Pathogenesis of Chagas disease cardiomyopathy

Abstract

Chagas disease, or American trypanosomiasis, is a parasitic infection caused by the flagellate protozoan Trypanosoma cruzi. Chagas disease is mainly affecting rural populations in Mexico and Central and South America. The World Health Organization estimates that 300 000 new cases of Chagas disease occur every year and approximately 20 000 deaths are attributable to Chagas. However, this organisation classified Chagas disease as a neglected tropical disease. The economic burden of this disease is significant. In many Latin American countries, the direct and indirect costs, including the cost of health care in dollars and loss of productivity, attributable to Chagas disease ranges from $40 million to in excess of $800 million per nation per annum. So, it remains a contemporary public health concern. In chronic phase, mortality is primarily due to the rhythm disturbances and congestive heart failure that result from the chronic inflammatory cardiomyopathy (CCC) due to the persistence presence of parasites in the heart tissue. Mechanisms underlying differential progression to CCC are still incompletely understood. In the last decades immunological proteomic genetic approaches lead to significant results which help to disperse the veil covering the knowledge of the pathogenic process. Here, we reported these significant progresses.

Key Words: Chagas; Pathogenesis; Cardiomyopathy; Susceptibility; Parasitic

INTRODUCTION

Chagas disease (American trypanosomiasis) caused by the protozoan Trypanosoma cruzi (T. cruzi) and transmitted by the reduviid bug (called “barbeiro” or “chupança” in Brazil) was discovered in 1909 by the Brazilian physician Carlos Chagas. Unfortunately, Chagas disease remains a neglected disease, with no vaccines available so far and only very few anti-parasitic drugs proven efficient for treating the acute phase of the disease.

CLINICAL AND EPIDEMIOLOGICAL ASPECTS OF CHAGAS DISEASE

Millions of people that were infected decades ago are still in need of appropriate treatment, since available antiparasitic drugs are toxic and have not shown yet a reliable effect on progression to symptomatic disease[1]. These millions of patients require the attention of the scientific community. Despite many vector control programs, Chagas disease is still a significant cause of morbidity and mortality in many countries of South and Central America, where it is estimated that 10 million people may be infected[2]. An important feature of this disease is the cardiac involvement in the chronic phase, the main cause of morbidity and mortality in about 10%-40% of individuals chronically infected with T. cruzi[3,4]. As a result, much research has been directed to its pathogenesis. Since the bulk of evidence indicates the inflammatory infiltrate is a significant effector of heart tissue damage. This review aims to summarize the major recent advances in the understanding of the immunopathogenesis of Chagas disease cardiomyopathy. Recent reviews on alternative pathogenic mechanisms can be seen in[5,6].

Clinical and experimental studies have shown the natural history of Chagas disease can be divided into acute and chronic phases. The acute phase can last from 4 to 12 wk and is characterized by easily detectable blood and tissue parasitism and acute myocarditis of variable intensity of severity[7]. About 10% of those infected individuals may show clinical manifestations that may vary from a flu-like disease to lethal fulminant myocarditis or infectious shock[8,9]. A strong innate and adaptive immune response partially controls T. cruzi-blood and tissue parasitism, and chronic, low-grade parasitism is established. After the acute phase, a long period, which can range from five years to three decades and last throughout the life of the individual, where the clinical manifestations remain missing – the so-called “indeterminate” clinical form (IND)[10]. After this asymptomatic period up to 30% of infected individuals develop chronic Chagas disease cardiomyopathy (CCC) while the remaining infected individuals may continue asymptomatic for life, in the so-called indeterminate form, or develop denervation and dilation of esophagus and colon[11]. The underlying causes for the differential progression of chronic T. cruzi infection are still unknown, but building evidence suggests a role for genetic host factors.

CCC is an inflammatory cardiomyopathy that can be accompanied by heart electric conduction defects, arrhythmias and thromboembolism[7]. Approximately 1/3 of individuals with cardiac disorders eventually develop life-threatening heart disease, such as severe dilated cardiomyopathy or arrhythmias. These patients may develop congestive heart failure, responsible for the high number of hospitalizations and significant mortality (52% in five years)[12]. Several studies suggest that heart failure due to CCC may have a worse prognosis with 50% shorter survival when compared to other cardiomyopathies of different etiologies such as ischemic cardiomyopathy and idiopathic dilated cardiomyopathy[12-14]. Significantly, a key difference between CCC and such cardiomyopathies is inflammation/myocarditis, present in greater intensity among CCC patients. In the absence of specific treatment, therapy for CCC is only supportive. In patients with refractory heart failure, the only available treatment is heart transplantation, while severe arrhythmia patients need pacemakers or implantable defibrillators.

The major histopathological feature attending dilated cardiomyopathy in CCC is the presence of a diffuse myocarditis, with intense cardiomyocyte damage and hypertrophy, and significant fibrosis, in the presence of very scarce T. cruzi forms[15,16]. Replacement by reparative fibrosis may be the main cause of pathologic ventricular remodeling[10,17,18]. Our group showed a significant correlation between myocarditis and ventricular dilation (manuscript in preparation) as well as fibrosis[19] in the Syrian hamster model of CCC. 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[20,21]. A direct role for heart parasitism has been proposed after the identification of T. cruzi antigen and DNA in CCC hearts by immunohistochemical and PCR techniques[15,22]. In addition, T. cruzi-specific CD8+ T cells have been isolated from endomyocardial biopsies of a CCC patient[23], 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[19,24]. However, the scarcity of T. cruzi in inflammatory lesions of CCC led early investigators to suggest that tissue damage had an autoimmune nature. However, regardless of the triggering antigen, the bulk of the evidence indicates the inflammatory infiltrate is a significant effector of heart tissue damage. In this paper, we will review the immunologic, transcriptomic/proteomic, and genetics studies of the pathogenesis of Chagas disease.

AUTOIMMUNITY

CCC is one of the few examples of post-infectious autoimmunity in humans, where infectious episodes with an established pathogen clearly triggers antigenic mimicry with host self-antigens, in association to target organ immune damage. CCC heart lesions are consistent with inflammation: a T cell/macrophage-rich myocarditis, fibrosis and heart fiber damage, in the presence of very scarce T. cruzi forms. Several studies have shown that T. cruzi DNA is found in myocardial tissue of essentially all CCC and IND cases[25,26]; the finding of T. cruzi DNA in hearts of IND patients argues against the hypothesis that local parasite presence is the trigger to myocardial inflammation in CCC. The discrepancy between the parasitism and inflammation suggested that tissue-damaging T cells were of autoimmune nature, possibly elicited by cross-reactive immune responses with T. cruzi parasite[6].

