untitled Drug Target Insights 2007: 2 61–69 61 ORIGINAL RESEARCH Correspondence: Maria de Nazaré C. Soeiro. Tel: 0055 21 2598-4331; Fax: 00 55 21 2260-4434; Email: soeiro@ioc.fi ocruz.br Please note that this article may not be used for commercial purposes. For further information please refer to the copyright statement at http://www.la-press.com/copyright.htm Electrocardiographic Findings in Acutely and Chronically T. cruzi-infected Mice Treated by a Phenyl-Substituted Analogue of Furamidine DB569 Elen M. de Souza, Gabriel M. Oliveira and Maria de Nazaré C. Soeiro Lab. Biologia Celular, DUBC, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brasil. Abstract: Aromatic diamidines have been successfully used to combat a wide range of parasites that cause important human infections. Recently we reported that a N-phenyl-substituted analogue of furamidine (DB569) exerts a micromolar trypanocidal activity against Trypanosoma cruzi in vitro. Since DB569 also reduces the cardiac parasitism and increases the survival rates of T. cruzi-infected mice, our present aim was to analyze the potential protection of DB569 in the devel- opment of altered cardiac electrical conduction system during acute and chronic T. cruzi infection. In our experimental model of acute infection (Swiss mice inoculated with Y strain of T. cruzi), the prevailing disorder observed in electrocar- diogram (ECG) analyses was sinus bradycardia. This ECG alteration was reverted in acutely infected mice treated with DB569. Interestingly, the DB569 treatment reduced signifi cantly the numbers of CD8+ T cells in the cardiac infi ltration. In addition, the noticed protection of DB569 in the ECG fi ndings of acutely-infected animals was further extended to the chronic infection. Our data suggest that the reversion to and further maintenance of normal ECG profi le in the DB569- treated infected animals may be associated with the reduced cardiac CD8+ lymphocyte infi ltration and parasitism that might be ultimately contributing to their increased survival rates. Keywords: electrocardiography, aromatic diamidines, therapy, infected mice, infl ammation, heart, Chagas’ disease. Introduction Trypanosoma cruzi is the ethiological agent of Chagas’ disease, which affects over 16–18 million people in endemic areas of South and Central America, leading to 50,000 deaths per year (WHO 2002). The disease has two phases: the acute, which appears shortly after the infection, and the chronic phase, which can develop in about one-third of the infected individuals after a silent period of years or decades called the indeterminate phase (Cunha-Neto et al. 2006). Although the main clinical manifestations of the Chagas disease include both the cardiac and the digestive alterations, the former is the most prominent: about 25% to 30% of the infected people will develop a progressive heart disease commonly displaying symptomatic ventricular arrhythmias, hyper- trophy; congestive heart failure; sinus bradycardia and tachycardia; thomboembolism and sudden death (Marin-Neto et al. 1999; Salles et al. 2003). Several hypotheses have been raised to explain the chronic chagasic cardiomyopathy (CCC) including parasite persistence (Golgher and Gazzinelli, 2004), micro- vascular dysfunction (Petkova et al. 2001), and imbalanced immune response with autoimmune impli- cations (Leon and Engman, 2003). The sudden cardiac death is one of the most common terminal events in Chagas’ disease with these patients usually presenting complex and sustained ventricular arrhythmia on ambulatory electrocardio- gram (ECG) (Maguire et al. 1987). The most frequent chagasic electrocardiographic abnormalities include (i) frequent sinus node dysfunction, (ii) high rate of involvement of the conduction system producing ECG alterations of the right bundle branch or left anterior hemiblock or both, (iii) high rate of ventricular arrhythmias, (iv) high frequency of atrioventricular