within it affect fat cell size (which can range from 25 to 250 mm).
A potential endocrine function of adipose tissue was first recognized
over two decades ago when it was reported that the serine protease,
adipsin was secreted by cultured 3T3-L1 adipocytes (Cook et al., 1987).
Subsequent investigations discovered additional adipokines, including
adiponectin originally known as Acrp30 (Scherer et al., 1995), leptin
(Zhang et al., 1994), resistin (Steppan et al., 2001), SAA3 (Lin et al., 2001),
omentin (Yang et al., 2006), visfatin (Fukuhara et al., 2005) and RBP4 (Yang
et al., 2005). These adipokines are critically important to the regulation of
energy homeostasis through effects on both central and peripheral tissues.
They also contribute to non-metabolic processes in the body such as the
immune response. The most adipocyte-specific adipokine is adiponectin
although other adipokines can also be synthesized by tissues other than
adipose tissue and/or by cells other than adipocytes.
10.2. ADIPONECTIN
Systemic energy homeostasis is maintained by the competing effects of a

number of different hormonal factors, some of which originate in adipose
tissue. These adipocyte-derived factors (adipokines) influence processes
such as food intake, energy expenditure and insulin sensitivity. Two
adipokines, resistin and adiponectin, have opposing effects on whole-
body glucose homeostasis (Combs et al., 2001; Rajala and Scherer, 2003).
Pharmacological doses of recombinant resistin hyperactivate gluconeo-
genesis through decreased hepatic insulin sensitivity.
Adiponectin is a hormone-like peptide that is almost exclusively pro-
duced by the adipocytes (Scherer et al., 1995). It is a 30-kDa molecule with
three defined domains. The N-terminus contains a hypervariable region,

which is commonly used as the antigenic site for species-specific antibody
generation. The collagenous stalk containing 22 GXY repeats is followed
by a globular domain at the C-terminus. Both intracellularly and extracel-
lularly, adiponectin exists in three different higher-order complexes: a
high molecular weight form (HMW; 12–36 mer), a low molecular weight
form (hexamer) and a trimeric form. The different complexes have dis-
tinct functions, and the ratio of HMW to the other forms serves as an
independent predicting factor of metabolic disorders. Total levels and
HMW ratio are decreased in obese patients and obese mouse models
suggesting that adiponectin, especially the HMW form, may be involved
in obesity-related disorders. Adiponectin modulates glucose and lipid
metabolism by exerting insulin-sensitizing effects. This may be due in
part to the increase in insulin sensitivity by the inhibition of hepatic
glucose output. In the normal metabolic state, adiponectin is present in
high concentrations in plasma, but there is also noted an inverse relation-
ship with body-fat mass, insulin resistance and type 2 diabetes mellitus.
Lower levels of circulating adiponectin are associated with increased
susceptibility to a variety of diseases associated with the metabolic syn-
drome, including diabetes, hypertension, obesity and a increase in the
expression of endothelin-1 (Yudkin, 2007).
There is an association between circulating adiponectin levels and
metabolic parameters that regulate insulin sensitivity in different patient
populations. For example, Arita et al. (1999) demonstrated decreased
plasma adiponectin concentrations in obese humans which was con-
firmed with obese animal models. The pattern of decreased adiponectin
secretion with increasing adiposity, though contrary to what is observed
for the majority of adipose-specific secretory proteins such as leptin, has
been well recognized. There is a reduction in the levels of adiponectin in
diabetics with coronary artery disease compared to diabetics without
coronary artery disease, and adiponectin levels in serum are negatively
correlated with basal metabolic rate, plasma glucose, insulin and serum
triglycerides (Hotta et al., 2000). Moreover, even a moderate weight loss
may be associated with significant increases in circulating adiponectin
levels (Yang et al., 2001) and an increase in insulin sensitivity. The para-
dox of why adiponectin levels tend to increase with decreasing adiposity
has never been adequately explained. After weight loss, the remaining
adipocytes may be more insulin sensitive and therefore secrete increased
amounts of adiponectin. Alternatively, adiponectin expression and/or
secretion may be directly or indirectly regulated by plasma insulin levels.
Supporting this view are the observations that insulin treatment of 3T3-L1
adipocytes results in significantly decreased adiponectin expression
(Fasshauer et al., 2002) and serum adiponectin levels are inversely pro-
portional to fasting insulin levels. Thus, it is likely that an inhibitory
feedback pathway exists to down-regulate the expression and secretion of

adiponectin in the obese.
Central adipose pads are the predominant sources of systemic adipo-
nectin in the lean state. The production of adiponectin by this tissue in the
obese state is reduced. Those with the highest levels of adiponectin had a
reduced risk of myocardial infarction compared with those with the
lowest adiponectin levels. This relationship persisted even when
controlling for several variables. Animal models have corroborated
these observations, demonstrating the importance of adiponectin for pre-
venting diet-induced progression of atherosclerosis. Life style changes
leading to improvements in insulin sensitivity such as weight reduction
and exercise will result in an increase in the level of plasma adiponectin.
The administration of peroxisome proliferator-activated receptor-g
(PPAR-g) agonists, such as the thiazolidinediones, increases adiponectin
secretion in cultured adipocytes and increases circulating adiponectin
levels in rodents and in patients with diabetes (Arita et al., 1999; Long
et al., 2010).
The mechanistic basis of the anti-atherosclerotic activity of adiponec-
tin has not been completely elucidated. It has been hypothesized that
adiponectin has inflammation-modulating activities and clinical studies
have demonstrated inverse associations between adiponectin levels and
serum markers of inflammation (Goldstein and Scalia, 2004; Ouchi et al.,
2003). Several studies have reported that the physiologically relevant, full-
length form of adiponectin has anti-inflammatory effects on both
endothelium and macrophages. However, it is unclear how or whether
adiponectin itself exerts anti-inflammatory properties. It has been demon-
strated that the synthesis of adiponectin by cultured adipocytes is inhib-
ited by inflammatory cytokines such as TNF-a (Ruan and Lodish, 2003).
This inhibition may be mediated in part by NFkB signalling. IkB kinase
inhibition leads to increased plasma adiponectin levels and an improve-
ment in systemic insulin sensitivity (Keller et al., 2003). The anti-inflam-
matory activity of adiponectin may be mediated in some instances by
activation of AMP-activated protein kinase (AMPK; Ouchi et al., 2000).
Recently, Holland and colleagues demonstrated that the broad spectrum
of effects attributed to adiponectin, including its anti-inflammatory, anti-
apoptotic and insulin-sensitizing actions are due to the adiponectin-
mediated stimulation of a potent ceramidase activity that leads to a
lowering of cellular ceramides and an increase in its degradation product,
sphingosine-1-phosphate (Holland et al., 2011). The ceramidase activity is
adiponectin receptor inherent or at least closely associated with these
receptors.
Chemokines positively control the secretion of leptin, suggesting a role
for these molecules in the regulation of adipose tissue. Importantly, leptin
is vital in immune cell differentiation and development. Targeting
chemokines may provide a novel therapeutic basis for the treatment of

obesity, diabetes and cachexia (Gerhardt et al., 2001). A high-fat diet
increases the expression of inflammatory genes, including the early
induction of MCP-1 and MCP-3 (Chen et al., 2005). Some of the proven
anti-atheromatous effects of adiponectin may be mediated by anti-inflam-
matory actions directly on the vasculature. Okamoto et al. (2008) recently
reported that adiponectin inhibits the production of CXCR-3 chemokine
ligands in macrophages and causes a reduction in T-lymphocyte recruit-
ment. Interestingly, Miller et al. (2010) have reported that IL-33 may play
a protective role in the development of adipose tissue inflammation, but
the relationship to infection is unclear. Recent studies have shown a direct
link between inflammation and diabetes and obesity.
Adiponectin has been reported to contribute in protecting against
cardiac hypertrophy and ischaemic heart disease. In mouse models,
adiponectin has been shown to protect against myocardial ischaemia–
reperfusion injury and overload- and adrenergically induced cardiac
myocyte hypertrophy by inhibiting hypertrophic signals via AMPK
(Ouchi et al., 2006; Shibata et al., 2004, 2005). Importantly, adiponectin
null mice have a cardiomyopathic phenotype (Ouchi et al., 2006; Shibata
et al., 2005; Shimano et al., 2010). Taken together, the current information
is consistent with the notion that adiponectin is anti-inflammatory and
that a reduction in adiponectin levels is proinflammatory.
10.3. ADIPOSE TISSUE AND INFECTION
The potential contribution of adipose tissue and the obese state to the
infectious process in general had been recently reviewed (Desruisseaux
et al., 2007). It has been appreciated that obese humans and animals have
difficulties responding to many types of infections including frank sepsis.
The first well-designed study to examine the possible relationship of
infection and adipose tissue was published by the Scherer laboratory
(Pajvani et al., 2005). In this study, it was demonstrated that injection of
LPS into mice that were rendered fatless, using the regulated fat apoptosis
murine model, did not result in the immediate death of mice as seen in
control mice with a normal component of adipose tissue (Pajvani et al.,
2005). This observation suggested that adipose tissue makes a significant
contribution during the acute phase response to infection. Interestingly,
during the recent H1N1 influenza epidemic, it was reported that in
individuals with increased BMI the morbidity rate was increased
(Tsatsanis et al., 2010), and this has been confirmed by studies in obese
mice (Karlsson et al., 2010). Responses to Staphylococcus aureus infection
have recently been studied by injection of S. aureus into the footpad of
the leptin receptor null mouse model of diabetes and obesity. Whereas
non-diabetic lean mice resolved this infection within 10 days, in the obese
mice the infection was prolonged and was associated with a significant
increase in the associated inflammatory response (Park et al., 2009). One
of the most intensively investigated areas in the interface between infec-
tion and adipose tissue has been in HIV/AIDS where receptors for the
virus have been reported on adipocytes and HIV-associated lipodystro-
phy has been described (Anuurad et al., 2010; Garrabou et al., 2011; Hazan
et al., 2002; Jan et al., 2004; Maurin et al., 2005; Mynarcik et al., 2002).
10.4. CHAGAS DISEASE AND ADIPOSE TISSUE
In the 1970s, Shoemaker and colleagues (Shoemaker and Hoffman, 1974;