Direct experimental evidence of autoimmunity in CCC and experimental models has been documented over the last 25 years and is reviewed by Marin-Neto et al[5]. Cross-reactive autoantibodies and T cells against several distinct host-T. cruzi antigenic pairs were described along the last decades (Table 1). In murine models of T. cruzi infection, our group[27] and others[28,29] have shown that cardiac myosin, the major heart protein, is an important target cardiac self-antigen. Cardiac myosin heavy chain is recognized by CD4+ T cells from T. cruzi-infected mice[27], and tolerance induction with cardiac myosin-rich heart extracts was shown to ameliorate ameliorate T. cruzi-induced chronic cardiac inflammation and fibrosis[30]. We have shown that CCC patients display serum IgG anti-cardiac myosin heavy chain autoantibodies that cross-reactively recognize T. cruzi membrane protein B13[31]. Furthermore, CD4+ T cell clones infiltrating hearts from CCC patients also cross-reactively recognize cardiac myosin and B13 protein[30,32]. We have also found restricted heterogeneity of T cell receptor (TCR) variable regions in CCC heart tissue, which is in line with an antigen-driven, possibly autoimmune response[33]. Moreover, in vitro priming with B13 or its synthetic peptides leads to the recovery of cardiac myosin cross-reactive T cell clones[34,35]. We identified one such T cell clone that cross reactively recognized one epitope in B13 protein and twelve distinct low homology peptides in cardiac myosin, indicative of a very degenerate TCR recognition pattern[34]. Molecular modeling of B13 and cardiac myosin epitopes identified shared TCR-exposed residues which could potentially explain the low-homology crossreactivity[36].

Table 1 Molecular mimicry after Trypanosoma cruzi infection.

Host component	T. cruzi antigen	Host	Molecular definition
Neurons, liver, Kidney, testis	?	M, R	Mab
Neurons	Sulphated glycolipids	H, R	Mab, sera
47 kDa neuron protein	FL 160	H, R	rDNA, AS
Heart and skeletal muscle	Microsomal fraction	H, M	Mab, Serum IgG
Smooth and striated muscle	150 kDa protein	H, M	Serum IgG
Human cardiac Myosin heavy chain	B13 protein	H	rDNA, Ab T cell clones
Human cardiac myosin heavy chain	Cruzipain	M	Ab
95 KDa myosin tail	T. cruzi cytoskeleton	M	Mab
Skeletal muscle Ca++ dependent SRA	SRA	H, Rb	AS, serum IgG
Glycosphingolipids	Glycosphingolipids	H, M	Serum IgG
MAP (brain)	MAP	H, M	rDNA, AS
Myelin basic protein	T. cruzi soluble extract	M	Serum IgG, T cells
28 kDa lymphocyte membrane protein	55 kDa membrane protein	H, M	Mab
23 kDa ribosomal protein	23 kDa ribosomal protein	H	Ab
Ribosomal P protein	Ribosomal P protein	H	rDNA, Ab, SP
38-kDa heart antigen	R13 peptide from ribosomal protein P1, P2	M	IgG1, IgG2
β1 adrenoreceptor M2 muscarinic receptor	Ribosomal P0 and P2 proteins	H	Ab, SP
β1-adrenoreceptor M2 cholinergic receptor	150 kDa protein	H, M	Mab
Cardiac muscarinic Acetylcholine receptors (mAChR)	?	H	Ab
Cardiac muscarinic Acetylcholine receptors (mAChR)	Cruzipain	M	Immunization with cruzipain
Cha antigen	SAPA, 36KDa TENU2845	M	Ab, T cells

M: Mouse; H: Human; Rb: Rabbit; R: Rat; AS; Antiserum; Ab: Patient antibody; Mab: Monoclonal antibody; rDNA: Recombinant DNA; SP: Synthetic peptides (adapted from Bilate and Cunha-Neto[6] and Cunha-Neto et al[120]). SRA: Cross-reacting antigen; MAP: Microtubule associated protein. T. Cruzi: Trypanosoma cruzi.

The existence of degenerate intramolecular recognition, with multiple low-homology, cross-reactive epitopes in a single autoantigenic protein may have implications in increasing the magnitude of the autoimmune response in CCC and other autoimmune diseases. Several mechanisms have been suggested to play a role in the triggering of autoimmunity after T. cruzi infection. Antigen exposure secondary to tissue damage, followed by sensitization in an appropriate inflammatory environment (i.e., bystander activation); antigenic or molecular mimicry, where T and B cells recognize parasite antigens that share structurally similar epitopes in host antigens, generating crossreactive autoimmune responses (Table 1); and polyclonal activation leading to autoantibody production. Autoimmune and T. cruzi-specific responses secondary to parasite persistence are not incompatible or mutually exclusive in Chagas disease, and a combination of these types of immune responses could be involved in the establishment of heart tissue lesions.

IMMUNOPATHOGENESIS OF CCC

Chagas cardiomyopathy is essentially a myocarditis. The inflammatory process, although more intense in the acute phase, is clinically silent but incessant in patients with the indeterminate and chronic phases of the disease[15].