block, (v) abnormal Q waves, ST- segment and T-waves and (vi) autonomic dysfunction (Salles et al. 2003; Garcia et al. 2005). It has been reported that most patients that present CCC display altered ECG (Rocha et al. 2003). In a recent study it has been demonstrated that patients with positive serology for T. cruzi and active infection, but with normal left ventricular cineangiography, showed 100% survival at 16 years follow-up, while the 62 De Souza et al Drug Target Insights 2007: 2 presence of apical aneurysm or left ventricular systolic dysfunction was along with 60% of mortality at 4 years follow-up (Dávila-Spinetti et al. 2005). The available chemotherapy against T.cruzi based on nitrofurans (nifurtimox–Bayer) and nitro- imidazoles (benznidazole–Roche) is unsatisfactory since both compounds are effective only against recent infections and have frequent toxic side effects besides the occurrence of drug resistance (reviewed in Coura and De Castro, 2002). Recent reviews clearly point to the need of fi nding more efficient and less toxic drugs. In this respect, aromatic diamidines represent a promising class of DNA-targeted anti-parasitic agents (reviewed in Soeiro et al. 2005; Wilson et al. 2005; Werbovetz, 2006). We have recently reported the high in vitro trypanocidal activity of DB569, a N-phenyl- substituted analogue of furamidine (De Souza et al. 2004). We also found that the treatment of acutely infected mice with DB569 signifi cantly reduced the cardiac parasitism and partially increased the survival rates (De Souza et al. 2006a). Since ECG alterations have also been reported in T.cruzi-infected mice (Bustamante et al. 2003, 2005; Zaidenberg et al. 2006) our aim in the present study was to evaluate the potential protection of DB569 in the development of cardiac alterations in both acutely and chronically T. cruzi infected mice. Materials and Methods Parasites Bloodstream trypomastigotes of the Y strain were harvested by heart puncture from T. cruzi-infected Swiss mice at the parasitaemia peak, as described previously (Meirelles et al. 1982). In vivo infection Male Swiss mice (25–30 g) were obtained from the Fundação Oswaldo Cruz (FIOCRUZ) animal facilities (Rio de Janeiro, Brazil). Mice were housed at maximum 8 per cage and kept in a conventional room at 20–24 °C under a 12/12-h light/dark cycle. The animals were provided with sterilized water and chow ad libitum. Infection was performed by intraperitoneal (i.p.) injection of 104 bloodstream trypomastigotes, and age-matched uninfected mice were maintained under identical conditions. All assays were performed 3 to 4 times and all procedures were carried out in accordance with the guidelines established by the FIOCRUZ Committee of Ethics for the Use of Animals (CEUA 0099/01). Experimental groups The animals were divided into the following groups: uninfected (non-infected and non-treated); untreated (infected with T. cruzi but non-treated); and treated (infected and treated with 20 mg/kg DB569). For analyses in the acute infection, at least 6 mice from each group were used at each different day post infection (dpi) until the 21st dpi. After one-year post infection, the surviving T. cruzi- infected animals were used as the chronic mice group. Drugs and treatment schedule The synthesis of DB569 has been reported previ- ously (Lansiaux et al. 2002). Stock solution (200 mg) of the diamidine was prepared in dimeth- ylsulfoxide and mice received 0.1 mL i.p. injection of 20 mg/kg/day DB569, starting at 3 dpi for 10 consecutive days as described (De Souza et al. 2006a). The infected and untreated mice group only received 0.1mL i.p. daily injection of phosphate- buffered saline (10 mM sodium phosphate, 0.015 M NaCl, pH 7.4) (PBS) as vehicle. Parasitaemia and mortality Parasitaemia was individually checked by direct microscopic counting of parasites in 5 µL of blood, as described before (De Souza et al. 2006a). Mortality