Shoemaker et al., 1970) demonstrated that T. cruzi parasitized adipose
tissue and Andrade and Silva (1995) subsequently demonstrated that
T. cruzi parasitized adipose tissue and the adipocyte (Andrade and
Silva, 1995). Buckner et al. (1999) demonstrated the detection of T. cruzi
in adipose tissue using special staining techniques. However, it was not
until the publication by Combs et al. (2005) that the potential impact of
parasitism of adipose tissue and the adipocyte was appreciated (Fig. 10.1).
Interest in the association between T. cruzi infection and adipose tissue
and diabetes has been a recent focus for several reasons. First is the
general belief, although not conclusively proven, proven that Chagas
disease may be associated with obesity and diabetes. The clinical–
epidemiologic evidence linking Chagas disease, obesity and diabetes is
unclear because published studies have been at variance and many of the
studies have not been subjected to rigorous statistical scrutiny (dos Santos
et al., 1999; Geraix et al., 2007; Guariento et al., 1993; Hidron et al., 2010;
Oliveira et al., 1993). Secondly, it is now well established that adipose
tissue and adipocytes are both targets of infection and a storage site from
which infection can arise later in life under circumstances of immunosup-
pression. Supporting this view, it was recently demonstrated that Rickett-
sia prowazekii, the cause of Brill–Zinsser disease (the relapsing form of
epidemic typhus), lives in adipocytes and adipose tissue and are a reser-
voir from which the infection can recrudesce and cause disease decades
later (Bechah et al., 2010). Thus, the parasitism of adipose tissue by T. cruzi
may create in some individuals a ‘‘low-grade’’ chronic inflammatory
state similar to what is observed in obesity (Ferrante, 2007; Weisberg
et al., 2003).
When mice are infected with T. cruzi, the plasma levels of adiponectin
are significantly reduced (Combs et al., 2005; Nagajyothi et al., 2010).
There is a concomitant reduction in expression of adiponectin and of
PPAR-g; both negatively regulate inflammation. Reduced levels of adipo-
nectin are often associated with insulin resistance, hyperglycaemia and
A C
LD
LD
LD
FIGURE 10.1 (A) Four representative scanning electron micrographs of 3T3-L1

adipocytes infected with T. cruzi. (B) Representative transmission electron micrographs
of 3T3-L1 adipocytes 48-h post-infection. Note the close proximity of parasites to lipid
droplets indicated by arrowheads. The picture on the top left corresponds to an unin-
fected cell. (C) Electron microscopy analysis of brown adipocytes at different magnifi-
cations. LD, lipid droplet. Arrows indicate intracellular amastigotes (4–5 mm in diameter)
(images from Combs et al., 2005).
obesity, that is, the metabolic syndrome. At 30 days post-infection, the

acute-phase reactants a-1 acid glycoprotein and SAA3, which are
expressed in adipocytes, were upregulated. The levels of resistin, a fat
cell-specific secretory factor with insulin-desensitizing properties, was
unchanged in adipose tissue obtained from T. cruzi (Brazil strain) mice.
Additionally, plasminogen activator inhibitor-1 levels were unaffected by
infection. Conversely, proinflammatory markers such as cytokines (TNF-
a, IL-1b, IFN-g) and chemokines and toll-like receptors (TLRs) were
markedly elevated in the adipose tissue from acutely infected mice, and
this elevation often persisted into the chronic phase (Combs et al., 2005).
Fifteen days after T. cruzi (Brazil strain) infection of CD-1 mice, there is
no peripheral parasitaemia and no mortality. There is a significant para-
site load in both brown and white adipose tissue as compared to other
organs such as the heart and spleen. At this early stage of infection, we
have demonstrated a significant influx of macrophages into adipose
tissue as determined by immunostaining with antibodies to macrophage
specific markers such as Iba-1and PCR analysis employing primers to
F4/80. There is also a reduction in fat mass as determined by magnetic

resonance imaging (Fig. 10.2). Concomitantly, there is a reduction in fat
content as determined by Oil red O staining and reduction in the size of
adipocytes. Western blot analysis indicates an increase in lipolysis
although apoptosis and necrosis may also be involved. This early process
may lead to release of parasites into the general circulation resulting in
increased peripheral parasitaemia. This ongoing process may represent a
mechanism by which low levels of parasites are continuously released
FIGURE 10.2 Representative transverse MRI of the abdominal region of a normal

control mouse with 15% total body fat (A), an infected mouse with a normal sized heart
and 7% total body fat (B) and an infected mouse with an enlarged heart and 3% total
body fat (C). The solid white arrows indicate the visceral and subcutaneous fat which
appears bright in these images. The spine is indicated for orientation. Total body fat was
determined using images spanning the entire mouse body. Three-dimensional recon-
structions of adipose tissue in an uninfected control mouse (D) and a chronically
infected mouse (E). MRI was performed using 9.4T Varian animal imaging system.
Transverse images of the mice were acquired from the tail to the neck. Images were
imported into Amira 3D visualization software. Image segmentation was performed, and
the adipose tissue is indicated in semi-transparent grey (spanning the base of the tail to
the neck). An image acquired at the level of the kidneys is included, and one of the
kidneys of each mouse is indicated and circled in white. The perirenal and visceral fat
depots are indicated. The images clearly show a reduction in adipose tissue mass in the
infected mouse.
into the circulation (at a level below detection by routine blood smear)
resulting in chronic infection. During the chronic phase of infection,
examination of adipose tissue reveals persistence of both macrophages
and parasites. Thus, adipose tissue is both an early sensor and target of
T. cruzi infection and a chronic reservoir from which infection can recru-
desce during periods of immunosuppression and/or lipoatrophic states.
Recently, Kosteli et al. (2010) have demonstrated that local lipolysis-
induced increases in fatty acids in adipose tissue lead to an increased
infiltration of immune cells, particularly macrophages. This offers a
potential explanation for the long-term effects we observe on some fat
pads after acute T. cruzi infection. The presence of parasites within chron-
ically infected fat pads leads to insulin resistance, associated with
increased lipolysis with chronically elevated local free fatty acid levels
that in turn will be triggering the observed increased infiltration of
macrophages.
Since adipose tissue is composed of many cell types, it was important
to determine if infection of adipocytes in the absence of other compound-
ing variables found in the tissue also resulted in an inflammatory pheno-
type. Indeed, T. cruzi infection of cultured adipocytes resulted in an
increased expression of chemokines, such as CCL2, CCL3, CCL5 and
CXCL10, as well as the cytokines TNF-a, IL-10 and interferon-g
(Nagajyothi et al., 2008). The expression of STAT3, an important down-
stream mediator of cytokine signalling, was also increased. TLR expres-
sion was increased (TLR-2 and -9), and there was evidence of activation of
components of the mitogen-activated protein kinase (MAPK) pathway,
such as ERK. Cyclin D1 expression was increased, and it is usually
upregulated by ERK and inversely regulated by caveolin-1 (Hulit et al.,
2000). Indeed, we demonstrated that infection resulted in a reduction in
the expression of caveolin-1 and the activation of ERK. Both of these
events increase the expression of cyclin D1. A reduction in caveolin-1
expression has also been demonstrated to be associated with an increased
proinflammatory cytokine response (Cohen et al., 2003, 2004). Interest-
ingly, T. cruzi infection activates the Notch pathway, which also regulates,
in part, the expression of cyclin D1 (Stahl et al., 2006).
T. cruzi infection of cultured adipocytes results in increased expression
of PI3 kinase and the activation of AKT, strongly suggesting that T. cruzi
infection induces the insulin/IGF-1 receptor pathway. This is an unex-
pected observation since the upregulation of proinflammatory pathways
is usually associated with a down-regulation of the insulin signal trans-
duction pathway (Ferrante, 2007; Hotamisligil, 2006). Whether other path-
ways influenced by insulin are affected is not known. Thus, T. cruzi
infection of cultured adipocytes as well as adipose tissue results in altera-
tions of several important pathways early in infection that persist well
into the chronic phase.
10.5. CHAGAS DISEASE AND GLYCAEMIA
We and others have demonstrated that T. cruzi infection of mice results in

