Trypanosoma cruzi-Derived Neurotrophic Factor: Role in Neural Repair and Neuroprotection

Marina V. Chuenkova^* and Mercio PereiraPerrin

Department of Pathology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA

*Corresponding Author:

Marina V. Chuenkova
Department of Pathology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA
E-mail: marina.chuenkova@tufts.edu

Received Date: 3 June 2010; Revised Date: 8 July 2010; Accepted Date: 26 July 2010

Visit for more related articles at Journal of Neuroinfectious Diseases

Abstract

Some patients infected with the parasite Try-panosoma cruzi develop chronic Chagas’ disease, whileothers remain asymptomatic for life. Although pathological mechanisms that govern disease progression remain unclear, the balance between degeneration and regeneration in the peripheral nervous system seems to contribute to the different clinical outcomes. This review focuses on certain new aspects of host-parasite interactions related to regeneration in the host nervous system induced by the trans-sialidase of T. cruzi, also known as a parasite-derived neurotrophic factor (PDNF). PDNF plays multiple roles in T. cruzi infection, ranging from immunosuppression to functional mimicry of mammalian neurotrophic factors and inhibition of apoptosis. PDNF affinity to neurotrophin Trk receptors provide sustained activation of cellular survival mechanisms resulting in neuroprotection and neuronal repair, resistance to cytotoxic insults and enhancement of neuritogenesis. Such unique PDNF-elicited regenerative responses likely prolong parasite persistence in infected tissues while reducing neuropathology in Chagas’ disease.

Keywords

chagas disease; Trypanosoma cruzi; peripheral nervous system; parasite-derived neurotrophic factor

Introduction

Despite recent progress in the control of Chagas’ disease, it is estimated that approximately 8 million people remain chronically infected with Trypanosoma cruzi, the etiological agent of the disease. Many of these infected individuals (30– 40%) will develop life-threatening heart or gastrointestinal pathology. Given that an additional 20% of the population is at risk of infection in endemic countries [57], Chagas’ disease (CD) remains a significant health problem in Latin America.

T. cruzi is transmitted to humans by hematophagousinsects of the Reduviidae family through the deposition of feces at bite sites. In addition, nonvectorial routes like blood transfusion, as well as mother-infant and oral ingestion are also important, particularly in nonendemic areas [57].

The initial infection causes transient flu-like symptoms and thus often escapes medical attention. Young children rarely (< 5%) develop severe inflammation of the heart or central nervous system (CNS). Survivors of the acute disease do not develop clinical symptoms for many years, and approximately 60–70% of them remain in the chronic indeterminate (asymptomatic) stage of the disease for life, while the other 30–40% will exhibit chronic symptomatic CD affecting peripheral nervous system (PNS) in the heart and digestive tract several years or even decades after the initial infection [6, 66].

Symptomatic CD can be fatal, but a vaccine for prevent-ing T. cruzi infection has not been developed yet, and try-panocidal drugs (benznidazole and nifurtimox) are effective only in acute stage and can cause serious side effects [72].

The reason for a long latent period between initial infection and clinical manifestations, and high proportion of asymptomatic patients, is not known; there is also no explanation for the absence of chronic disease in CNS. Analysis of T. cruzi interaction with CNS and PNS in the heart and gastrointestinal tract (GI) can provide some clues for the understanding of CD progression from benign to pathological form and ideas for possible therapeutic intervention.

Neuropathology in Chagas’ disease

Molecular mechanisms underlying pathogenesis in CD remain unclear. It is thought that direct cell parasitism, acute inflammation, autoimmune reactions, or neuronal damage are primary causes of disease pathogenesis [22, 37, 67]. The neurogenic hypothesis states that the severe neuronal loss in the heart and GI of Chagasic patients defines the transition from asymptomatic to symptomatic CD. The foundation to neurogenic theory was laid by the pioneering work of Koberle¨ who detected striking neuronal depopulation in autonomous nervous system of CD patients with cardiomyopathy, megaesophagus, and megacolon [35]. Multiple pathological studies in humans and experimental animals have confirmed this finding by demonstrating extensive destruction of cardiac parasympathetic ganglia [9, 22, 23, 34, 47, 58], which is thought to allow unopposed sympathetic activation resulting in progressive myocardial damage [22, 23]. However, lesions in sympathetic postganglionic fibers in the sinus node and the myocardium have also been detected in patients with cardiac form of CD and in experimental animals [34, 59] and it was shown that reduction in both cholinergic and noradrenergic cardiac nerves paralleled development of the acute myocarditis at the end of the acute phase of experimental CD [47, 58].