Immunological dynamics of the acute phase of T. cruzi infection

Data from animal models show that inflammatory cytokines play a central role in acute T. cruzi infection (Table 2). Shortly after the acute infection starts, T. cruzi components - including its DNA and membrane glycoconjugates - trigger innate immunity via Toll-like receptors 2, 4 and 9 in macrophages and dendritic cells[37]. Upon activation, such cells secrete proinflammatory cytokines and chemokines, express costimulatory receptors, and increase endocytosis and intracellular killing of parasites through release of reactive oxygen and nitrogen species. Proinflammatory cytokines, such as interleukin (IL)-1, IL-6, IL-12, IL-18, IL-27 and tumor necrosis factor (TNF)-α are promptly released, and further activate other inflammatory cells[6,38]. Macrophages and dendritic cells that have endocytosed the parasite subsequently elicit a strong T cell and antibody response against T. cruzi. Interferon-γ (IFN-γ)-producing T. cruzi-specific T cells are thus generated[6], that migrate together with other blood leukocytes to sites of T. cruzi-induced inflammation in response to chemokines such as CCL2, CCL3, CCL4, CCL5, and CXCL10, participating in the immune response against the parasite[39]. These inflammatory T cells and antibody responses lead to control - but not complete elimination - of tissue and blood parasitism. On the other hand, the blockade of CCR5 with Met-RANTES significantly decreased the intensity of cardiac inflammatory infiltrate, suggesting that lymphocyte migration to the myocardium in acute infection is dependent of CCR5 ligands[40]. A recent study has shown that Beagle dogs at the acute phase of T. cruzi infection presented higher mRNA expression of CCL1 and CXCL9 than uninfected animals[41]. Some studies have evaluated the role of chemokines in experimental T. cruzi infection in rat. Holtzman rats immunized with a CCL4-encoding DNA vaccine had enhanced heart inflammation but unchanged heart parasite load when infected with T. cruzi, suggesting that CCL4 could be recruiting regulatory cells to the infected tissue and, hence, regulating the intensity of the inflammatory response to the infection[42]. On the other hand, the vaccination with CCL3 and CCL5 increased heart parasitism and decreased local IFN-γ production, but did not influence the intensity of inflammation suggesting the chemokines are relevant for the control of parasitism, and may play a differential role in inflammatory cell recruitment during acute T. cruzi infection in rats[43].

Table 2 Cytokine and chemokine expression in Chagas disease and animal models.

Cytokines/chemokines	Phase (acute/chronic/IND/severe/moderate CCC)	Host (mouse/human)	Organ/cell type	Reference
IFN-γ	Severe CCC	Human	Mononuclear cells	[54,56]
IFN-γ	Severe CCC	Human	Myocardium	[60,61]
IFN-γ	Severe CCC	Human	Heart-infiltrating T cells	[54]
IFN-γ	IND, Severe CCC	Human	Plasma	[52,54,55]
TNF-α	Severe CCC	Human	Mononuclear cells	[54, 56]
TNF-α	Severe CCC	Human	Heart-infiltrating T cells	[54]
TNF-α	Severe CCC	Human	Myocardium	[60, 61]
TNF-α	IND and Severe CCC	Human	Plasma	[52,54,55]
IFN-γ	Acute/chronic	Mouse	Heart	[121-123]
TNF-α	Acute/chronic	Mouse	Heart	[124]
IL-6	Severe CCC	Human	HEART-infiltrating T cells	[54,60,61]
IL-2	Severe CCC	Human	Heart-infiltrating T cells	[54,60,61]
IL-4	Severe CCC	Human	Heart-infiltrating T cells	[54,60,61]
IL-10	Severe CCC	Human	Heart-infiltrating T cells	[54,60,61]
IL-7	Severe CCC	Human	Myocardium	[62]
IL-15	Severe CCC	Human	Myocardium	[62]
IL-12	Acute	Mouse	Mononuclear cells	[125]
IL-18	Acute	Mouse	Mononuclear cells	[126]
IL-10	Acute	Mouse	Mononuclear cells	[44-46]
TGF-β	Acute	Mouse	Mononuclear cells	[44-46]
IL-17	Chronic	Mouse	Mononuclear cells	[47]
CCL2, CXCL10, CXCL9 (mRNA)	Severe CCC	Human	Myocardium	[59]
CCR2, CXCR3 (mRNA)	Severe CCC	Human	Myocardium	[59]
CCR5, CXCR3	Severe CCC, IND	Human	Mononuclear cells	[63]
CCL5, CXCL9, CXCL10	Chronic	Mouse	Cardiomyocytes	[127]
CCR5	Chronic	Mouse	Heart	[40,128]
CCL5, CCL4, CXCR3 (mRNA)	Chronic	Dog	Heart	[41]

CCC: Chronic Chagas disease cardiomyopathy; IND: Indeterminate clinical form; IFN: Interferon; TNF: Tumor necrosis factor; IL: Interleukin.

On the other hand, some cytokines are associated with susceptibility to T. cruzi infection, as IL-10 and TGF-β[44-46]. Recent data show that IL-17 controls the resistance to T. cruzi infection in mice, with increased levels resulting in enhancement of myocarditis, premature mortality and decreased parasite load in the heart. Neutralization of IL-17 neutralization resulted in increased production of IL-12, IFN-γ and TNF-α and enhanced specific type 1 chemokine and chemokine receptors expression[47]. In experimental T. cruzi infection, CD4⁺CD25⁺GITR⁺Foxp3⁺ T cells migrate to the heart of T. cruzi infected mice, and the treatment with anti-GITR enhanced the myocarditis, with increased migration of CD4⁺, CD8⁺, and CCR5⁺ leukocytes, TNF-α production, tissue parasitism leading to increased mortality, suggesting that GITR⁺Foxp3⁺ regulatory T cells may control the heart inflammation, parasite replication, and host resistance in the acute phase of infection[48].

The Syrian hamster model of T. cruzi infection reproduces the range of different outcomes of human Chagas disease[19,49]. During acute T. cruzi infection, hamsters displaying high cardiac parasitism also showed increased expression of TNF-α, IFN-γ, IL-10, and CCL3 mRNA, as well as acute phase signs such as weight loss, vomiting and diarrhea, while animals showing low cardiac parasitism displayed a modest increase in cytokine/chemokine mRNA and no acute phase signs, suggesting that cardiac parasitism was apparently related to the increased expression of cytokines and chemokines[6].

Few immunological studies have been performed with acutely infected patients. Acutely infected children have shown increased expression inflammatory cytokines, such as circulating IL-6 and TNF-α[50] and have also displayed increased production of IFN-γ by mononuclear cells[51].

Cytokines and chemokines in the chronic phase of T. cruzi infection

Inflammatory cytokines are produced along the chronic phase of Chagas disease (Table 2). Increased levels of plasma TNF-α and peripheral blood mononuclear cell-produced IFN-γ are detected even in infected individuals in the indeterminate form of the Chagas’ disease[52-54], probably in response to parasite persistence. The subsets of patients that develop Chagas cardiomyopathy display an array of immunological alterations consistent with an exacerbated Th1 immune response. CCC patients display increased circulating levels of TNF-α and CCL2 compared to individuals in the indeterminate form of the Chagas disease, or those with electrocardiogram (ECG) abnormalities alone but no ventricular dysfunction[52,55]. Additionally, CCC patients show an increased number of the IFN-γ -producing CCR5⁺ CXCR3⁺ CD4⁺ and CD8⁺ T cells in the peripheral blood, with reduced numbers of IL-10-producing and CD4+CD25+FOXP3⁺ regulatory T cells[54,56,57] as compared to patients in the indeterminate form of Chagas disease. Recent studies also have shown that FOXP3⁺ cells are significantly more abundant in myocardial sections from IND than in CCC patients or non-infected individuals, suggesting that regulatory T cells are less abundant in CCC than IND hearts[58]. Given the reduced levels of peripheral blood CD4+CD25+FOXP3⁺ regulatory T cells among CCC patients, this could indicate such cells may play a role in the control of the myocardial inflammatory process among chronic Chagas disease patients.