severe hypoglycaemia (Combs et al., 2005; Holscher et al., 2000). Acute
infection of CD-1 mice with the Brazil strain of T. cruzi is usually asso-
ciated with severe hypoglycaemia and generally correlated with mortality
(Combs et al., 2005). It has been suggested that the hypoglycaemia was the
result of ‘‘cytokine storm’’ and reduced food intake. Interestingly, the
metabolic response to bacterial sepsis is often associated with hypergly-
caemia, insulin resistance, profound negative nitrogen balance and the
diversion of protein from skeletal muscle to splanchnic tissues. Thus, the
response to T. cruzi infection differs from that generally observed in
bacterial sepsis. It is possible that there is an effect on glucose metabolism
due to invasion of the liver by the parasite. During acute infection, glucose
levels in all of the T. cruzi-infected mice were below those measured in the
control mice. Even though the baseline glucose levels in the infected
animals were lower, the oral glucose tolerance test indicated a relatively
normal ability to clear ingested glucose despite the high degree of inflam-
mation associated with this infection (Combs et al., 2005). The decreased
insulin levels observed 30 days post-infection in the mouse model of
T. cruzi infection are consistent with a physiological response to very
low glucose levels during that time (Combs et al., 2005).
Observational studies in people and case reports are suggestive that that
the incidence of diabetes may be increased in the chagasic population (dos
Santos et al., 1999; Guariento et al., 1993; Oliveira et al., 1993). One such
study demonstrated a significant reduction in insulin among chronically
infected individuals (dos Santos et al., 1999). However, the data in this
report could also be interpreted to reflect weight loss or illness rather than
pancreatic b-cell destruction. The notion that T. cruzi could cause diabetes is
not entirely new since it has been known that this parasite can invade any
cell type including those of the pancreas. When streptozotocin-induced
diabetes was produced in mice which were then infected with T. cruzi,
they displayed higher parasitaemia levels and mortality rates (Tanowitz
et al., 1988). After insulin was administered, the glucose levels returned to
normal and the parasitaemia levels and mortality rates were reduced.
Mice carrying a defective leptin receptor gene (db/db mice) are meta-
bolically challenged in that they are hyperglycaemic, obese and have low
levels of adiponectin. They are bred on a FVB background. When these
mice are infected with the Brazil strain of T. cruzi, they have a high
peripheral parasitaemia and tissue parasitism and suffer 100% mortality.
These mice also displayed an upregulation of the inflammatory pathway
as well as an increase in myocardial pathology, and a large numbers of
parasite pseudocysts. In genetically modified db/db mice, (NSE-Rb db/db
mice), central leptin signalling is reconstituted only in the brain, which is
sufficient to correct the metabolic defects (de Luca et al., 2005). They are
lean and normoglycaemic. In order to determine the consequences of the
lack of leptin signalling on infection in the absence of metabolic dysregu-
lation, we infected these mice with the Brazil strain and found a minimal
transient peripheral parasitaemia and tissue parasitism and no mortality.
The myocardium was virtually devoid of parasites. The observation in the
NSE-Rb db/db mice was similar to that observed in the wild-type FVB
mice (Nagajyothi et al., 2010). Thus, the restoration of the metabolic
dysfunction was sufficient to control the Brazil strain infection. More
recently, we observed that when we infected the NSE-Rb db/db mice
with the virulent Tulahuen strain the mortality was 100% (unpublished
observations). This is an example demonstrating that both the strain of
mouse and parasite are important in the final outcome of infection. These
findings suggest that leptin resistance in individuals with obesity and
diabetes mellitus may have adverse consequences in T. cruzi infection.
10.6. CONCLUSIONS
There is a close association between adipocytes and glucose metabolism.

The small numbers of studies that have examined T. cruzi infection and
adipocytes and glucose metabolism have given us increased insight into
the pathogenesis of Chagas disease but have also raised interesting ques-
tions that require more research. For example, what are the precise roles
of the adipocyte and leptin signalling on T. cruzi infection? Since adipo-
nectin null mice have a cardiomyopathic phenotype, could the T. cruzi-
induced reduction in adiponectin expression contribute to the cardiomy-
opathy of Chagas disease?
ACKNOWLEDGEMENTS
This study was supported by grants from the United States National Institutes of Health
National Institutes of Health (Grants R01-AI-076248, R01-HL-73732 and R21-AI-06538 to
H. B. T.; Grants R01-DK55758, R01-CA112023, RC1 DK086629 and P01-DK088761 to P. E.
S.; Grants P60-DK020541 and PO1-DK-26687 to S. C. C.); Einstein Diabetes Center (pilot grant
to H. B. T.); Conselho Nacional de Desenvolvimento Cientı́fico e Tecnologico (grant to F. S.
M.) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (grant to F. S. M.).
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INDEX
A anti-T. cruzi antibodies, 137–138

autoantibodies, 138–139
Actin, 51–52
autoreactive T cells, 139–140
Adipose tissue description, 134
adipocytes, 236 molecular mimicry, 138
adiponectin mononuclear cell infiltrate, 137
animal models, 239
pathological evidence, 130, 133
anti-atherosclerotic activity, 239 polyclonal activation, 138
cardiac hypertrophy, 240 molecular mimicry
chemokines, 239–240 B13 peptide, 141–142
description, 237–238
cardiac myosin, 141
systemic energy homeostasis, 237 HKTC, 140
weight loss, 238–239 neuron-T. cruzi antibodies, 140–141
CD
T. cruzi infection, 130, 131
cultured adipocytes, 244
cyclin D1 expression, 244 B
obesity and diabetes, 241
peripheral parasitaemia, 242–244 Bioactive lipids, T. cruzi infection
proinflammatory markers, 241–242 eicosanoid synthesis, vertebrates
T. cruzi detection, 241 AA metabolism, 4–5
3T3-L1 adipocytes, 242 COX pathway, AA metabolism, 7–10
Western blot analysis, 242–244 cytochrome P-450 monooxygenases, 7
description, 237 LO pathway, 6–7
endocrine function, 237 mammals, 4
infection, 240–241 endogenous regulation, CD
lipids, 237 acute infection, 13–16
T. cruzi, 236 chronic infection, 16
Arachidonic acid (AA) metabolism fatty acid deficiency, 12–13
COX pathway insect vectors, 16–17
cell surface receptors, 9–10 humans, 3–4
enzymes, 7–8 metabolism and eicosanoid biosynthetic
PGH2, 7 pathways
PGs and thromboxane, 9 G-protein-coupled receptors, 12
cytochrome P-450 pathway, 7 kinetoplasts, 10
LO pathway PGF2a, 11
5-LO, 6 PLA1-and PLA2, 10–11
LTA4, 6–7 TcOYE and TbPGFS, 11–12
metabolic pathways, 4 pharmacological intervention
PKC activation, 5 COX isoforms, 18
Autoimmunity indomethacin-amides, 18
CCC (see Chronic Chagas disease NSAIDs, 17–18
cardiomyopathy) parasitaemia, 18–19
Chagas disease platelet-activating factor, 19–20
antigen exposure, 138 stages, 3–4
251
252 Index
Bioactive lipids, T. cruzi infection (cont.) T. cruzi trypomastigotes, 111–112

transgenic/knockout mice phenotypes chronic infection, eicosanoids
ASA and ATL, 20–21 T-lymphocytes, 16
Chagas disease, 20 TXA2, 16
COX inhibitors, 20 DCs, 117–118
host-parasite interdependence, 21 fatty acid deficiency, 12–13
indomethacin-amide derivatives, glycaemia
21–22 diabetes, 245
leukocyte migration, 23 hypoglycaemia, 245
5-LO null mice, 22–23 leptin receptor gene, 245–246
TXA2 receptors, 22 myocardium, 245–246
Bradykinin receptors (BKRs), 108–112 infection-associated vasculopathy
cardiac remodelling, 106
C endothelin, 105–106
thromboxane (TXA2), 106–107
CCC. See Chronic chagas disease
trans-sialidase (TS), 107–108
cardiomyopathy
insect vectors, eicosanoids, 16–17
CCM. See Chronic chagasic myocardiopathy
interstitial oedema, kinin system
CD. See Chagas’ disease
Dm28c trypomastigotes, 112
Chagas’ disease (CD). See also Adipose
hamster cheek pouch (HCP), 112–113
tissue; Gap junctions and Chagas’
neutrophils, 113–114
disease; Inflammation and Chagas’
proinflammatory activities, 115
disease
‘‘steady-state’’ tissues, 113
ACE
neuronal damage
antigen-presenting cells (APCs),
acute inflammation, 202–204
114–116
autoimmunity, 205
P. gingivalis, 116–117
parasitism, 202
TH1 polarization, 116–117
neuroregeneration
acute infection, eicosanoids
age-related degeneration, 207, 209
immunomodulators, 15–16
asymptomatic and chronic
inflammatory cytokines and lipid
patients, 206
mediators, 14–15
functional improvement, heart and
lipoxins (LXAs), 15
colon, 206
LTC4 treatment, 14–15
glial population, heart and GI tract,
PGE2, 13
206–207
PGF2a, 13
immune-privileged CNS, 207
prostaglandin release, 14
neurodegeneration mechanism, 207,
quorum sensing, 14
208
ANS involment, pathogenesis
neuronal plasticity, PNS, 206
GI neurodegeneration, 200–202
PDNF, 207
neurodegeneration, heart, 198–200
TS/PDNF, 209–220
neurogenic theory, 197
Chemokines, inflammation mediators
BKRs
CCL3/CCL5 and CCR1/CCR5 receptors,
angiotensin-converting enzyme
184–185
(ACE), 109
CCR5 receptor, 183–184
BK1R upregulation, 109
CXC ligand 9 and 2, 181–183
cruzipain, 110
Foxp3, transcription factor, 183–184
human umbilical vein endothelial cells
IFN-g and TNF-a, NO production, 181–183
(HUVECs), 110–111
infected and non infected,
‘‘kininogenases’’, 109–110
comparison, 183
kinins, 108–109
levels, 183
parasite invasion, 111
Met-RANTES, 181–183
pseudocysts, 108
Index 253
transmembrane-spanning G-protein- connexin gene family, 64, 65