In Chagasic GI disease, neuronal destruction is even more drastic [48]. Loss of 50 to 90% of nerve fibers in myenteric and submucosal plexuses and reduction in enteric glial cell population correspond to striking luminal enlargement and muscular hypertrophy of the esophagus and colon [20, 36, 46, 53]. The aperistalsis thus developed leads to organ obstruction and stagnated food passage resulting in megasyndromes (megaesophagus and megacolon) and weight loss [49].

In contrast to the prominent damage of the PNS, T. cruzi invades CNS without noticeable deleterious effects. Trypo-mastigotes are frequently found in the cerebrospinal fluid, brain, and spinal cord in the absence of neurological symp-toms [32, 56], unless patients are severely immunocompro-mised [26]. In experimental CD, even in younger animals, which are usually very susceptible to T. cruzi infection, CNS is largely preserved regardless of the parasite load, presence of amastigote nests inside astrocytes, and strong inflamma-tory response [9, 10, 45]. Such silent infection is contrary to that in the heart, colon, and esophagus, and it does not produce chronic symptoms [56]. The molecular mechanisms underlying opposite effects of T. cruzi invasion of CNS and PNS remain unresolved.

PNS is most vulnerable to damage in the acute stage of the infection characterized by high parasitemia and tissue parasitism. Parasites found in neuronal ganglia, Schwann cells, and enteric glia [47, 73] destroy cells directly and induce cytotoxic immune responses, mediated by NO production and oxidative burst resulting in myocardial damage and lesions in enteric nervous plexuses [55, 64].

Although acute disease symptoms could in some cases be dangerous, they normally resolve, and Chagasic individ-uals advance to an indeterminate, asymptomatic stage, with many of them showing normal electrocardiogram and X-rays of the heart, esophagus, and colon [20, 53, 70]. In fact, it was demonstrated that the rate of age-related neuronal degeneration in the colon and heart of Chagasic patients was decreased compared to that of noninfected age-matched individuals [35], suggesting enhanced neuronal survival. In experimental CD functional improvement in the heart and colon was associated with reinnervation of muscle fibers, collateral sproutings of damaged nerves, and axonal regrowth, and with an increase in the number of enteric glia [43, 45, 53]. For most of infected patients the extent of recovery is such that they remain free of symptoms for the rest of their lives, despite retaining pathogenic T. cruzi. It is thus likely that neuroregeneration is involved in the mechanisms that prevent manifestations of chronic disease.

It is also possible that T. cruzi infection, traditionally viewed as an entirely detrimental process to the host, can elicit specific reparative/survival responses in neuronal tis-sues. This possibility received experimental support with the findings with the T. cruzi trans-sialidase.

T. cruzi trans-sialidase/parasite-derived neurotrophicfactor (PDNF)

Catalytic and immunologic properties

A T. cruzi surface antigen, trans-sialidase/PDNF, plays mul-tiple roles in parasite invasion of mammalian host. As an enzyme it catalyzes the transfer of α-2,3-linked sialic acid to terminal β-Gal residues [54, 62], which can protect blood-stream trypomastigotes from complement lysis [7] and pro-mote parasite invasion [50, 68].

PDNF is attached to trypomastigote surface by GPI anchor and is copiously shed as a soluble factor into the extracellular space and bloodstream [1, 60, 63]. Soluble PDNF was shown to remodel surface of immune cells and augment T. cruzi immunosuppresion in the acute phase of CD by inhibiting CD8+ lymphocytes cytotoxicity and promoting apoptosis of T cells [29, 51].