was checked daily until 30 dpi and expressed as percentage of cumulative mortality (%CM) (De Souza et al. 2006a). Histopathological analysis At 8, 15, 21 dpi and after one year of infection, hearts were removed, cut longitudinally, rinsed in ice-cold PBS and fi xed in Millonig-Rosman solu- tion (10% formaldehyde in phosphate-buffered saline). The tissues were then dehydrated and embedded in paraffi n. Sections (3 µm) stained by routine hematoxylin-eosin (HE) were analyzed by light microscopy. The number of amastigote nests and of inflammatory infiltrates (more than 10 mononuclear cells) was determined in at least 30 fi elds (total magnifi cation, 40X) for each slide. The 63 Electrocardiographic Findings in T. cruzi-infected Mice Treated by DB569 Drug Target Insights 2007: 2 mean number of amastigotes’ nests or infl ammatory infi ltrates per fi eld was obtained from at least three mice per group with three sections from each mouse. Flow Cytometry analysis of cardiac CD4+ and CD8+ T cells To evaluate the levels of cardiac CD8+ and CD4+ T cells, three mice of each experimental group were sacrifi ced at the peak of cardiac infl ammation (15 dpi) (De Souza et al. 2006a), the hearts quickly removed and their infl ammatory cells isolated for fl ow cytometry analysis as reported, with minor modifi cations (Marino et al. 2004). Briefl y, to isolate mononuclear cells invading the cardiac tissue, hearts were fragmented (1 to 2 mm) and submitted to seven enzymatic dissociations employing 0.2% collagenase. The recovered cells were washed in PBS, fi xed for 20 min with 1% parafomaldehyde and labeled with phycoerythrin (PE)-conjugated anti-CD4 and Cy-Chrome- conjugated anti-CD8 mAb (Pharmingen/BD Biosciences, San Diego, CA). Negative controls for specifi c labeling were prepared from isotype- matched mAbs (Pharmingen/BD Biosciences). A total of 10,000 events were acquired using a FACSCalibur fl ow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with Cell Quest software. The data analysis was performed in the WinMDI software (Multiple Document Interface Flow Cytometry Application, v2.8, by Joseph Trotter, Scripps Research Institute, San Diego, CA, USA). Electrocardiography ECG recording and analysis were performed in uninfected, acutely (8, 15, 21 dpi) and chronically (after 1 year post infection) T. cruzi-infected mice submitted or not to the DB569 therapy, as reported (Garcia et al. 2005; Zeidenberg et al. 2006). Briefl y, mice were placed under stable sedation with diaz- epan (20 mg/kg, i.p.), fi xed in the supine position, and eight-lead ECGs were recorded from 18-gauge needle electrodes subcutaneously implanted in each limb and two electrodes at precordial posi- tions lead II. The electrocardiographic tracing was obtained with standard lead (dipolar lead DII), recording with amplitude set to give 2 mV/1s. ECGs were recorded by using a band-pass fi ltering (Bio Amp—AD Instruments, Hastings, United Kingdom) between 0.1 and 100 Hz. Supplementary amplifi cation and analog-digital conversion was performed with a Powerlab 16S instrument (AD Instruments, Hastings). Digital recordings (16 bit, 4 kHz/channel) were analyzed with the Scope (version v3.6.10) program (AD Instruments). The signal-averaged ECG (SAECG) was calculated by using the mouse SAECG extension (version 1.2) program (AD Instruments) and a template- matching algorithm. ECG parameters were evalu- ated in the acute and chronic chagasic mice by using the following standard criteria: (i) the heart rate was monitored by beats/minute (bpm), and (ii) the variation at P wave and PQ, QRS and QT intervals were measured at milliseconds (ms). Statistical analysis Statistical analysis was carried out using the vari- ance (ANOVA), with the level of signifi cance set at p ≤ 0.05. The data are representative of 2–4 experiments run in duplicate. Results DB569 (Fig. 1) is effective towards both the intra- cellular and trypomastigote