coupled serpentine receptors, dilated cardiomyopathy, 66
181–183 loss and coupling
Chronic chagas disease cardiomyopathy amiodarone, 71
(CCC) beating rate, 66
heart-specific inflammatory lesions calcium concentrations, 68–71
myocarditis, fibrosis and venticular confocal microscopy, 68, 72
dilation, 134–135 connexin 43 protein and mRNA
T. cruzi, 135 expression, 70
immunopathogenesis Cx43 immunofluorescence, 66–68
cytokines and chemokines, 135 immunostaining, 68
familial aggregation, 137 intercalated disc discontinuity,
IFN-g-mediated chronic myocardial 72–74
inflammation, 136–137 murine models, 71–74
inflammatory infiltrate, heart lesions, remodelling, connexin, 72
136 T. cruzi infection, 67
PBMC, 135–136 transforming growth factor-b (TGF-b),
Chronic chagasic myocardiopathy (CCM) 71–72
autoimmunity, 103–104 microarray experiments and gene
parasite antigens, 108 expression
T. cruzi antigens, 103 cadherins (Cdh), 74–76
Cyclooxygenase (COX) HRD genes, 74–76, 77
biological responses, cell surface hybridization, Duke oligonucleotide
receptors, 9–10 arrays, 74–76
enzymes protein-protein interactions, 65
COX-1 and COX-2, 7–8 T. cruzi infection, 74, 75
human, 7 Gastrointestinal (GI) neurodegeneration
PGH2 generation, 8 enteric neurons, 200–201
PGs and thromboxane synthesis, 9 excitatory transmission, 201
ICC and EGCs, 201–202
E neuronal ganglia, extrinsic and
intrinsic, 200
Eicosanoids NO-donor drugs, 201
description, 4 oesophagus and colon, 200
endogenous regulation, Chagas diseases preganglionic lesions and dorsal cell
acute infection, 13–16 reduction, 201
chronic infection, 16
insect vectors, 16–17 H
synthesis and AA metabolism
COX pathway, 7–10 Host cell invasion, T. cruzi
cytochrome P-450 pathway, 7 cardiomyocytes, 35–36
LO pathway, 6–7 cytoskeleton
metabolic pathways, 4–5 actin, 51–52
Endothelin microtubules, 52–53
ET-1 cytosolic residence establishment, 41–42
inflammation mediators, 185–186 extracellular matrix binding, 38
synthesis, 176 gp82/gp90, metacyclic trypomastigotes
proinflammatory activities, 174 antisense oligonucleotides, 40
gastric mucin, 40
G recognition and signalling, 41
gp85/TS, tissue-specific homing
Gap junctions and Chagas’ disease FLY domain, 38–39
cardiac myocytes, 65–66, 78 neurotrophin receptors, 39–40
254 Index
Host cell invasion, T. cruzi (cont.) L

lysosome pathways
autophagy, 47–48 LAMPs. See Lysosome-associated
exocytosis, 42–46 membrane proteins
plasma membrane invagination, 46–47 Lipid mediators, inflammation
mediators, 185
trypomastigotes, 43
parasitophorous vacuole membrane Lysosome-associated membrane proteins
disruption and cytosolic localization (LAMPs), 50
Lysosome pathways, T. cruzi host cell
LAMPs, 50
LYT1, 49–50 invasion
TC-TOX, 49–50 autophagy, 47–48
phosphatidylinositol-3-kinases exocytosis
Ca2+ signalling, 44–46
(PI3 kinases), 48–49
recognition and adhesion, 37–38 live cell imaging, 42
reversible invasion, 53–54 synaptotagmin VIII, 43–44
trypomastigotes plasma membrane invagination, 46–47
trypomastigotes, 43
description, 36–37
pulse-chase experiments, 36–37
M
replicative amastigotes formation, 34
Microtubules, 52–53
I
N
Inflammation and Chagas’ disease
defined, 172 Neurodegeneration, heart
and immune mechanisms parasympathetic dysautonomia, 198
acute murine models, 173–174 dilatation, left ventricle and apical
human chagastic cardiomyopathy, 175 aneurism, 198–199
morbidity, 174–175 impaired heart rate regulation, 198
parameters evaluation, 173–174 left ventricular systolic dysfunction, 198
parasite and inocula, virulent strains, mAChRs, 198
174–175 malignant ventricular
pathogenesis, 175–176 tachyarrhythmias and
rats, heart disease, 175 fibrillation, 198
rodents, mice, 173–174 prognosis and mortality, 199
timeline, T. cruzi infection, 174 vagal anti-inflammatory signals, 199
immunosuppressant drugs, 172–173 sympathetic dysfunction
mediators, tissue damage adrenergic blockers, 199–200
chemokines, 181–185 chagasic cardiomyopathy, 200
endothelin, 185–186 noradrenergic innervation, 200
inflammation, 177
P
interconnectivity, acute and chronic
phases, 181 Parasite-derived neurotrophic factor
lipid mediators, 185 (PDNF), Trk receptors
pathogenesis, T. cruzi infection, 178 cholinergic and adrenergic phenotypes,
protection mediators induction, 215–216
CD4+ T and CD8+ T cells, 177 functional similarity, 213–214
chemokines production, 177 invasion, T. cruzi, 217–218
IL-12 stimulation, 176 MAPK/Erk activation and
innate immune cells interaction, 176 differentiation, 214–215
non-TLR receptors, 176 neuronal regeneration
T. cruzi, protozoan parasite, 172–173 Akt phosphorylation, 213
tissue integrity and function, 172 CREB, 213
Index 255
NTs, 213 pro-oxidant status, seropositive

receptor-mediated signalling, human, 160–161
211–213 respiratory complexes, 155
tyrosine kinases, 211 T. cruzi infection and Chagas disease
neurotrophic activity mapping, 216 cytokines and chemokines, 156–157
PI3K/Akt activation and survival, 214 gene expression profiling, 158
synergy, neuropoietic cytokines, 215 inflammatory infiltrate, 158
and T. cruzi receptor-independent innate and adaptive immune
intracellular signalling responses, 157
blocked apoptosis, 218 vaccination approach, 157
host pro-survival, 218–219 toll-like and NOD-receptors, 154–155
parasite entrance, 218
Ser-and Thr-containing motifs, T
phosphorylation, 220
and substrate sites, Akt kinase, 217, 219 Trans-sialidase (TS)
TNF-a, apoptosis, 219–220 description, 209–210
Peripheral blood mononuclear cell (PBMC), infective bloodstream trypomastigotes,
135–136 210
LTR/SAPA, 210
R parasite attachment, 210
T. cruzi, 210–211
Reactive oxygen species (ROS) signalling
Trypanosoma cruzi (T. cruzi). See also
cardiomyocytes, 156
Bioactive lipids, T. cruzi infection
cytokine responses
cell-cell coupling, 67
inhibition, NOX, 159
Chagas’ disease (see Chagas’ disease)
macrophages and DCs, 158–159
connexin43, 69
NF-kB activation, 159–160
host cell invasion (see Host cell invasion,
PARP-1 signals, DNA repair, 159–160
T. cruzi)
pro-inflammatory mediators and
infection
chemotactic factors, 158–159
DCs activation, 117–118
redox regulation, 158–159
inflammatory cytokines, 156–158
defined, 154
microcirculation, 86–88
endothelial activation, 155
molecular mimicry, 131
GTPases, 163
invasion, cardiovascular cells, 108–112
in vitro assay systems/animal models,
junctional proteins, 74
154–155
life cycle, 35
inflammatory responses and pathogen
ROS signalling (see Reactive oxygen
control, 162–163
species (ROS) signalling)
large-scale randomized trials testing, 163
Trypomastigotes
mitochondria
inhibition, 45
degeneration, 155
interstitial oedema, 112–114
dysfunction, 156
metacyclic, 35, 40–41
NOS isoforms, 154
T. cruzi, 36–37, 51–53
NOX homologs, 154
tissue culture-derived, 44
oxidative stress and cardiac dysfunction
TS. See Trans-sialidase
antioxidant reserve and anti-
inflammatory responses, 160–161 V
foetal genes, re-expression, 161–162
hypertrophic markers and collagen Vasculature, Chagas disease
deposition, 161–162 dogs
myocardial hypertrophy, 162 coronary perfusion, 89
pro-oxidant and pro-inflammatory degenerative change, ECs, 88–89
mediators, 161 description, 88–89
256 Index
Vasculature, Chagas disease (cont.) in vitro

interaction, ECs and effector immune cytokines secretion, 91–92
cells, 88–89 IL-1b and CSF-1 factors, 91–92
history microvascular perfusion and cyclic
acute myocarditis and chronic AMP metabolism, 91
cardiomyopathy, 84 molecular paradigm, pathogenesis, 92
alergic phenomenon, ischaemic signal-regulated kinases 1 and 2, 92
lesion, 85 microcirculation, T. cruzi infection
alterations, 84–85 BALB/c mice immunization, 86
decapillarization, 85–86 cardiomyopathies, 87
malignant ventricular ECs activation, 88
tachyarrhythmias, 86 in vivo visualization, 87
pathology, 84–85 T. cruzi-infected mouse, 87
vascular lesions, 84–85 vasoactive peptides and eicosanoids
humans differentiation, ET-1 and TXA2, 91
abnormal perfusion, 93 endothelin-1(ET-1), 89–90
anatomy and histotopographical TXA2, 90–91
studies, 92–93 vasoconstrictor and receptors, 90
biopsies, 93–94 Vasculopathy, Chagas disease
denervation and myocardial ACE, 114–117
perfusion, 94 bradykinin receptors, 108–112
dilatations, arterioles and capillaries, DCs activation, 117–118
93–94 infection-associated, 105–108
vascular changes, heart apex, 92–93 interstitial oedema, 112–114
vasospasm, 93
CONTENTS OF VOLUMES
IN THIS SERIES
Volume 41 M. Albonico, D.W.T. Cromption, and