PDNF consists of N-terminal catalytic domain, con-nected through a lectin-like region to a C-terminus with variable number of 12 amino acids repeats in tandem (long tandem repeat, LTR) [8], also called SAPA (shed acute phase antigen) [28]. LTR/SAPA is not required for sialic acid transfer, but it is highly immunogenic and contributes to parasite immune evasion by inducing abnormal polyclonal B cell activation and nonspecific Ig secretion characteristic for the acute phase of Chagas’ disease [30, 31]; furthermore, it upregulates IL-6 production in various cell types [61]. Consistent with PDNF functioning as an immunosuppressor, heterologous expression of PDNF in Leishmania major enhanced parasite virulence [5].

However, a relatively small subset of TS/PDNF proteins has enzymatic activity—of 1400 PDNF gene family mem-bers, only 15, produced by invasive trypomastigotes, func-tion as trans-sialidases [4, 27]. Trypomastigotes also express PDNF molecules without catalytic activity, due to a single mutation of Tyr342 to His [19].

Unique subsets of PDNF molecules lacking catalytic activity and LTR/SAPA domain belong to intracellular amastigotes, while no TS was detected for insect epimastig-ote forms [4].

Such structural diversity among multiple members of TS/PDNF family possibly resulted in some of them exhibit-ing biological activities unrelated to the release and acquisi-tion of sialyl residues.

Interaction with Trk receptor tyrosine kinases

The discovery of PDNF specific affinity to Trk receptors of mammalian neurotrophins pointed to a new direction in T. cruzi research, suggesting a possible mechanism for theparasite involvement in regeneration of mammalian nervous tissue.

The development and maintenance of mammalian PNS and CNS critically depends on the neurotrophins (NTs) nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT3). NTs activate their respective Trk tyrosine kinase receptors TrkA, B, and C, and downstream pathways of PI3kinase/Akt kinase, PLCγ, and mitogen-activated protein kinases (MAPK) to control survival, axon, and dendrite outgrowth, plasticity and neurotransmission. As a result, they protect neurons from toxic insults and facilitate neural repair [33]. Deficit of endogenously generated NTs causes neuronal apoptosis and degeneration, and is implicated in stroke, trauma, and neurodegenerative conditions such as Parkinson, Alzheimer, and Huntington diseases, and amyotrophic lateral sclerosis [25].

Activation of Trk signaling and survival of neurons

Similar to neurotrophic factors, PDNF affinity to Trk receptors was shown to support DRG, hippocampal, and cerebellar neurons and a variety of neuronal cells through trophic deficiency, oxidative stress, and neurotoxic insults—pathological conditions that typically lead to neurodegeneration and apoptosis [11, 12, 14, 15, 75].

Resistance to apoptotic death induced by PDNF in neuronal cells is a direct result of activating Trk downstream signaling via PI3K/Akt and Ras/MAPK/Erk (Figure 1). Thus TrkA-dependent activation of Akt kinase by PDNF is followed by inhibition of pro-apoptotic GSK-3 kinase, upregulation of mitochondrial anti-apoptotic Bcl-2, and reduced ROS formation [12, 14], while induction of MAPK/Erk signaling prevented activation of caspase-3 and cleavage of caspase-3 substrate PARP (poly-ADP-ribose polymerase), a DNA repair enzyme [18]. This chain of events leads to enhanced neuronal survival and resistance to exogenous insults, such as neurotoxin MPTP that causes symptoms and pathology in nigrostriatal neurons analogous to Parkinson disease [14].

Sustained activation of Erk by PDNF also correlated with increased neuritogenesis in DRG neurons and differentiation of neuronal PC12 cells to sympathetic phenotype [13], underlined by activation of cAMP-response-element- (CRE-) binding protein CREB and CRE-dependent transcription [16, 17].