forms of T. cruzi, presenting IC50 values at low-micromolar doses in vitro (De Souza et al. 2004). This furamidine analogue also decreases mouse mortality and the levels of alanine aminotransferase and creatinine, indicating a protective role respectively against hepatic and renal lesions caused by the parasite infection (De Souza et al. 2006a). Since it signifi - cantly decreases cardiac parasitism to a level similar to that found for benznidazole treatment (De Souza et al. 2006a), we decided to investigate the potential protection of DB569 for the develop- ment of cardiac electrophysiologic abnormalities during acute and chronic T. cruzi infection. We confi rmed our previous data showing that while the diamidine therapy slightly reduces the parasi- taemia levels (Fig. 2A), DB569 significantly controls the cardiac parasitism, reducing in 95% the number of parasite nests (p ≤ 0.049) at the 15th dpi (Fig. 2B and G), as compared to untreated animals (Fig. 2B and F). In addition, the fl ow cytometry analysis revealed that DB569 decreased in 40% the frequency of cardiac infi ltrating CD8+ T cells (Fig. 2E), as compared to the untreated animals (Fig. 2D). However, histological analysis of heart samples at the 15th dpi, which represent the peak of both cardiac parasite load as well as infl ammation in our experimental model (De Souza 64 De Souza et al Drug Target Insights 2007: 2 et al. 2006a), showed non-signifi cant reduction (p ≤ 0.5) of the infl ammation (Fig. 2C, H–I). Inter- estingly, the scarce parasite’ nests found in heart tissues of drug-treated mice (Fig. 2G, asterisks) were frequently associated to infl ammatory cells (Fig. 2G, arrow). Next we analyzed the electrocar- diogram profiles in uninfected, untreated and DB569-treated mice (Fig. 3A). No differences among the groups were observed at the 8th dpi (Fig. 3A–B). However, at 15 and 21 dpi, we noticed sinus bradycardia as the prevailing ECG disorder found in untreated mice as compared to uninfected group, as detected by low heart rates (Fig. 3A,C–D). While uninfected mice exhibited values ranging from 630 to 670 beats/min (bpm), untreated and DB569-treated animals, displayed decreased heart frequencies, reaching 496 and 501 bpm, respec- tively (Fig. 3A and C). Interestingly, although decreased cardiac frequencies were maintained in the untreated mice at the 21st dpi, DB569- treatment reverted (p ≤ 0.01) this ECG alteration, reaching values similar to the counterpart- uninfected group (Fig. 3A and D). As diamidine treated group showed 40% reduction of the mortality compared to non-treated mice (Fig. 3E), we further investigated if the protection exerted by DB569 during the acute infection would be maintained in the chronic phase. We again observed lower cardiac frequency as the prevailing electrocardiographic change found in the chronic untreated group. We also found that DB569- treated mice reverted the cardiac alterations found in untreated animals (p ≤ 0.01), showing ECG profi les similar to uninfected mice and indicating that they sustained normal ECG profi les (Fig. 4A–B) as recovered at the 21st dpi in the acutely-infected mice. Discussion The lack of an effective therapy for Chagas’ disease associated to the toxicity and side effects of the available drugs (nifurtimox and beznidazole) justify the investigation of different natural as well as synthetic compounds that could substitute the current chemotherapy (Coura and de Castro, 2002). The well-known activity of aromatic diamidines towards many pathogens in both in vitro and in vivo studies (reviewed in Soeiro et al. 2005) prompted us to verify the effect of furamidine and its N-phenyl-substitute analogue, DB569. Moreover, the superior activity of DB569 as compared to furamidine even after short periods of in vitro treat- ment, without exerting toxicity towards mammalian cells (De Souza et al. 2004), impelled further analysis of the DB569 activity during T. cruzi infec- tion in vivo (De Souza et al. 2006a). As electrocar- diographic studies give important information regarding aspects of cardiac function, representing a useful tool to measure the severity and prognosis of the chagasic cardiopathy (Salles et al. 2003), here we sought to evaluate the potential protection of Figure 1. Structure of the DB569. 