L. Savioli
Drug Resistance in Malaria Parasites of
Animals and Man DNA Vaocines: Technology and
W. Peters Applications as Anti-parasite and
Anti-microbial Agents
Molecular Pathobiology and Antigenic J.B. Alarcon, G.W. Wainem and
Variation of Pneumocystis carinii D.P. McManus
Y. Nakamura and M. Wada
Ascariasis in China
P. Weidono, Z. Xianmin and Volume 43
D.W.T. Crompton
Genetic Exchange in the
The Generation and Expression of Trypanosomatidae
Immunity to Trichinella spiralis in W. Gibson and J. Stevens
Laboratory Rodents
R.G. Bell The Host-Parasite Relationship in
Neosporosis
Population Biology of Parasitic A. Hemphill
Nematodes: Application of
Genetic Markers Proteases of Protozoan Parasites
T.J.C. Anderson, M.S. Blouin and P.J. Rosenthal
R.M. Brech Proteinases and Associated Genes of
Schistosomiasis in Cattle Parasitic Helminths
J. De Bont and J. Vercruysse J. Tort, P.J. Brindley, D. Knox, K.H. Wolfe,
and J.P. Dalton
Parasitic Fungi and their
Volume 42 Interaction with the Insect
Immune System
The Southern Cone Initiative Against A. Vilcinskas and P. Götz
Chagas Disease
C.J. Schofield and J.C.P. Dias
Phytomonas and Other Trypanosomatid
Parasites of Plants and Fruit Volume 44
E.P. Camargo
Cell Biology of Leishmania
Paragonimiasis and the Genus B. Handman
Paragonimus
Immunity and Vaccine Development in
D. Blair, Z.-B. Xu, and T. Agatsuma
the Bovine Theilerioses
Immunology and Biochemistry of N. Boulter and R. Hall
Hymenolepis diminuta
The Distribution of Schistosoma bovis
J. Anreassen, E.M. Bennet-Jenkins, and
Sonaino, 1876 in Relation to
C. Bryant
Intermediate Host Mollusc-Parasite
Control Strategies for Human Intestinal Relationships
Nematode Infections H. Moné, G. Mouahid, and S. Morand
257
258 Contents of Volumes in This Series
The Larvae of Monogenea Satellites, Space, Time and the African

(Platyhelminthes) Trypanosomiases
I.D. Whittington, L.A. Chisholm, and D.J. Rogers
K. Rohde Earth Observation, Geographic
Sealice on Salmonids: Their Biology Information Systems and
and Control Plasmodium falciparum Malaria in
A.W. Pike and S.L. Wadsworth Sub-Saharan Africa
S.I. Hay, J. Omumbo, M. Craig, and
R.W. Snow
Volume 45
Ticks and Tick-borne Disease Systems in
The Biology of some Intraerythrocytic Space and from Space
Parasites of Fishes, Amphibia S.E. Randolph
and Reptiles
The Potential of Geographical
A.J. Davies and M.R.L. Johnston
Information Systems (GIS) and
The Range and Biological Activity of FMR Remote Sensing in the Epidemiology
Famide-related Peptides and and Control of Human Helminth
Classical Neurotransmitters Infections
in Nematodes S. Brooker and E. Michael
D. Brownlee, L. Holden-Dye, and R.
Advances in Satellite Remote Sensing of
Walker
Environmental Variables for
The Immunobiology of Gastrointestinal Epidemiological Applications
Nematode Infections in Ruminants S.J. Goetz, S.D. Prince, and J. Small
A. Balic, V.M. Bowles, and E.N.T.
Forecasting Diseases Risk for Increased
Meeusen
Epidemic Preparedness in Public
Health
Volume 46 M.F. Myers, D.J. Rogers, J. Cox, A.
Flauhalt, and S.I. Hay
Host-Parasite Interactions in
Acanthocephala: A Morphological Education, Outreach and the Future of
Approach Remote Sensing in Human Health
H. Taraschewski B.L. Woods, L.R. Beck, B.M. Lobitz, and
M.R. Bobo
Eicosanoids in Parasites and Parasitic
Infections
A. Daugschies and A. Joachim
Volume 48
The Molecular Evolution of
Volume 47 Trypanosomatidae
J.R. Stevens, H.A. Noyes, C.J. Schofield,
An Overview of Remote Sensing and and W. Gibson
Geodesy for Epidemiology and
Public Health Application Transovarial Transmission in the
S.I. Hay Microsporidia
A.M. Dunn, R.S. Terry, and J.E. Smith
Linking Remote Sensing, Land Cover
and Disease Adhesive Secretions in the
P.J. Curran, P.M. Atkinson, G.M. Foody, Platyhelminthes
and E.J. Milton I.D. Whittington and B.W. Cribb
Spatial Statistics and Geographic The Use of Ultrasound in Schistosomiasis

C.F.R. Hatz
Information Systems in
Epidemiology and Public Health Ascaris and Ascariasis
T.P. Robinson D.W.T. Crompton
Contents of Volumes in This Series 259
Volume 49 Volume 52
Antigenic Variation in Trypanosomes: The Ecology of Fish Parasites with
Enhanced Phenotypic Variation in a Particular Reference to
Eukaryotic Parasite Helminth Parasites and their
H.D. Barry and R. McCulloch Salmonid Fish Hosts in Welsh
Rivers: A Review of Some of the
The Epidemiology and Control of Human
Central Questions
African Trypanosomiasis
J.D. Thomas
J. Pépin and H.A. Méda
Biology of the Schistosome Genus
Apoptosis and Parasitism: from the
Trichobilharzia
Parasite to the Host Immune
P. Horák, L. Kolárová, and C.M. Adema
Response
G.A. DosReis and M.A. Barcinski The Consequences of Reducing
Transmission of Plasmodium
Biology of Echinostomes Except
falciparum in Africa
Echinostoma
R.W. Snow and K. Marsh
B. Fried
Cytokine-Mediated Host Responses
during Schistosome Infections:
Volume 50 Walking the Fine Line Between
The Malaria-Infected Red Blood Cell: Immunological Control and
Structural and Functional Changes Immunopathology
B.M. Cooke, N. Mohandas, and R.L. K.F. Hoffmann, T.A. Wynn, and D.W.
Coppel Dunne
Schistosomiasis in the Mekong Region:

Epidemiology and Phytogeography
S.W. Attwood Volume 53
Molecular Aspects of Sexual Interactions between Tsetse
Development and Reproduction in and Trypanosomes with
Nematodes and Schistosomes Implications for the Control of
P.R. Boag, S.E. Newton, and R.B. Gasser Trypanosomiasis
S. Aksoy, W.C. Gibson, and M.J. Lehane
Antiparasitic Properties of Medicinal
Plants and Other Naturally Enzymes Involved in the Biogenesis of
Occurring Products the Nematode Cuticle
S. Tagboto and S. Townson A.P. Page and A.D. Winter
Diagnosis of Human Filariases (Except
Volume 51 Onchocerciasis)
M. Walther and R. Muller
Aspects of Human Parasites in which
Surgical Intervention May Be
Important
D.A. Meyer and B. Fried Volume 54
Electron-transfer Complexes in Ascaris Introduction – Phylogenies,
Mitochondria Phylogenetics, Parasites and the
K. Kita and S. Takamiya Evolution of Parasitism
D.T.J. Littlewood
Cestode Parasites: Application of In Vivo
and In Vitro Models for Studies of the Cryptic Organelles in Parasitic Protists
Host-Parasite Relationship and Fungi
M. Siles-Lucas and A. Hemphill B.A.P. Williams and P.J. Keeling
Phylogenetic Insights into the Evolution The Mitochondrial Genomics of Parasitic

of Parasitism in Hymenoptera Nematodes of Socio-Economic
J.B. Whitfield Importance: Recent Progress, and
Implications for Population Genetics
Nematoda: Genes, Genomes and the
and Systematics
Evolution of Parasitism
M. Hu, N.B. Chilton, and R.B. Gasser
M.L. Blaxter
The Cytoskeleton and Motility in
Life Cycle Evolution in the Digenea: A
Apicomplexan Invasion
New Perspective from Phylogeny
R.E. Fowler, G. Margos, and G.H. Mitchell
T.H. Cribb, R.A. Bray, P.D. Olson, and
D.T.J. Littlewood
Progress in Malaria Research: The Case Volume 57
for Phylogenetics Canine Leishmaniasis
S.M. Rich and F.J. Ayala J. Alvar, C. Cañavate, R. Molina, J.
Phylogenies, the Comparative Moreno, and J. Nieto
Method and Parasite Evolutionary Sexual Biology of Schistosomes
Ecology H. Moné and J. Boissier
S. Morand and R. Poulin
Review of the Trematode Genus Ribeiroia
Recent Results in Cophylogeny Mapping (Psilostomidae): Ecology, Life
M.A. Charleston History, and Pathogenesis with
Inference of Viral Evolutionary Rates Special Emphasis on the Amphibian
from Molecular Sequences Malformation Problem
A. Drummond, O.G. Pybus, and A. P.T.J. Johnson, D.R. Sutherland,
Rambaut J.M. Kinsella and K.B. Lunde
Detecting Adaptive Molecular Evolution: The Trichuris muris System: A Paradigm