The exacerbated Th1 response observed in the peripheral blood of CCC patients is reflected on the nature of the inflammatory infiltrate found in their heart tissue[59]. Mononuclear cells infiltrating CCC heart tissue express IFN-γ, TNF-α, IL-6, with lower levels of IL-2, IL-4 and IL-10[54,60,61]. The survival cytokines IL-7 and IL-15 also show increased expression in CCC heart tissue, and may be the underlying reason for the predominance of CD8+ T cells, which express increased levels of IL-15Rα and γ_c chain receptor[62]. Real-time quantitative PCR analysis in human CCC myocardium showed that the gene expression levels of IFN-γ-inducible chemokines CCL2, CXCL10 and CXCL9, as well as the chemokine receptors CCR2 and CXCR3, were selectively upregulated[59]. Corroborating these data, we were able to detect mononuclear cells that express CXCR3, CCR5, CXCL9 and CCL5 in the myocardium of CCC patients using confocal immunofluorescence assays. We also observed abundantly increased mRNA expression of the CCR5 and CXCR3 as well as several of their chemokine ligands in the myocardium CCC patients. The intensity of the myocardial infiltrate correlated with chemokine mRNA expression. Significantly, a recent study of chemokine expression in the heart of chronically T. cruzi-infected Beagle dogs with the cardiac form also observed increased mRNA expression of CXCR3, CCL4 and CCL5 as compared to those in the indeterminate form[41]. The increased expression of chemokines and their receptors in CCC heart tissue are in line with the increased amounts of CD4⁺ and CD8⁺ T cells expressing CXCR3/CCR5 and co-expressing IFN-γ/TNF-α observed in peripheral blood from CCC patients, as compared to T. cruzi-seropositive IND individuals[63]. Taken together, these findings suggests that the recruitment of inflammatory cells to the CCC myocardium, a key pathogenetic event, may indeed be driven by CC and CXC chemokines.

Given the major role of Th1/inflammatory infiltrate in CCC heart, we tried to dampen the inflammatory response in an attempt to reduce the heart damage. Using the hamster model of dilated cardiomyopathy of human CCC, it was observed that TNF-α blockade at the chronic phase (7 mo post infection) with the soluble receptor Etanercept paradoxically worsened cardiac function[64] in the absence of increased parasitism, direct drug toxicity or increased myocarditis. These results suggest a beneficial role for residual TNF-α signaling in Chagas disease cardiomyopathy, also suggesting that TNF-α antagonism in the chronic phase of the T. cruzi infection worsens experimental cardiomyopathy – playing a cautionary note on cytokine-blocking intervention in human Chagas disease.

Non-immunological pathogenic effects of cytokines and chemokines in CCC

Our group and others found significant evidence of non-inflammatory cytokine and chemokine effects on cardiomyocytes and other myocardial cell types, in addition to the inflammatory effects of cytokines and chemokines. Immunohistochemical analysis of cardiac biopsies in CCC patients showed phosphorylated Smad2, a component of the TGF-β signaling pathway, as well as fibronectin, a marker of fibrosis[65], suggesting that TGF-β could play a role in the induction of fibrosis in cardiac tissue of patients with CCC.

Significant IFN-γ signaling was observed in the myocardium of CCC patients, including genes that are not ordinarily expressed by inflammatory cells. In vitro experiments have shown that IFN-γ- alone or in combination with CCL2 - may induce deep changes in the cardiomyocyte gene expression program, including induction of atrial natriuretic factor and of the hypertrophic gene expression program[59]. IL-18 and the CCR7 ligand CCL21, upregulated in CCC myocardium, induce cardiomyocyte hypertrophy and molecules involved in the fibrotic process[66,67]. We can thus hypothesize that, apart from the direct inflammatory damage, non-immunological effects of several mediators locally produced in the myocardium, like IFN-γ, TNF-α, IL-18, CCL2 and CCL21, may play a significant pathogenic role in CCC, by modulating gene and protein expression of cardiomyocytes and fibrocytes in pathways essential to the development of CCC, like hypertrophy and fibrosis. T cell migration to the myocardium, and non-immunological effects of chemokines and other mediators are thus prime candidates for intervention in Chagas disease. Understanding the importance of these pathways in the pathogenesis may be instrumental for the development of more adequate therapy for chronic Chagas disease cardiomyopathy.

LARGE-SCALE GENE AND PROTEIN EXPRESSION PROFILING IN CCC

In recent years, new-generation high-throughput technologies, including next-generation sequencing technology and mass spectrometry methods, have been widely applied in solving biological problems, especially in human diseases field. These data driven and large-scale research model enable the multi-level study of human diseases from the perspectives of genomics (the quantitative study of genes, regulatory and non-coding sequences), transcriptomics (RNA and gene expression) and proteomics (protein expression) levels. This type of investigation typically yield data that identify pathways involved in the pathogenesis of particular diseases. In addition, results of this type may provide novel targets for chemotherapy.

Several studies have applied global gene expression analysis with DNA microarrays in tissue derived from infected animals or in myocardial tissue from CCC patients to gain insight into the molecular events associated with Chagas disease. Cunha-Neto et al[59] analyzed the gene expression profiling of CCC myocardial tissues of CCC, idiopathic dilated cardiomyopathy (DCM) and heart donor by a 10 368 element cDNA microarray. The cardiac hypertrophy signature was shown to be up-regulated in both CCC and DCM heart tissue. Moreover, lipid metabolism and mitochondrial oxidative phosphorylation genes were selectively up-regulated in myocardial tissue of the tested CCC patients. Immune response- related genes and IFN-γ-induced genes were prominently up-regulated only in CCC patients[59].