Recent data demonstrated that PDNF also mediates T. cruzi interaction with TrkC receptor. PDNF similar tonatural TrkC ligand NT3 induces activation of MAPK/Erk signaling and promotes differentiation and survival of neuronal and glial cells [71]. TrkC-expressing Schwann and enteric glial cells, which myelinate neurons in PNS and modulate neuronal homeostasis and neurotransmission in the gut [65, 76], undergo regeneration in the course of T. cruzi infection in humans and animals [21, 53]. ThusPDNF activation of TrkC widens the scope for possible role of T. cruzi in the repair of infected nervous tissues. It would be broadened even further by T. cruzi activation of TrkB [74]; however, live trypomastigotes and parasite-isolated PDNF did not recognize TrkB receptor in the conditions they bind TrkA and TrkC [71].

Induction of cholinergic and adrenergic phenotypes in neuronal cells

T. cruzi residence in PNS and affinity to TrkA and TrkCcould underlie such an important aspect of nervous tissue functional integrity as neurotransmitter release, which is imbalanced in acute CD [22, 38, 42, 45]. PDNF activation of TrkA and downstream Akt and Erk1/2 signaling increased expression of cholinergic locus genes, choline acetyltransferase (ChAT), and vesicular acetylcholine (ACh) transporter (VAChT)—two key components of ACh synthetic cascade that define cholinergic phenotype in neurons [2].

Such PDNF activity also stimulated acquisition of adrenergic phenotype in ventral mesenchephalic neurons and neuronal PC12 cells [17], mimicking NGF and other neurotrophic factors, which can restore both cholinergic and adrenergic neuronal populations in NS [41]. PDNF, via upregulation of MAPK/Erk and CRE-dependent transcription, induced activation of tyrosine hydroxylase (TH)—the rate-limiting enzyme in the biosynthesis of catecholamines, increasing TH-catalyzed conversion of tyrosine to dopamine precursor L-DOPA [17] (Figure 1).

Such PDNF activity could be instrumental in restoring ChAT expression, ACh and catecholamine levels in later stages of T. cruzi infection [23, 42, 44], and regeneration of sympathetic neurons.

Promotion of cell invasion

PDNF interaction with TrkA induced endocytosis of receptor-PDNF complexes [75], and T. cruzi successfully exploits this mechanism for cell invasion. Trypomastigotes enter neuronal cells, Schwann cells, and other cells in Trk-dependable manner, which in addition requires receptor-mediated signaling, as inhibition of Trk-dependent signal transduction abrogated parasite invasion, reduced parasite load and inflammatory responses, and attenuated experimental CD [24].

The dependence of T. cruzi invasion on intact TrkA-mediated signaling is in line with the other data describing signaling pathways downstream of Trk receptor tyrosine kinase 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 [52, 69], and PI3K-mediated signaling, which induced accumulation of membrane PIP3, mobilization of intracellular Ca2+ stores, and formation of parasitophorous vacuole [3]. Therefore, T. cruzi activation of TrkA tyrosine kinase prior to using it as a vehicle for cell entry is a powerful mechanism to provide conditions for efficient cell invasion.

Anti-infective anti-Trk receptor antibody

The importance of Trk receptors in T. cruzi invasion is fur-ther underlined by the discovery that patients with asymp-tomatic CD produce specific antibody against Trk receptors, which blocked T. cruzi cell invasion [39, 40]. Passive immu-nization of mice with these autoantibodies reduced para-sitemia and inflammation in the heart and protected from lethal T. cruzi infection [39]. Isolated from asymptomatic patients these autoantibodies might function as a defense mechanism to control T. cruzi invasion of nervous tissues.

Conclusion

Growth factors play important roles as intercellular signaling molecules throughout mammalian nervous system, taking part in numerous functions, including neuronal regeneration. As a parasite-derived growth factor, the T. cruzi TS/PDNF induces survival and resistance to cytotoxic stimuli in neuronal cells, possibly subserving neural repair and structural and functional recovery in the PNS of the heart and GI in Chagas’ disease. The realization that T. cruzi can promote along with destructive, regenerative processes in infected tissues offers a new framework for studies of molecular pathogenesis that may suggest future therapeutic opportunities to prevent progression from asymptomatic to pathological CD.