Additional Tools for the of Resistance and Susceptibility to
Parasitologist Intestinal Nematode Infection
J.O. McInerney, D.T.J. Littlewood, and L.J. Cliffe and R.K. Grencis
C.J. Creevey Scabies: New Future for a Neglected
Disease
Volume 55 S.F. Walton, D.C. Holt, B.J. Currie, and
D.J. Kemp
Contents of Volumes 28–52
Cumulative Subject Indexes for Volumes
28–52
Contributors to Volumes 28–52
Volume 58
Leishmania spp.: On the Interactions they
Establish with Antigen-Presenting
Volume 56 Cells of their Mammalian Hosts
J.-C. Antoine, E. Prina, N. Courret, and
Glycoinositolphospholipid from
T. Lang
Trypanosoma cruzi: Structure,
Biosynthesis and Immunobiology Variation in Giardia: Implications
J.O. Previato, R. Wait, C. Jones, for Taxonomy and Epidemiology
G.A. DosReis, A.R. Todeschini, N. R.C.A. Thompson and P.T. Monis
Heise and L.M. Previata
Recent Advances in the Biology of
Biodiversity and Evolution of the Echinostoma species in the
Myxozoa ‘‘revolutum’’ Group
E.U. Canning and B. Okamura B. Fried and T.K. Graczyk
Human Hookworm Infection in the Volume 61

21st Century
S. Brooker, J. Bethony, and P.J. Hotez Control of Human Parasitic
Diseases: Context and Overview
The Curious Life-Style of the David H. Molyneux
Parasitic Stages of Gnathiid Isopods
N.J. Smit and A.J. Davies Malaria Chemotherapy
Peter Winstanley and Stephen Ward
Insecticide-Treated Nets
Volume 59 Jenny Hill, Jo Lines, and Mark Rowland
Genes and Susceptibility to Control of Chagas Disease
Leishmaniasis Yoichi Yamagata and
Emanuela Handman, Colleen Elso, and Jun Nakagawa
Simon Foote
Human African Trypanosomiasis:
Cryptosporidium and Cryptosporidiosis Epidemiology and Control
R.C.A. Thompson, M.E. Olson, G. Zhu, E.M. Fèvre, K. Picozzi, J. Jannin,
S. Enomoto, Mitchell S. Abrahamsen S.C. Welburn and I. Maudlin
and N.S. Hijjawi
Chemotherapy in the Treatment and
Ichthyophthirius multifiliis Fouquet and Control of Leishmaniasis
Ichthyophthiriosis in Freshwater Jorge Alvar, Simon Croft, and
Teleosts Piero Olliaro
R.A. Matthews
Dracunculiasis (Guinea Worm Disease)
Biology of the Phylum Nematomorpha Eradication
B. Hanelt, F. Thomas, and A. Schmidt- Ernesto Ruiz-Tiben and Donald
Rhaesa R. Hopkins
Intervention for the Control of Soil-
Volume 60 Transmitted Helminthiasis in the
Community
Sulfur-Containing Amino Acid
Marco Albonico, Antonio Montresor, D.W.
Metabolism in Parasitic Protozoa T. Crompton, and Lorenzo Savioli
Tomoyoshi Nozaki, Vahab Ali, and
Masaharu Tokoro Control of Onchocerciasis
Boakye A. Boatin and Frank O. Richards,
The Use and Implications of Ribosomal Jr.
DNA Sequencing for the
Discrimination of Digenean Species Lymphatic Filariasis: Treatment, Control
Matthew J. Nolan and Thomas H. Cribb and Elimination
Eric A. Ottesen
Advances and Trends in the Molecular
Systematics of the Parasitic Control of Cystic Echinococcosis/
Platyhelminthes Hydatidosis: 1863–2002
Peter D. Olson and Vasyl V. Tkach P.S. Craig and E. Larrieu
Wolbachia Bacterial Endosymbionts of Control of Taenia solium Cysticercosis/
Filarial Nematodes Taeniosis
Mark J. Taylor, Claudio Bandi, and Achim Arve Lee Willingham III and
Hoerauf Dirk Engels
The Biology of Avian Eimeria with an Implementation of Human
Emphasis on their Control by Schistosomiasis Control: Challenges
Vaccination and Prospects
Martin W. Shirley, Adrian L. Smith, and Alan Fenwick, David Rollinson, and
Fiona M. Tomley Vaughan Southgate
Volume 62 Targeting of Toxic Compounds to the

Trypanosome’s Interior
Models for Vectors and Vector-Borne Michael P. Barrett and Ian H. Gilbert
Diseases
D.J. Rogers Making Sense of the Schistosome
Surface
Global Environmental Data for Patrick J. Skelly and R. Alan Wilson
Mapping Infectious Disease
Distribution Immunology and Pathology of
S.I. Hay, A.J. Tatem, A.J. Graham, Intestinal Trematodes in Their
S.J. Goetz, and D.J. Rogers Definitive Hosts
Rafael Toledo, José-Guillermo Esteban, and
Issues of Scale and Uncertainty in Bernard Fried
the Global Remote Sensing of
Disease Systematics and Epidemiology of
P.M. Atkinson and A.J. Graham Trichinella
Edoardo Pozio and K. Darwin Murrell
Determining Global Population
Distribution: Methods, Applications
and Data
D.L. Balk, U. Deichmann, G. Yetman,
Volume 64
F. Pozzi, S.I. Hay, Leishmania and the Leishmaniases:
and A. Nelson A Parasite Genetic Update and
Defining the Global Spatial Limits of Advances in Taxonomy,
Malaria Transmission in 2005 Epidemiology and Pathogenicity
C.A. Guerra, R.W. Snow and S.I. Hay in Humans
Anne-Laure Bañuls, Mallorie Hide and
The Global Distribution of Yellow Fever Franck Prugnolle
and Dengue
D.J. Rogers, A.J. Wilson, S.I. Hay, and Human Waterborne Trematode and
A.J. Graham Protozoan Infections
Thaddeus K. Graczyk and Bernard Fried
Global Epidemiology, Ecology and
Control of Soil-Transmitted Helminth The Biology of Gyrodctylid
Infections Monogeneans: The ‘‘Russian-Doll
S. Brooker, A.C.A. Clements and Killers’’
D.A.P. Bundy T.A. Bakke, J. Cable, and P.D. Harris
Tick-borne Disease Systems: Mapping Human Genetic Diversity and the

Geographic and Phylogenetic Space Epidemiology of Parasitic
S.E. Randolph and D.J. Rogers and Other Transmissible Diseases
Michel Tibayrenc
Global Transport Networks and
Infectious Disease Spread
A.J. Tatem, D.J. Rogers and S.I. Hay Volume 65
Climate Change and Vector-Borne ABO Blood Group Phenotypes and
Diseases Plasmodium falciparum Malaria:
D.J. Rogers and S.E. Randolph Unlocking a Pivotal Mechanism
Marı́a-Paz Loscertales, Stephen Owens,
James O’Donnell, James Bunn, Xavier
Bosch-Capblanch, and Bernard J. Brabin
Volume 63
Structure and Content of the Entamoeba
Phylogenetic Analyses of Parasites in the histolytica Genome
New Millennium C. G. Clark, U. C. M. Alsmark, M.
David A. Morrison Tazreiter, Y. Saito-Nakano, V. Ali,
S. Marion, C. Weber, C. Mukherjee, Volume 67

I. Bruchhaus, E. Tannich, M. Leippe, Introduction
T. Sicheritz-Ponten, P. G. Foster, Irwin W. Sherman
J. Samuelson, C. J. Noël, R. P. Hirt,
T. M. Embley, C. A. Gilchrist, An Introduction to Malaria Parasites
B. J. Mann, U. Singh, J. P. Ackers, Irwin W. Sherman
S. Bhattacharya, A. Bhattacharya, The Early Years
A. Lohia, N. Guillén, M. Duchêne, Irwin W. Sherman
T. Nozaki, and N. Hall
Show Me the Money
Epidemiological Modelling for Irwin W. Sherman
Monitoring and Evaluation of
Lymphatic Filariasis Control In Vivo and In Vitro Models
Edwin Michael, Mwele N. Malecela- Irwin W. Sherman
Lazaro, and James W. Kazura Malaria Pigment
The Role of Helminth Infections in Irwin W. Sherman
Carcinogenesis Chloroquine and Hemozoin
David A. Mayer and Bernard Fried
Irwin W. Sherman
A Review of the Biology of the
Isoenzymes
Parasitic Copepod Lernaeocera
branchialis (L., 1767)(Copepoda: Irwin W. Sherman
Pennellidae The Road to the Plasmodium falciparum
Adam J. Brooker, Andrew P. Shinn, and Genome
James E. Bron Irwin W. Sherman
Carbohydrate Metabolism
Irwin W. Sherman
Volume 66 Pyrimidines and the Mitochondrion