Several studies evaluating large-scale gene expression using animal models infected with T. cruzi have also been conducted. In a study, C57Bl/6 mice chronically infected with the Colombian strain of T. cruzi (8 mo post-infection) showed a number of genes up-regulated in the myocardium that probably play a role in modulating inflammation and fibrosis. Pathways of genes significantly up-regulated in infected hearts prominently include immune response and related terms (e.g. inflammatory response, intracellular signaling cascade, and chemokine and cytokine receptor activity). In addition, up-regulated pathways include phosphate transport, cell proliferation, and actin binding (e.g. Arp2/3 protein complex and actin filament organization, cytoskeleton, and membrane ruffling). Pathways containing an overrepresentation of down-regulated genes included mitochondrion, enzymatic activity of several types, and tyrosine kinase signaling[68]. Garg et al[69], in a time course experiment (up to 100 d post-infection), showed an up-regulation of genes associated with inflammation and IFN-γ-induced immune response; expression of extracellular matrix proteins and repressed structural and contractile genes, such as troponins, suggestive of active reparative and remodeling reactions following injury to the myocardium; and a depression of genes encoding mitochondrial oxidative phosphorylation complexes I and IV in T. cruzi-infected murine hearts. Mukherjee et al[70], using a single time point (100 d post-infection), reported that the heart from infected C57BL/6 mice showed symptoms of chronic inflammation, vasculitis, fibrosis and an up-regulation of atrial natriuretic peptide precursor, a strong indicator of cardiac pathogenesis[71]. In this study, it was found that the extracellular matrix genes, especially those associated with fibrosis, were up-regulated in the chronic disease model, similar to that observed at day 37 post-infection[69]. These changes in structural components could possibly be initiated in response to myocardial injuries by invading and replicating parasites, and it is suggestive of active reparative and remodeling reactions. Moreover, Mukherjee et al[72] examined transcriptome changes in CD-1 mice infected with the Brazil strain of T. cruzi through the various stages of the disease (30, 60, 90, 120, 150 and 180 d post-infection). Several key genes belonging to important biological processes and pathways were found to be deregulated, including inflammatory mediators, growth factors, cell cycle, apoptotic and cytoskeletal genes.

Although these studies provided important insights into the molecular events associated with the pathogenesis of CCC at the organ level, the response of cardiomyocytes themselves to infection at different time points could provide further details. Goldenberg et al[73] employed microarrays (7624 annotated unigenes) to examine infected cardiac myocyte cultures 48 h post-infection. Major categories of affected genes included those involved in immune response (e.g. CXCL12 was down-regulated, while CXCL16, CCL7, CXCL1 AND CXCL16 were up-regulated), extracellular matrix and cell adhesion. These findings on infected cardiac myocytes in culture reveal that alterations in cardiac gene expression described in Chagas disease are the consequence of both direct infection of the myocytes themselves as well as resulting from the presence of other cell types in the myocardium and systemic effects of the infection.

Since proteins are the ultimate biological determinants of disease phenotype, studies have used proteomic analysis to get a better understanding of pathophysiological mechanisms of diseases, including cardiomyopathies, and to find new biomarkers for diagnosis and prognosis[74-77]. Several studies mapping the proteins expressed by the parasite T. cruzi were performed[78-86]; however, few studies have examined the proteins in cardiac tissue infected by the parasite. In one study, the protein expression profile in the human CCC myocardium were analyzed by two-dimensional electrophoresis and mass spectrometry[87]. Authors identified 246 proteins in the CCC myocardium samples, corresponding to 112 distinct proteins. Along with structural/contractile and metabolism/energy metabolism proteins, proteins involved in apoptosis (e.g. caspase 8, caspase 2), immune system (T cell receptor beta chain, granzyme A, HLA class I) and stress processes (including several oxidative stress proteins) were identified. The identification of active caspase isoforms and oxidative stress proteins may indicate the occurrence of active apoptosis and significant oxidative stress in CCC myocardium. Differential protein expression analysis between CCC and DCM cardiac samples identified significantly reduced expression of proteins involved in the generation of cytoplasmic ATP, such as creatine kinase M (Teixeira et al[88] unpublished observations), suggesting the myocardium of CCC patients may be under energy deprivation. T. cruzi–infected outbred hamsters reproduce the range of different outcomes of Chagas disease noted in humans[19]. While in the acute phase of infection, 50% of animals displayed weight loss and signs of acute-phase infection (APS) (e.g., lethargy, vomiting, and diarrhea). Proteomic analysis of these animals showed that T. cruzi infection and the presence of APS were associated to distinct myocardial expression of contractile, stress response, and metabolism proteins. The study has shown the up-regulation of selective forms of the structural/contractile proteins (ACTC), muscle-specific intermediate filament (DES), proteins involved in cardiac myofibril assembly (MLC2), and proteins involved in actin cytoskeleton regulation (MYL3) in animals with APS but not in animals without APS, this may be associated with the greater cardiac parasitism observed among infected animals with APS. Moreover, the increased expression of stress response proteins, such as CRYAB, HSPA5, and HSPA9, in infected animals, compared with uninfected controls, might be associated with a stress response triggered by T. cruzi infection[89].

The discovery of biomarkers associated with a particular disease or disease stage could also give valuable insights into the basic biology of the host–parasite interface. Technologies such as surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry can be applied to discover biomarkers associated with parasitic diseases. Such biomarkers can represent host proteins, fragments of host proteins or parasite proteins that appear in body fluids or tissues following infection. Individual biomarkers or biomarker patterns/signatures not only have diagnostic utility (e.g. in active disease, prognosis, tests of cure) but can also provide unique insights into the mechanisms underlying host responses and pathogenesis[90]. SELDI-TOF analysis identified several host protein fragments found almost exclusively in sera from subjects with Chagas Disease[90]. These include both N- and C-terminal fragments of human apolipoprotein A-I, a C-terminal (desArg) truncation of C3a anaphylatoxin, and an N-terminal fragment of fibronectin. In a western blot format, the anti-sera directed against the truncated form of human fibronectin were found to be both sensitive and specific for the diagnosis of latent Chagas disease, indicating the antigens were biomarkers[90]. Such protein biomarkers not only can reveal new targets for therapies and vaccines but could also suggest new diagnostic tests.

In conclusion, the results of these types of studies may yield data identifying pathways involved in the pathogenesis of particular diseases. In addition, results of this type may provide novel targets for chemotherapy or diagnosis for Chagas disease. Considering the role of proteins and genes that were found differentially expressed in the cited studies, we can hypothesize that cytokine-induced modulation of cardiomyocyte gene/protein expression may be a novel disease mechanism in CCC, in addition to direct inflammatory damage, that in turn, could contribute to the severe outcome commonly observed among CCC patients.