65 Electrocardiographic Findings in T. cruzi-infected Mice Treated by DB569 Drug Target Insights 2007: 2 Figure 2. Treatment of T. cruzi-infected mice (104 Y strain/mice) with DB569 from 3 to 12 dpi: (A) Kinetics of parasitaemia, (♦) Untreated (infected and non-treated) and (■) DB-treated (infected and treated with 20 mg/kg/day DB569); (B) Number of amastigote’ nests and (C) Infl amatory infi ltration within the heart of untreated and DB-treated groups at 15th dpi; Cardiac CD4+ and CD8+ T cells expression measured by fl ow cytometry analysis of (D) untreated and (E) DB-treated T. cruzi-infected mice at 15th dpi. The lymphocyte gate was determined ac- cording to expression of CD4+ and CD8+ in FSC × SSC dot plot in infected animals; Histological patterns in heart sections at 15th dpi (F-I) of untreated (F and H) and DB-treated (G and I) T. cruzi-infected mice. Note the presence of parasite nests (F and G, asterisks) and the intensity of the infl ammatory process (F-I, arrow). Bar = 50 µM. p indicate signifi cant differences between DB-treated and untreated groups. r means the ratio between CD4+ and CD8+ cardiac cells). 66 De Souza et al Drug Target Insights 2007: 2 Figure 3. Electrocardiographic fi nding during acute phase of T. cruzi-infection, were verifi ed by the variation at heart rate (beats/min) and P wave and PQ, QRS and QT intervals (ms) at uninfected (white), untreated (black) and DB-treated (gray) groups: (A) Electrocardiographic tracing at 8°dpi, 15°dpi and 21°dpi of all groups; Electrocardiogram values at 8°dpi (B), 15°dpi (C) and at 21°dpi (D). Cumulative mortality curve of untreated (♦) and DB-treated (■) T. cruzi-infected groups accompanied until 30 dpi (E). Asterisks indicate signifi cant differences of DB569-treated in relation to the untreated group for electrocardiographic fi nding, p ≤ 0.01. Untreated 0 2 -1 0 2 -1 0 2 -1 100 200 400 600 800 Treated 0 2 -1 Uninfected 0 2 -1 0 2 -1 0 2 -1 100 200 400 600 800 8°dpi 15°dpi 21°dpi A 0 100 200 300 400 500 600 700 800 P PQ QRS QT freq 8 days pos-infection uninfected untreated DB-20mg/kg 0 100 200 300 400 500 600 700 800 P PQ QRS QT freq 15 days pos-infection uninfected untreated DB-20mg/kg B C D 0 100 200 300 400 500 600 700 800 P PQ QRS QT freq 21 days pos-infection uninfected untreated DB-20mg/kg * 0 10 20 30 40 50 60 70 80 90 100 0 6 12 18 24 30 Dpi % untreated treated E 67 Electrocardiographic Findings in T. cruzi-infected Mice Treated by DB569 Drug Target Insights 2007: 2 0 2 -1 0 2 -1 0 2 -1 100 200 400 600 800 1 year pos-infection Uninfected Untreated Treated A 0 100 200 300 400 500 600 700 800 P PQ QRS QT f req 1 year pos-infection Uninf ected Untreated DB-20mg/kg B * Figure 4. Electrocardiographic fi nding at chronic phase after one year of T. cruzi infection were verifi ed by the variation at heart rate (beats/ min) and P wave and PQ, QRS and QT intervals (ms) at uninfected (white), untreated (black) and DB-treated (gray) groups: (A) electrocar- diographic tracing and (B) electrocardiographic parameters of all groups. Asterisks indicate signifi cant differences of DB569-treated in relation to the untreated group, p ≤ 0.01. DB569 in the development of ECG alterations in both acute and chronic murine T. cruzi infection. As previously reported, our data confi rmed that T. cruzi-infected mouse represents an interesting model for analyzing ECG alterations due to para- site infection, displaying electrocardiographic abnormalities (Rivarola et al. 2005; Zaidenberg et al. 2006). In our present experimental model, these abnormalities were mostly related to a