References

1. R. Agusti, A. S. Couto, O. E. Campetella, A. C. Frasch, and R. M. de Lederkremer, The trans-sialidase of Trypanosoma cruzi is anchored by two different lipids, Glycobiology, 7 (1997), pp. 731– 735.
2. N. Akpan, K. Caradonna, M. V. Chuenkova, and M. PereiraPerrin, Chagas’ disease parasite-derived neurotrophic factor activates cholinergic gene expression in neuronal PC12 cells, Brain Res, 1217 (2008), pp. 195–202.
3. L. O. Andrade and N. W. Andrews, The Trypanosoma cruzi-hostcell interplay: location, invasion, retention, Nat Rev Microbiol, 3 (2005), pp. 819–823.
4. J. A. Atwood, III, D. B. Weatherly, T. A. Minning, B. Bundy, C. Cavola, F. R. Opperdoes, R. Orlando, and R. L. Tarleton, The Trypanosoma cruzi proteome, Science, 309 (2005), pp. 473–476.
5. M. Belen Carrillo, W. Gao, M. Herrera, J. Alroy, J. B. Moore, S. M. Beverley, and M. A. Pereira, Heterologous expression of Trypanosoma cruzi trans-sialidase in Leishmania major enhances virulence, Infect Immun, 68 (2000), pp. 2728–2734.
6. A. Biolo, A. L. Ribeiro, and N. Clausell, Chagas cardiomyopathy– where do we stand after a hundred years?, Prog Cardiovasc Dis, 52 (2010), pp. 300–316.
7. C. A. Buscaglia, V. A. Campo, A. C. Frasch, and J. M. Di Noia, Trypanosoma cruzi surface mucins: host-dependent coat diversity, Nat Rev Microbiol, 4 (2006), pp. 229–236.
8. A. Buschiazzo, M. F. Amaya, M. L. Cremona, A. C. Frasch, and P. M. Alzari, The crystal structure and mode of action of transsialidase, a key enzyme in Trypanosoma cruzi pathogenesis, Mol Cell, 10 (2002), pp. 757–768.
9. E. R. Camargos, D. J. Franco, C. M. Garcia, A. P. Dutra, A. L. Teixeira, Jr., E. Chiari, and C. R. Machado, Infection with different Trypanosoma cruzi populations in rats: myocarditis, cardiac sympathetic denervation, and involvement of digestive organs, Am J Trop Med Hyg, 62 (2000), pp. 604–612.
10. K. Caradonna and M. PereiraPerrin, Preferential brain homing following intranasal administration of Trypanosoma cruzi, Infect Immun, 77 (2009), pp. 1349–1356.
11. M. V. Chuenkova, F. B. Furnari,W. K. Cavenee, andM. A. Pereira, Trypanosoma cruzi trans-sialidase: a potent and specific survival factor for human Schwann cells by means of phosphatidylinositol 3-kinase/Akt signaling, Proc Natl Acad Sci USA, 98 (2001), pp. 9936–9941.
12. M. V. Chuenkova and M. A. Pereira, A trypanosomal protein synergizes with the cytokines ciliary neurotrophic factor and leukemia inhibitory factor to prevent apoptosis of neuronal cells, Mol Biol Cell, 11 (2000), pp. 1487–1498.
13. _______, The T. cruzi trans-sialidase induces PC12 cell differentiation via MAPK/ERK pathway, Neuroreport, 12 (2001), pp. 3715– 3718.
14. _______, PDNF, a human parasite-derived mimic of neurotrophic factors, prevents caspase activation, free radical formation, and death of dopaminergic cells exposed to the Parkinsonism-inducing neurotoxin MPP+, Brain Res Mol Brain Res, 119 (2003), pp. 50– 61.
15. M. V. Chuenkova and M. PereiraPerrin, Chagas’ disease parasite promotes neuron survival and differentiation through TrkA nerve growth factor receptor, J Neurochem, 91 (2004), pp. 385–394.