SEVERAL GENE POLYMORPHISMS WERE ASSOCIATED TO AN HIGHEST RISK FOR CCC

The first report suggesting that host genetic factors may be involved in disease susceptibility to CCC progression was done by Zicker et al[91]. Indeed, a case-control study was conducted in a population survey in Goiânia, Brazil, including 247 cases of Chagas’ heart disease, 345 asymptomatic seropositive subjects and 529 seronegative subjects. Increasing age and male sex were consistently and significantly related to an increased risk of electrocardiographic abnormalities. No association was found between length of residence in an endemic area, physical activity, and electrocardiographic abnormalities. A clear significant concordance of clinical types with sharing the same household has been described. Comparing seronegative to seropositive subjects, it was shown that males were at greater risk of any electrocardiographic alteration and left anterior hemiblock compared to females. An increasing risk of ventricular premature beats with age was clearer for seropositive than for seronegative subjects. Subjects with history of heart disease were at an increased risk of electrocardiographic abnormalities. These findings suggest a possible geographical clustering or a familial aggregation of cases of Chagas’ heart disease. Several genetic studies have been performed, so far, in order to identify genetic factors that control human susceptibility to severe diseases. All these studies were based on case control studies. These approaches have leaded to significant results that were partially confirmed in independent studies. We will summarize all these studies.

Regarding the Major Histocompatibility Complex (MHC) genes, several studies show contradictory results that vary with the population studied. Fernandez-Mestre et al[92] demonstrate the first evidence of association between Chagas disease and HLA genetic susceptibility when analysed HLA class II alleles in a sample of 67 serologically positive individuals with and without cardiomyopathy and compare with 156 healthy controls of similar ethnic origin. The comparison of DRB1 and DQB1 allele frequencies among the patients and control subjects showed a decreased frequency of DRB1*14 and DQB1*0303 in the patients, suggesting independent protective effects to the chronic infection in that population. Allele frequencies comparison between patients with and without cardiomyopathy showed a higher frequency of DRB1*01, DRB1*08 and DQB1*0501 and a decreased frequency of DRB1*1501 in the patients with arrhythmia and congestive heart failure[92]. These results suggest that HLA Class II genes may be associated with the development of a chronic infection and with heart damage in Chagas’ disease.

Later on, Layrisse et al[93] have shown, on the same population, a strong association of HLA class I gene to cardiac damage. HLAC*03 allele showed increased frequencies among patients with cardiac damage compared to asymptomatic subjects.

At the same time, Deghaide et al[94] have characterised a Brazilian population including 176 patients presenting with pure cardiomyopathy with heart failure (n = 60), cardiomyopathy without heart failure (n = 18), pure digestive tract manifestations (n = 25), cardiac plus digestive disease (n = 40), and asymptomatic patients with positive serology for chronic T. cruzi infection (n = 33) and non infected individuals (n = 448). Serologic HLA class II analysis showed that HLA-DQ1 conferred susceptibility to, while HLA-DQ7 antigen conferred protection against the development of the disease in the total group of patients. Oligonucleotide typing has shown that HLA-DQB1*06 alleles were underrepresented in the total group and in the subgroups presenting with pure digestive or cardiac disease, conferring closely similar relative risks and preventive fractions. Asymptomatic patients showed a significant increase of HLA-DQB1*0302 specificity[94].

These data were confirmed at study in a Venezuelan population by Colorado et al[95] that studied 35 asymptomatic cases and 72 symptomatic cases. The asymptomatic population (33.68%) consisted of apparently healthy individuals having normal, borderline, or abnormal ECG, without cardiomegaly. The symptomatic population was divided in 2 distinct groups, group B (33.68%) included patients with either arrhythmia-related symptoms, five or more extrasystoles per minute, or embolic episodes as first symptom; and group C (32.63%) by patients with overt congestive heart failure[95]. Most had severe cardiomegaly and arrhythmias. Statistical analysis confirmed the significant increment of the DRB1*01 DQB1*0501 haplotype (P = 0.03). The DPB1*0401 allele frequency is also significantly increased in patients with heart disease (groups B + C) (P = 0.009, OR = 6.0) while DPB1*0101 frequency is higher among the asymptomatic group (P = 0.04, OR = 0.32) compared with individuals of group C. The DPB1*0401 allele in homozygous form or in combination with allele DPB1*2301 or 3901, was found present more often in patients of groups B and C. Thus, the combination of two of these three alleles, sharing specific sequence motifs in positions 8, 9, 76, and 84-87 confers a relative risk of 6.55 to develop cardiomyopathy in seropositive patients (P = 0.041). Furthermore, 32% of the cardiomyopathics have either DRB1*01 DQB1*0501 and/or DPB1*0401/*0401, 0401/*2301, or* 0401/*3901 compared with 9% of the seropositive asymptomatics (P = 0.006, OR = 5.0).

Rodriguez-Perez et al[96] have studied 54 Mexican patients (27 asymptomatic subjects and 27 patients with chronic cardiomyopathy) and 169 healthy individuals. The whole group of patients showed increased frequencies of TNFA-308A allele when compared to healthy controls (P = 0.008, OR = 3.03). Similar results were obtained when CCC patients were compared to asymptomatic individuals (P = 0.0002) or healthy controls (P = 4 × 10⁻⁷, OR = 7.02). Similar results were obtained when they took into account the AG genotype[96].

Similarly, Campelo et al[97] have conducted on evaluation of genetic susceptibility to chronic disease in relationship of five microsatellite polymorphisms in a series of Brazilian Chagasic patients stratified according to the clinical form of disease presentation, i.e., cardiac, digestive, digestive plus cardiac, or indeterminate form. A total of 162 chronic patients including 54 patients with cardiomyopathy with heart failure, 17 patients with cardiomyopathy without heart failure, 25 patients with pure digestive manifestations, 33 patients with digestive plus cardiac manifestations in 33 patients and 33 other patients characterized by indeterminate form. An additional group of 221 negative serology subjects was also included. After patient stratification according to the clinical form, the frequencies of several TNF microsatellite alleles were significantly increased in each clinical group. The relative risks associated with the susceptibility alleles ranged from 1.674 (P = 0.04) (TNFa2 in digestive plus cardiac form) to 10.21 (P = 0.010) (TNFd7 in digestive plus cardiac form), indicating that the individuals who possess these susceptibility alleles have almost 2 to 10 times higher risk of developing a given form of chronic disease if infected. On the other hand, some other alleles are associated to protection[97]. Exact tests done haplotype frequencies revealed significant differences when patients were considered as a whole or stratified according to the clinical variant were compared to controls. All these results suggest that this chromosomal region is associated with susceptibility to or resistance against CCC forms.