remarkable decrease of the cardiac frequency, as also reported by others (Bustamante et al. 2005). Our results showed that DB569 considerably reduced the ECG alterations in both acutely and chronically T. cruzi-infected mice similarly as described in other studies performed with different drugs (Lo Presti et al. 2004; Rivarola et al. 2005; Garcia et al. 2005; Zaidenberg et al. 2006). Interestingly, the DB569 protection was directly related to the levels of cardiac cytotoxic T cells, since the treatment also decreased the levels of CD8+ cells found in the infl amed heart and partially recovered the CD4+/CD8+ ratio that was 0.37 and 0.50 in untreated mice and DB569 treated mice, respectively. This is an interesting fi nding since it 68 De Souza et al Drug Target Insights 2007: 2 has been reported that in acutely and chronically T. cruzi-infected patients and during the murine infection, cardiac CD4+/CD8+ T cell ratio shows a predominance of CD8+ T cells suggestive of an immunologic imbalance response (Higuchi et al. 1997; Marino et al. 2004; Fuenmayor et al. 2005; Henriques-Pons et al. 2005; Cunha-Neto et al. 2006). The parasitism control and decreased CD8+ T cells levels in the myocardium of DB569-treated group was concomitantly found with an unaltered ECG profi le that might be ultimately contributing to their increased survival rates. This therapeutic effect of DB569 on the bradyarrhythmia could be related to trypanocidal effect of the diamidine and/or the modulation of the host immunological response to the parasite. In fact, the regulation of immune response in T. cruzi-infected mice by other trypanocidal drugs, such as benznidazole, has already been reported (Olivieri et al. 2006; Romanha et al. 2002), and our previous in vitro data showed that parasite elimination was more effective after treatment of infected phagocytic cells as compared to cardiac ones (De Souza et al. 2004). Additionally, as DB569 induces apoptosis in T. cruzi (De Souza et al. 2006b), the presence of apoptotic parasites in cardiac tissues could contribute for regulation of the host infl ammatory orchestra (Fadok et al. 2000; DosReis et al. 2005). Although the mechanisms leading to chronic chagasic cardiomyopathy (CCC) are still unknown, the initial cardiac damage caused by the parasite seems to play a critical role for the progression of the disease (Tarleton, 2001). Moreover, specifi c trypanocidal treatment seems to be associated with delay in human heart disease progression (Viotti et al. 2006). In addition, although accumulated data highlighted the concept that tissue damage is direct or indirectly induced by T. cruzi itself and thus the control of parasite load by a host effective infl am- matory response could represent a prerequisite to arrest the evolution of the disease, an excessive or imbalanced infl ammation (as an auto reactivity response) can also contribute to the genesis of the cardiac damage (reviewed in Dutra et al. 2005). In fact, chronic chagasic patients submitted to benz- nidazole therapy although not parasitologically cured, presented a marked reduction in the occur- rence of ECG changes and a lower frequency of deterioration in their clinical conditions (Viotti et al. 1994). Then, despite the genesis of CCC, the current concept suggests that parasite eradication is a requisite to arrest the evolution of the Chagas disease pointing to the urgent need for the discovery and development of safe and effective therapy for the chagasic patients. In this context, our results suggest that diamidines are a potential class of compounds to be employed further in vitro and in vivo studies for Chagas’ disease treatment. Acknowledgments MNCS and EMDS would like to thank Conselho Nacional Desenvolvimento Científi co e Tecnoló- gico (CNPq); Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and PAPES IV/FIOCRUZ for fi nancial support. The authors thank Dr. Vinicius Cotta- de-Almeida (LBC/IOC/FIOCRUZ) for the careful revision. The authors express special appreciation to Dr. David Boykin (Department of Chemistry, Georgia State University, Atlanta, USA) for manu- script revision and for supplying the DB569. References Armenti, A. 2006. Long-term cardiac outcomes of treating chronic Chagas disease with benznidazole versus no treatment: a nonrandomized trial. Ann. Intern. Med., 144:724–34. Bustamante, J.M., Rivarola, H.W. and Fretes, R. et al. 2005. Weekly elec- trocardiographic pattern in mice infected with two different Trypano- soma cruzi strains. Int. J. Cardiol., 102:211–7. Bustamante, J.M., Rivarola, H.W. and Fernandez, A.R. et al. 2003. Try- panosoma cruzi reinfections provoke synergistic effect and cardiac beta-adrenergic receptors’ dysfunction in the acute phase of experi- mental Chagas’ disease. Exp. Parasitol., 103:136–42. Coura, R.J. and De Castro, S.L. 2002. A critical review on Chagas’ disease chemotherapy. Mem. Inst. Oswaldo. Cruz., 97:3–24. Dávila-Spinetti, D.F., Colmenarez-Mendoza, H.J. and Lobo-Vielma, L. 2005. Mechanisms responsible for myocardial damage progression in Chronic Chagas disease. Rev. Esp. Cardiol., 58:1007–09. De Souza, E.M., Lansiaux, A. and Bailly, C. et al. 2004. Phenyl substitution of furamidine markedly potentiates its anti-parasitic activity against Trypanosoma cruzi and Leishmania amazonensis. Biochem. Phar- macol., 68:593–600. De Souza, E.M., Oliveira, G.M. and Boykin, D.W. et al. 2006a. Trypano- cidal activity of the phenyl-substituted analogue of furamidine DB569 against Trypanosoma cruzi infection in vivo. J. Antimicrob Che- mother, 58:610–4. De Souza, E.M., Menna-Barreto, R. and Araujo-Jorge, T.C. et al. 2006b. Antiparasitic activity of aromatic diamidines is related to apoptosis- like death in Trypanosoma cruzi. Parasitology, 133:75–9. DosReis, G.A., Freire-de-Lima, C.G. and Nunes, M.P. et al. 2005. The importance of aberrant T-cell responses in Chagas disease. Trends Parasitol, 21:237–43. Dutra, W.O., Rocha, M.O.C. and Teixeira, M.M. 2005. The clinical im- munology of human Chagas disease. Trends in Parasitology, 21:581–7. Cunha-Neto, E., Bilate, A.M. and Kenneth, V. et al. 2006. Induction of cardiac autoimmunity in Chagas heart disease: A case for molecular mimicry. Autoimmunity, 39:41–54. 69 Electrocardiographic Findings in T. cruzi-infected Mice Treated by DB569 Drug Target Insights 2007: 2 Fadok, V.A., Bratton, D.L. and Rose, D.M. et al. 2000. A receptor for phospha- tidylserine-specifi c clearance of apoptotic cells. Nature, 405:85–90. Fuenmayor, C., Higuchi, M.L. and Carrasco, H. et al. 2005. Acute Chagas’ disease: immunohistochemical characteristics of T-cell infi ltrate and its relationship with T. cruzi parasitic antigens. Acta. Cardiol., 60:33–7. Garcia, S., Ramos, C.O. and Senra, J.F. et al. 2005. Treatment with benz- nidazole during the chronic phase of experimental Chagas’ disease decreases cardiac alterations. Antimicrob Agents Chemother., 49:1521–8. Golgher, D. and Gazzinelli, R.T. 2004. Innate and acquired immunity in the pathogenesis of Chagas disease. Autoimmunity, 37:399–409. Henriques-Pons, A., Olivieri, B.P. and Oliveira, G.M. et al. 2005. Experi- mental infection with Trypanosoma cruzi increases the population of CD8(+), but not CD4(+), immunoglobulin G Fc receptor-positive T lymphocytes Infect. Immun., 73:5048–52. Higuchi, M.D., Ries, M.M. and Aiello, V.D. et al. 1997. Association of an increase in CD8+ T cells with the presence of Trypanosoma cruzi antigens in chronic, human, chagasic myocarditis. Am. J. Trop. Med. Hyg., 56:485–9. Lansiaux, A.F., Tanious, Z. and Mishal, L. et al. 2002. Distribution of fur- amidine analogues in tumor cells: targeting of the nucleus or mito- chondria depending on the amidine substitution. Cancer Res., 62:7219–29. Leon, J.S. and Engman, D.M. 2003. The signifi cance of autoimmunity in the pathogenesis of Chagas heart disease. Front Biosci., 8:315–22. Lo