Irwin W. Sherman
Strain Theory of Malaria: The First
50 Years The Road to Atovaquone
F. Ellis McKenzie,* David L. Smith, Irwin W. Sherman
Wendy P. O’Meara, and Eleanor The Ring Road to the Apicoplast
M. Riley
Irwin W. Sherman
Advances and Trends in the Molecular
Ribosomes and Ribosomal Ribonucleic
Systematics of Anisakid Nematodes,
Acid Synthesis
with Implications for their
Irwin W. Sherman
Evolutionary Ecology and
Host–Parasite Co-evolutionary De Novo Synthesis of Pyrimidines
Processes and Folates
Simonetta Mattiucci and Giuseppe Irwin W. Sherman
Nascetti
Salvage of Purines
Atopic Disorders and Parasitic Infections Irwin W. Sherman
Aditya Reddy and Bernard Fried
Polyamines
Heartworm Disease in Animals and Irwin W. Sherman
Humans
John W. McCall, Claudio Genchi, Laura H. New Permeability Pathways
Kramer, Jorge Guerrero, and and Transport
Luigi Venco Irwin W. Sherman
Hemoglobinases Tracking Transmission of the Zoonosis

Irwin W. Sherman Toxoplasma gondii
Judith E. Smith
Erythrocyte Surface Membrane Proteins
Irwin W. Sherman Parasites and Biological Invasions
Alison M. Dunn
Trafficking
Irwin W. Sherman Zoonoses in Wildlife: Integrating Ecology
into Management
Erythrocyte Membrane Lipids Fiona Mathews
Irwin W. Sherman
Understanding the Interaction
Invasion of Erythrocytes Between an Obligate Hyperparasitic
Irwin W. Sherman Bacterium, Pasteuria penetrans
Vitamins and Anti-Oxidant Defenses and its Obligate Plant-Parasitic
Irwin W. Sherman Nematode Host, Meloidogyne spp.
Keith G. Davies
Shocks and Clocks
Irwin W. Sherman Host–Parasite Relations and Implications
for Control
Transcriptomes, Proteomes Alan Fenwick
and Data Mining
Irwin W. Sherman Onchocerca–Simulium Interactions and the
Population and Evolutionary Biology
Mosquito Interactions of Onchocerca volvulus
Irwin W. Sherman Marı́a-Gloria Basáñez, Thomas
S. Churcher, and Marı́a-Eugenia Grillet
Volume 68 Microsporidians as Evolution-Proof
Agents of Malaria Control?
HLA-Mediated Control of HIV and HIV
Jacob C. Koella, Lena Lorenz, and Irka
Adaptation to HLA
Bargielowski
Rebecca P. Payne, Philippa C. Matthews,
Julia G. Prado, and Philip J. R. Goulder
An Evolutionary Perspective on
Volume 69
Parasitism as a Cause of Cancer The Biology of the Caecal Trematode
Paul W. Ewald Zygocotyle lunata
Bernard Fried, Jane E. Huffman, Shamus
Invasion of the Body Snatchers:
Keeler, and Robert C. Peoples
The Diversity and Evolution of
Manipulative Strategies in Fasciola, Lymnaeids and Human
Host–Parasite Interactions Fascioliasis, with a Global
Thierry Lefévre, Shelley A. Adamo, David Overview on Disease Transmission,
G. Biron, Dorothée Missé, David Epidemiology, Evolutionary
Hughes, and Frédéric Thomas Genetics, Molecular Epidemiology
and Control
Evolutionary Drivers of Parasite-Induced
Santiago Mas-Coma, Marı́a Adela Valero,
Changes in Insect Life-History Traits:
and Marı́a Dolores Bargues
From Theory to Underlying
Mechanisms Recent Advances in the Biology of
Hilary Hurd Echinostomes
Rafael Toledo, José-Guillermo Esteban, and
Ecological Immunology of a Tapeworms’
Bernard Fried
Interaction with its Two Consecutive
Hosts Peptidases of Trematodes
Katrin Hammerschmidt and Martin Kašný, Libor Mikeš, Vladimı́r
Joachim Kurtz Hampl, Jan Dvořák,
Components of Asobara Venoms and their

Conor R. Caffrey, John P. Dalton, and
Effects on Hosts
Petr Horák
Sébastien J.M. Moreau, Sophie Vinchon,
Potential Contribution of Anas Cherqui, and Geneviève Prévost
Sero-Epidemiological Analysis
Strategies of Avoidance of Host Immune
for Monitoring Malaria
Defenses in Asobara Species
Control and Elimination:
Geneviève Prévost, Géraldine Doury,
Historical and Current
Alix D.N. Mabiala-Moundoungou,
Perspectives
Anas Cherqui, and Patrice Eslin
Chris Drakeley and Jackie Cook
Evolution of Host Resistance and
Parasitoid Counter-Resistance
Volume 70 Alex R. Kraaijeveld and H. Charles
J. Godfray
Ecology and Life History Evolution of
Frugivorous Drosophila Parasitoids Local, Geographic and Phylogenetic
Frédéric Fleury, Patricia Gibert, Scales of Coevolution in Drosophila–
Nicolas Ris, and Roland Allemand Parasitoid Interactions
S. Dupas, A. Dubuffet, Y. Carton, and
Decision-Making Dynamics in
M. Poirié
Parasitoids of Drosophila
Andra Thiel and Thomas S. Hoffmeister Drosophila–Parasitoid Communities as
Model Systems for Host–Wolbachia
Dynamic Use of Fruit Odours to Locate
Interactions
Host Larvae: Individual Learning,
Fabrice Vavre, Laurence Mouton, and
Physiological State and Genetic
Bart A. Pannebakker
Variability as Adaptive
Mechanisms A Virus-Shaping Reproductive Strategy
Laure Kaiser, Aude Couty, and in a Drosophila Parasitoid
Raquel Perez-Maluf Julien Varaldi, Sabine Patot,
Maxime Nardin, and Sylvain Gandon
The Role of Melanization and Cytotoxic
By-Products in the Cellular Immune
Responses of Drosophila Against
Parasitic Wasps Volume 71
A. Nappi, M. Poirié, and Y. Carton
Cryptosporidiosis in Southeast
Virulence Factors and Strategies of Asia: What’s out There?
Leptopilina spp.: Selective Responses Yvonne A.L. Lim, Aaron R. Jex,
in Drosophila Hosts Huw V. Smith, and Robin B. Gasser
Mark J. Lee, Marta E. Kalamarz,
Indira Paddibhatla, Chiyedza Small, Human Schistosomiasis in the Economic
Roma Rajwani, and Shubha Govind Community of West African States:
Epidemiology and Control
Variation of Leptopilina boulardi Success in Héléne Moné, Moudachirou Ibikounlé,
Drosophila Hosts: What is Inside the Achille Massougbodji, and Gabriel
Black Box? Mouahid
A. Dubuffet, D. Colinet, C. Anselme,
S. Dupas, Y. Carton, and M. Poirié The Rise and Fall of Human
Oesophagostomiasis
Immune Resistance of Drosophila Hosts A.M. Polderman, M. Eberhard, S. Baeta,
Against Asobara Parasitoids: Cellular Robin B. Gasser, L. van Lieshout,
Aspects P. Magnussen, A. Olsen, N.
Patrice Eslin, Geneviève Prévost, Spannbrucker, J. Ziem,
Sébastien Havard, and Géraldine Doury and J. Horton
Volume 72 Combating Taenia solium Cysticercosis

in Southeast Asia: An Opportunity
Important Helminth Infections in for Improving Human Health and
Southeast Asia: Diversity, Potential Livestock Production Links
for Control and Prospects for A. Lee Willingham III, Hai-Wei Wu, James
Elimination Conlan, and Fadjar Satrija
Jürg Utzinger, Robert Bergquist, Remigio
Olveda, and Xiao-Nong Zhou Echinococcosis with Particular Reference
to Southeast Asia
Escalating the Global Fight Against Donald P. McManus
Neglected Tropical Diseases Through
Interventions in the Asia Pacific Food-Borne Trematodiases in Southeast
Region Asia: Epidemiology, Pathology,
Peter J. Hotez and John P. Ehrenberg Clinical Manifestation and Control
Banchob Sripa, Sasithorn Kaewkes,
Coordinating Research on Neglected Pewpan M. Intapan, Wanchai
Parasitic Diseases in Southeast Asia Maleewong, and Paul J. Brindley
Through Networking
Remi Olveda, Lydia Leonardo, Feng Zheng, Helminth Infections of the Central
Banchob Sripa, Robert Bergquist, and Nervous System Occurring in
Xiao-Nong Zhou Southeast Asia and the Far East
Shan Lv, Yi Zhang, Peter Steinmann,
Neglected Diseases and Ethnic Minorities Xiao-Nong Zhou, and Jürg Utzinger
in the Western Pacific Region:
Exploring the Links Less Common Parasitic Infections in
Alexander Schratz, Martha Fernanda Southeast Asia that can Produce
Pineda, Liberty G. Reforma, Nicole M. Outbreaks
Fox, Tuan Le Anh, L. Tommaso Peter Odermatt, Shan Lv, and Somphou
Cavalli-Sforza, Mackenzie K. Sayasone
Henderson, Raymond Mendoza, Jürg
Utzinger, John P. Ehrenberg, and Ah
Sian Tee Volume 73
Controlling Schistosomiasis in Southeast Concepts in Research Capabilities
Asia: A Tale of Two Countries Strengthening: Positive Experiences
Robert Bergquist and Marcel Tanner of Network Approaches by TDR in
the People’s Republic of China and
Schistosomiasis Japonica: Control and Eastern Asia
Research Needs Xiao-Nong Zhou, Steven Wayling, and
Xiao-Nong Zhou, Robert Bergquist, Robert Bergquist
Lydia Leonardo, Guo-Jing Yang,
Kun Yang, M. Sudomo, and Multiparasitism: A Neglected Reality on
Remigio Olveda Global, Regional and Local Scale
Peter Steinmann, Jürg Utzinger, Zun-Wei
Schistosoma mekongi in Cambodia and Du, and Xiao-Nong Zhou
Lao People’s Democratic Republic
Sinuon Muth, Somphou Sayasone, Health Metrics for Helminthic Infections
Sophie Odermatt-Biays, Samlane Charles H. King
Phompida, Socheat Duong, and Implementing a Geospatial Health Data
Peter Odermatt Infrastructure for Control of Asian
Elimination of Lymphatic Filariasis in Schistosomiasis in the People’s
Southeast Asia Republic of China and the
Mohammad Sudomo, Sombat Chayabejara, Philippines
Duong Socheat, Leda Hernandez, John B. Malone, Guo-Jing Yang, Lydia
Wei-Ping Wu, and Robert Bergquist Leonardo, and Xiao-Nong Zhou
The Regional Network for Asian Studies on the Parasitology,