Several other investigations were done by other groups to determine the role of the TNF gene in the human susceptibility to the Chagas disease chronic forms. Drigo et al[98] have analyzed the allele distribution of the TNFA microsatellite and TNFA-308 promoter polymorphisms in 166 CCC patients compared to 80 asymptomatic individuals employed as control group. The comparisons of TNFA microsatellite and TNFA-308 promoter allele frequencies between the two groups do not lead to detection of statistical difference. This lack of association was also found in a Peruvian population including 85 serologically positive chagasic individuals and in 87 healthy controls[99].

So, these three studies lead to conflicting results. According to these three previous studies we performed a meta-analysis on the TNFA-308 polymorphism that leads to significant association (Table 3 and 4). The fact that significant association was also detected into meta analysis is indicating that this chromosomal region is involved into the genetic control of human susceptibility to CCC forms. These discrepancies between the three studies may be due either to modest clinical group size or to differing genetic backgrounds. According to this hypothesis into Mexican population the TNFA-308 polymorphism may be in strong disequilibrium with the functional variant. This local linkage disequilibrium map may be different from one population to the other one. As soon as, this linkage disequilibrium decreases the association with the TNFA-308 polymorphism may be lost.

Table 3 Case control studies done on the TNFA-308 polymorphism.

Study name	Cases No. of chr carrying the TNFA-308T2 allele	Cases Total No. of chromosome	Controls No. of chr carrying the TNFA-308T2 allele	Controls Total No. of chromosome
Rodriguez-Perez et al[96]	13	108	14	324
Drigo et al[98]	37	332	16	160
Beraun et al[99]	5	66	4	104

Even though Drigo et al[100] didn’t confirm the previous association, they studied putative correlation between this polymorphism and differential survival of severe CCC patients. On an independent population done in Brazil, including 42 patients with severe ventricular dysfunction (left ventricular ejection fraction ≤ 40%), Drigo et al[100] have shown that patients (16) positive for TNFA-308A allele display a significantly shorter survival time compared to those (26) carrying other allele. The median survival times were 2.9 (positive patients) and 8 mo (negative patients) (P = 0.020, hazard ratio = 2.28).

A Colombian study was based on 130 patients with cardiomyopathy and 130 asymptomatic subjects on IL1B genes[101]. A significant difference was detected for the IL1B+5810GA polymorphism. The IL1B+5810GG genotype frequency was higher in the CCC patient group than in the asymptomatic group (P = 0.03, OR = 2.64)[101]. Differences in the distribution of the allele frequencies confirmed the association. A haplotype covering the IL1A, IL1B and IL1RN genes was also associated to protection. Putative association for the IL1 antagonist gene (IL1RN) was described on a Mexican population[102]. This study included 86 individuals seropositive for T. cruzi (58 CCC patients and 28 asymptomatics), 50 seronegative individuals with idiopathic dilated cardiomyopathy and 109 healthy individuals. An increased IL-1RN.4CC genotype frequency was detected when T. cruzi-infected infected patients were compared to idiopathic dilated cardiomyopathy patients (P = 0.028, OR = 11.46). A similar increase was also detected between CCC patients and controls (P = 0.011, OR = 3.6)[102].

In Brazil, a study on the IL10 gene was performed. One hundred fifty-five patients in the chronic phase of the disease were enrolled in this study as well as 43 individuals without Chagas disease[103]. The IL10-1082G/A polymorphism, which correlates with lower expression of IL-10, was associated with the development of Chagas disease cardiomyopathy. Comparison of genotype distribution, between the indeterminate group and CCC patients, showed an association (P < 0.01, OR = 1.6)[103]. The frequency of the IL10-1082A allele (associated with lower expression of IL-10) was higher in the severe patient group than in the indeterminate group (P < 0.01, OR = 0.84)[103]. An independent study suggests that an epistasis between MHC and IL-10 is associated to susceptibility/resistance to Chagas’ disease[104].

A study performed on Colombian population on IL12B gene has detected an association with the polymorphism IL12B+1188A/C located in the 3’UTR region of this gene[105]. Two hundred seronegative individuals and 260 serologically positive patients (130 with Chagas cardiomyopathy and 130 asymptomatic) were enrolled. The IL12B+1188CC genotype was significantly increased among cardiomyopathic patients as compared to asymptomatic individuals (P = 0.005, OR = 3.39). In addition, the authors observed that the IL12B+1188C allele was present at significantly higher frequency in CCC patients (P = 0.008, OR = 1.69) than in asymptomatic ones[105]. However, this polymorphism failed to discriminate the seropositive patients from the control individuals[105].

Ramasawmy et al[106] performed an analysis of the following TLR1, TLR2, TLR4, TLR5, TLR9, and MAL/TIRAP genes, on a Brazilian cohort. One hundred and sixty-nine patients with cardiomyopathy and 76 asymptomatic individuals were investigated. Comparison of the asymptomatic and CCC groups with respect to genotype distribution for each SNP showed a statistically significant difference only for the MAL/TIRAPS180L variant[106]. Indeed, the frequency of homozygosity for the MAL/TIRAP180S allele was significantly higher among patients with CCC than among asymptomatic patients (P = 0.0004, OR = 3.1), whereas while the percentage of subjects homozygous for the MAL/TIRAP180L allele was similar in both groups. The percentage of subjects heterozygous for MAL/TIRAPS180L among patients with CCC was significantly different from the percentage found on asymptomatic patients (P = 0.0004, OR = 3.1)[106].

The same authors have also four other candidate genes. Ramasawmy et al[107] assessed CCL2-2518A/G variants in 245 Brazilian individuals, all of whom were infected with T. cruzi. One hundred sixty-nine patients had CCC, and 76 were asymptomatic. Genotype distributions were different between the CCC group and the asymptomatic group (p=0.009)[107]. When the frequency of genotypes possessing at least 1 CCL2-2518A allele was compared with the genotype CCL2-2518A GG between CCC and asymptomatic groups, the observed difference reached statistical significance (P = 0.004, OR = 3.4)[107]. Thus, The A allele seems to confer susceptibility to CCC (P = 0.001, OR = 1.9).