Presti, M.S., Rivarola, H.W. and Bustamante, J.M. et al. 2004. Thio- ridazine treatment prevents cardiopathy in Trypanosoma cruzi in- fected mice. Int. J. Antimicrob Agents, 23:634–6. Maguire, J.H., Hoff, R. and Sherlock, I. et al. 1987. Cardiac morbidity and mortality due to Chagas’ disease: prospective electrocardiographic study of a Brazilian community. Circulation, 75:1140–5. Marin-Neto, J.A., Simoes, M.V. and Sarabanda, A.V. 1999. Chagas’ heart disease. Arq. Bras. Cardiol., 72:247–80. Marino, A.P., da Silva, A. and dos Santos, P. et al. 2004. Regulated on activation, normal T cell expressed and secreted (RANTES) antago- nist (Met-RANTES) controls the early phase of Trypanosoma cruzi- elicited myocarditis Circulation, 110:1443–9. Meirelles, M.N., Chiari, E. and de Souza, W. 1982. Interaction of blood- stream, tissue culture-derived and axenic culture-derived trypomas- tigotes of Trypanosoma cruzi with macrophages. Acta Trop, 39:195–203. Olivieri, B.P., Cotta-De-Almeida, V. and Araujo-Jorge, T. 2002. Benznida- zole treatment following acute Trypanosoma cruzi infection triggers CD8+ T-cell expansion and promotes resistance to reinfection. Antimicrob Agents Chemother, 46:3790–6. Olivieri, B.P., Farias-De-Oliveira, D.A. and Araujo-Jorge, T.C. et al. 2005. Benznidazole therapy in Trypanosoma cruzi-infected mice blocks thymic involution and apoptosis of CD4+CD8+ double-positive thymocytes. Antimicrob Agents Chemother, 49:1981–7. Olivieri, B.P., de Souza, A.P. and Cotta-de-Almeida, V. et al. 2006. Try- panosoma cruzi: Alteration in the lymphoid compartments following interruption of infection by early acute benznidazole therapy in mice. Exp. Parasitol., 114:228–34. Petkova, S.B., Huang, H. and Factor, S.M. et al. 2001. The role of endo- thelin in the pathogenesis of Chagas’ disease. Int. J. Parasitol., 31: 499–511. Rivarola, H.W., Bustamante, J.M. and Lo Presti, S. et al. 2005. Trypano- soma cruzi: chemotherapeutic effects of clomipramine in mice infected with an isolate obtained from an endemic area. Exp. Para- sitol., 111:80–6. Rocha, M.O., Ribeiro, A.L. and Teixeira, M.M. 2003. Clinical management of chronic Chagas cardiomyopathy. Front Biosci., 8:44–54. Romanha, A.J., Alves, R.O. and Murta, S.M. et al. 2002. Experimental chemotherapy against Trypanosoma cruzi infection: essential role of endogenous interferon-gamma in mediating parasitologic cure. J. Infect. Dis., 186:823–8. Salles, G., Xavier, S. and Sousa, A. et al. 2003. Prognostic value of QT interval parameters for mortality risk stratifi cation in Chagas’ disease: results of a long-term follow-up study. Circulation, 108:305–12. Soeiro, M.N., De Souza, E.M. and Stephens, C.E. et al. 2005. Aromatic diamidines as antiparasitic agents. Expert Opin. Investig. Drugs, 14:957–72. Tarleton, R.L. 2001. Parasite persistence in the aetiology of Chagas disease Int. J. Parasitol., 31:550–4. Viotti, R., Vigliano, C. and Armenti, H. et al. 1994.Treatment of chronic Chagas’ disease with benznidazole: clinical and serologic evolution of patients with long-term follow-up. Am. Heart J., 127:151–62. Viotti, R., Vigliano, C. and Lococo, B. et al. 2006. Long-term cardiac outcomes of treating chronic Chagas disease with benznidazole versus no treatment: a nonrandomized trial. Ann. Intern. Med., 144:724–34. Werbovetz, K. 2006. Diamidines as antitrypanosomal, antileishmanial and antimalarial agents. Curr. Opin. Investig. Drugs., 7:147–57. Wilson, W.D., Nguyen, B. and Tanious, F.A. et al. 2005. Dications that target the DNA minor groove: compound design and preparation, DNA interactions, cellular distribution and biological activity. Curr. Med. Chem. Anti. Cancer Agents, 5:389–408. World Health Organization. 2002. Control of Chagas disease. WHO Tech- nical Report Series 905:109. Zaidenberg, A., Luong, T. and Lirussi, D. et al. 2006. Treatment of experi- mental chronic Chagas disease with trifl uralin. Basic Clin. Pharma- col. Toxicol., 98:351–6.