Schistosomiasis and Other Phylogeography and the Evolution of
Helminth Zoonoses (RNASþ ): Host–Parasite Interactions for the
Target Diseases in Face of Snail Intermediate Hosts of Medically
Climate Change Important Trematode Genera in
Guo-Jing Yang, Jürg Utzinger, Shan Lv, Southeast Asia
Ying-Jun Qian, Shi-Zhu Li, Qiang Stephen W. Attwood
Wang, Robert Bergquist, Penelope
Vounatsou, Wei Li, Kun Yang, and
Xiao-Nong Zhou
Volume 74
Social Science Implications for Control of
Helminth Infections in Southeast The Many Roads to Parasitism: A Tale of
Asia Convergence
Lisa M. Vandemark, Tie-Wu Jia, and Robert Poulin
Xiao-Nong Zhou Malaria Distribution, Prevalence, Drug
Towards Improved Diagnosis of Zoonotic Resistance and Control in Indonesia
Trematode Infections in Southeast Iqbal R.F. Elyazar, Simon I. Hay, and
Asia J. Kevin Baird
Maria Vang Johansen, Paiboon Cytogenetics and Chromosomes of
Sithithaworn, Robert Bergquist, and Tapeworms (Platyhelminthes,
Jürg Utzinger Cestoda)
The Drugs We Have and the Drugs We Marta Špakulová, Martina Orosová, and
Need Against Major Helminth John S. Mackiewicz
Infections Soil-Transmitted Helminths of Humans
Jennifer Keiser and Jürg Utzinger in Southeast Asia—Towards
Research and Development of Integrated Control
Antischistosomal Drugs in the Aaron R. Jex, Yvonne A.L. Lim, Jeffrey
People’s Republic of China: Bethony, Peter J. Hotez, Neil D. Young,
A 60-Year Review and Robin B. Gasser
Shu-Hua Xiao, Jennifer Keiser, Ming-Gang The Applications of Model-Based
Chen, Marcel Tanner, and Jürg Geostatistics in Helminth
Utzinger Epidemiology and Control
Control of Important Helminthic Ricardo J. Soares Magalhães, Archie C.A.
Infections: Vaccine Development as Clements, Anand P. Patil, Peter W.
Part of the Solution Gething, and Simon Brooker
Robert Bergquist and Sara Lustigman
Our Wormy World: Genomics,
Proteomics and Transcriptomics in Volume 75
East and Southeast Asia Epidemiology of American
Jun Chuan, Zheng Feng, Paul J. Trypanosomiasis (Chagas Disease)
Brindley, Donald P. McManus, Louis V. Kirchhoff
Zeguang Han, Peng Jianxin,
and Wei Hu Acute and Congenital Chagas Disease
Caryn Bern, Diana L. Martin, and
Advances in Metabolic Profiling of Robert H. Gilman
Experimental Nematode and
Trematode Infections Cell-Based Therapy in Chagas Disease
Yulan Wang, Jia V. Li, Jasmina Saric, Antonio C. Campos de Carvalho,
Jennifer Keiser, Junfang Wu, Jürg Adriana B. Carvalho, and
Utzinger, and Elaine Holmes Regina C.S. Goldenberg
Targeting Trypanosoma cruzi Sterol Advances in Imaging of Animal Models

14a-Demethylase (CYP51) of Chagas Disease
Galina I. Lepesheva, Fernando Villalta, Linda A. Jelicks and Herbert B. Tanowitz
and Michael R. Waterman
The Genome and Its Implications
Experimental Chemotherapy and Santuza M. Teixeira, Najib M. El-Sayed,
Approaches to Drug Discovery for and Patrı́cia R. Araújo
Trypanosoma cruzi Infection
Genetic Techniques in Trypanosoma cruzi
Frederick S. Buckner
Martin C. Taylor, Huan Huang, and
Vaccine Development Against John M. Kelly
Trypanosoma cruzi and Chagas
Nuclear Structure of Trypanosoma cruzi
Disease
Sergio Schenkman, Bruno dos Santos
Juan C. Vázquez-Chagoyán,
Pascoalino, and Sheila C. Nardelli
Shivali Gupta, and
Nisha Jain Garg Aspects of Trypanosoma cruzi Stage
Differentiation
Genetic Epidemiology of
Samuel Goldenberg and Andrea Rodrigues
Chagas Disease
Ávila
Sarah Williams-Blangero,
John L. VandeBerg, John Blangero, The Role of Acidocalcisomes in the Stress
and Rodrigo Corrêa-Oliveira Response of Trypanosoma cruzi
Roberto Docampo, Veronica Jimenez,
Kissing Bugs. The Vectors
Sharon King-Keller, Zhu-hong Li, and
of Chagas
Silvia N.J. Moreno
Lori Stevens, Patricia L. Dorn,
Justin O. Schmidt, John H. Klotz, Signal Transduction in Trypanosoma cruzi
David Lucero, and Stephen A. Klotz Huan Huang

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SERIES EDITORS

First edition 2011

For information on all Academic Press publications

Printed and bound in UK

Antonio Carlos Campos de Carvalho

Maria de Narareth Meirelles

Nisha Jain Garg

Luciana Ribeiro Garzoni

Regina Coeli Goldenberg

Luciana Gabriel Nogueira

Priscila Camillo Teixeira

Chagas disease or American trypanosomiasis is caused by the hemofla-

completion of the genomes of other strains will continue to add to our

Chagas disease in the setting of immune suppression has been documen-

organelle which the author of this chapter, was instrumental in describing

Contents 1.1. Introduction 3

Advances in Parasitology, Volume 76 # 2011 Elsevier Ltd.

Abstract Chagas disease is caused by Trypanosoma cruzi, a protozoan para-

ATL aspirin-triggered lipoxin

1.2. EICOSANOID SYNTHESIS IN VERTEBRATES

Phospholipids (PL) Phosphatidylinositides

Epoxides PGs H(P)ETEs

Inflammation, vascular tone, haemostasis, broncheconstriction

In addition to its role in eicosanoid synthesis, AA itself is capable of

1.2.2. The LO pathway of AA metabolism

cysteine residue in its structure; therefore, they are often referred to as

1.2.3. The cytochrome P-450 pathway of AA metabolism

1.2.4. The COX pathway of AA metabolism

1.2.4.1. The COX family of enzymes

synthesis is required to keep the PG/TX synthesis operative. This short

1.2.4.2. Synthesis of PGs and thromboxane

1.2.4.3. The biological responses to eicosanoids are mediated by cell

dioxin-responsive elements—that activate the expression of a panel of

1.3. LIPID METABOLISM AND EICOSANOID BIOSYNTHETIC

Kinetoplasts have specialized, adaptable biosynthetic pathways for lipids

life stages of T. cruzi (trypomastigote and amastigote). Moreover, the

et al., 2004), they have failed to corroborate the relationship between

1.4. ENDOGENOUS REGULATION OF EICOSANOIDS DURING

It is now appreciated that the release of eicosanoids during infection with

by apoptosis. This compromises host defence and favours chronic disease

1.4.1. Acute infection

Prostaglandin release from the parasite, primarily TXA2, also appears

by protozoa, as demonstrated in several studies (Henderson and Chi, 1998;

system or whether it hijacks the host PG metabolic pathway in its favour.

1.4.2. Chronic infection

1.4.3. Insect vectors

1.5. LESSONS FROM PHARMACOLOGICAL MANIPULATION

1.5.1. Pharmacological intervention

receptor antagonist or the 5-LO inhibitor nordihydroguaiaretic acid was

produce a PAF-like lipid with all the biological properties of mammalian-

1.5.2. Phenotypes of transgenic/knockout mice

determined whether the target gene (TcCYP51) of these indomethacin-

results were obtained in mice treated with MK886, an inhibitor of LT

Contents 2.1. Introduction 34

Advances in Parasitology, Volume 76 # 2011 Elsevier Ltd.

Abstract One of the more accepted concepts in our understanding of the

et al., 1991; Buckner et al., 1999; Caradonna and PereiraPerrin, 2009;

2.2. GENERAL FEATURES OF TRYPANOSOMA CRUZI

2.2.1. Trypanosoma cruzi trypomastigotes actively invade

studies that have incorporated live cell imaging techniques, as well as

2.2.2. Host recognition and adhesion

2.2.3. Binding host cell extracellular matrix

2.2.4. Role of gp85/TS in tissue-specific homing