Ramasawmy et al[108] have also focused their efforts on UAP56 genes which encodes BAT1 protein. For the polymorphism UAP56-22G/C, a significant difference in frequency between the CCC patients (154) and the asymptomatic patients (76) was revealed at the genotype level (P = 0.004, OR = 4.7). The UAP56-22C allele seems to confer susceptibility to CCC.

Lymphotoxin-α protein (encodes by LTA gene) is a proinflammatory cytokine which also induces adhesion molecules and cytokines from vascular endothelial cells and vascular smooth-muscle cells, which may contribute to the inflammation process. The putative involvement of this gene has been also investigated by Ramasawmy et al[109]. The LTA+80C/C genotype was significantly more common in patients with CCC (169) than asymptomatic patients (76) (P = 0.006, OR = 3.1)[109]. A significant difference in the frequencies of alleles LTA+80A and LTA+80C between patients with CCC and asymptomatic patients (P = 0.007, OR = 1.7) was also observed were compared. Similar results were obtained with the polymorphism LTA+252A/G. The LTA+252G allele was associated with susceptibility to CCC (P = 0.006, OR = 1.7)[109]. Authors performed also an haplotype analysis based on these two polymorphisms. The LTA+80C-LTA+252G haplotype was associated with susceptibility to CCC (P = 0.018), whereas, LTA+80A-LTA+252A haplotype was more common in the asymptomatic patients (P = 0.009), suggesting that it provides protection against CCC[109].

Finally this group has looked at the IKBL gene which encodes an inhibitor of NF-κB. Ramasawmy et al[110] provide evidence that two variants (IKBL-62A/T and IKBL-262A/G) in the promoter region of the IKBL gene are associated to susceptibility to develop CCC. Indeed, Genotype distributions for both IKBL-62A/T and IKBL-262A/G differed between the CCC patients and asymptomatic subjects (P = 0.025 and P = 0.03, respectively)[110]. Subjects homozygous for the IKBL-62A allele, had three-fold risk of developing CCC compared with those carrying the IKBL-62TT genotype (P = 0.0095, OR = 2.9). Similar trend was observed for the IKBL-262A homozygotes (P = 0.005, OR = 2.7). An haplotype analysis led to the identification of a susceptibility haplotype (IKBL-262A IKBL-62A) more frequent in CCC patients (P = 0.0014, OR = 2.1)[110].

As mentioned before, Cunha-Neto et al[59] characterized gene expression profiles of human Chagas’ cardiomyopathy and dilated cardiomyopathy to identify selective disease pathways. Among these altered genes we may notice the CCR5 gene[59]. Putative implication of the CCR5 gene in the development of chronic disease was investigated on 85 seropositive and 87 seronegative Peruvian individuals[111]. No differences in the distribution of CCR5-59029 promoter genotype or phenotype frequencies between chagasic patients and controls. The authors observed that the CCR5-59029A/G genotype was significantly increased in asymptomatic compare to cardiomyopathic patients (P = 0.02, OR = 0.33)[111]. In addition, the presence of the CCR5-59029G allele was also increased in asymptomatics when compared with cardiomyopathics (P = 0.02, OR = 0.35)[111]. The authors suggest that the CCR5-59029 promoter polymorphism may be involved in a differential susceptibility to chagasic cardiomyopathy. An independent study, performed in Venezuela, indicates that this polymorphism is also associated to Chagas disease. However, the associated genotype was not the same one[112].

Torres et al[113] performed two independent studies on 240 chagasic patients and 199 controls from Colombia; and 74 chagasic patients and 85 controls from Peru. For the polymorphism, MIF-173G/C, a statistically significant difference was detected between patients and controls in the Colombian cohort (P = 0.006, OR = 1.6). Similar association was found in the Peruvian cohort (P = 0.003, OR = 2.4). A meta-analysis of the Colombian and Peruvian cohorts has demonstrated that the MIF-173C allele confers a risk effect in Chagas patients Moreover, a dose effect for the susceptibility allele was observed (P = 0.004)[113].

All these positive association studies support the idea that host genetic factors may be involved in disease susceptibility to CCC progression. It identified main genes and pathways involved into the control of this susceptibility. However these studies were conducted on a limited number of SNPs. So, the real functional variants are probably not identified. Moreover, some studies lead to unreplicated results that will need to be confirmed. Lacks of association were also reported for TLR2[114], TLR4[114], PTPN22[115], angiotensin-converting enzyme[116], NRAMP1[117], NOS2[118], IL4[119] and IL4R (weak association[119]) genes. These lacks of association may be due to the limited number of tested SNPs and to the cohort size which remained relatively limited. It is now essential to set up a whole genome genetic study using new technologies on large study populations characterized by an acute phenotype. The identification of susceptibility genes and functional variants will be useful to understand the pathogenic process, for diagnosis and theranostic.

Even if twenty thousand deaths attributable to Chagas disease occur annually, it is still considered as a neglected disease. However, it remains a contemporary public health concern, even more so because of the threat of its re-emergence in some endemic areas where it was thought to have been controlled and emergence in non-endemic areas. Unfortunately, the mechanisms underlying differential progression to CCC are still incompletely understood.

Chagas disease is not a single disease, but rather 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, and genetic susceptibility factors have been identified. T cell migration to the myocardium and ensuing inflammation, non-immunological effects of cytokines and chemokines in the myocardium, and genetic components are clearly key events. It is likely that the progression to overt inflammatory dilated cardiomyopathy may result from the combined effect and inadequate counter regulation of expression of relevant genes such as those mentioned above. It is possible to speculate that the combined effect of several gene polymorphisms, together non-inflammatory events such as differential myocardial resilience to damage, could generate the observed distinct disease forms, progression rates, and gender differences. It is likely that the persistence of a parasite which induces strong innate immunity and proinflammatory cytokines may continuously boost the production of potentially pathogenic 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 chemokines. 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 pathophysiological role by causing direct inflammatory damage, as well as modulating cardiac cell gene expression. Functional agonistic autoantibodies directed against adrenergic or cholinergic receptors may 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.

CONCLUSION

Large-scale gene and protein profiling in CCC are promising new avenues of research that may identify additional important disease pathways. Identification of the mechanisms involved into the host-pathogen interactions at the establishment and chronic phases of the disease will lead to a better understanding of the immunological and pathological processes and facilitate the development of new drugs. It will also allow an earlier detection of susceptible subjects who will require stronger medical survey in order to avoid the development of severe cardiomyopathy. It will be also benefit for the understanding of the common mechanisms to the other dilated cardiomyopathies.