16. _______, A synthetic peptide modeled on PDNF, Chagas’ disease parasite neurotrophic factor, promotes survival and differentiation of neuronal cells through TrkA receptor, Biochemistry, 44 (2005), pp. 15685–15694.
17. _______, Enhancement of tyrosine hydroxylase expression and activity by Trypanosoma cruzi parasite-derived neurotrophic factor, Brain Res, 1099 (2006), pp. 167–175.
18. K. K. Cole and J. R. Perez-Polo, Poly(ADP-ribose) polymerase inhibition prevents both apoptotic-like delayed neuronal death and necrosis after H2O2 injury, J Neurochem, 82 (2002), pp. 19–29.
19. M. L. Cremona, D. O. S´anchez, A. C. Frasch, and O. Campetella, A single tyrosine differentiates active and inactive Trypanosoma cruzi trans-sialidases, Gene, 160 (1995), pp. 123–128.
20. A. B. da Silveira, M. A. Freitas, E. C. de Oliveira, S. G. Neto, A. O. Luquetti, and J. B. Furness, et al., Neuronal plasticity of the enteric nervous system is correlated with chagasic megacolon development, Parasitology, 135 (2008), pp. 1337–1342.
21. _______, Glial fibrillary acidic protein and S-100 colocalization in the enteroglial cells in dilated and nondilated portions of colon from chagasic patients, Hum Pathol, 40 (2009), pp. 244–251.
22. D. F. D´avila, J. H. Donis, A. Torres, and J. A. Ferrer, A modified and unifying neurogenic hypothesis can explain the natural history of chronic Chagas heart disease, Int J Cardiol, 96 (2004), pp. 191– 195.
23. D. F. D´avila, C. F. Gottberg, A. Torres, G. Holzhaker, R. Barrios, P. Ramoni, and J. H. Donis, Cardiac sympathetic-parasympathetic balance in rats with experimentally-induced acute chagasic myocarditis, Rev Inst Med Trop Sao Paulo, 37 (1995), pp. 155– 159.
24. M. de Melo-Jorge and M. PereiraPerrin, The Chagas’ disease parasite Trypanosoma cruzi exploits nerve growth factor receptor TrkA to infect mammalian hosts, Cell Host Microbe, 1 (2007), pp. 251–261.
25. H. P. Deigner, U. Haberkorn, and R. Kinscherf, Apoptosis modulators in the therapy of neurodegenerative diseases, Expert Opin Investig Drugs, 9 (2000), pp. 747–764.
26. C. A. Diazgranados, C. H. Saavedra-Trujillo, M. Mantilla, S. L. Valderrama, C. Alquichire, and C. Franco-Paredes, Chagasic encephalitis in HIV patients: common presentation of an evolving epidemiological and clinical association, Lancet Infect Dis, 9 (2009), pp. 324–330.
27. N. M. El-Sayed, P. J. Myler, D. C. Bartholomeu, D. Nilsson, G. Aggarwal, and A. N. Tran, et al., The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease, Science, 309 (2005), pp. 409–415.
28. A. C. Frasch, Functional diversity in the trans-sialidase and mucin families in Trypanosoma cruzi, Parasitol Today, 16 (2000), pp. 282–286.
29. L. Freire-de Lima, F. Alisson-Silva, S. T. Carvalho, C. M. Takiya, M. M. Rodrigues, and G. A. DosReis, et al., Trypanosoma cruzi subverts host cell sialylation and may compromise antigen-specific CD8+ T cell responses, J Biol Chem, 285 (2010), pp. 13388– 13396.
30. W. Gao and M. A. Pereira, Trypanosoma cruzi trans-sialidase potentiates T cell activation through antigen-presenting cells: role of IL-6 and Bruton’s tyrosine kinase, Eur J Immunol, 31 (2001), pp. 1503–1512.
31. W. Gao, H. H. Wortis, and M. A. Pereira, The Trypanosoma cruzi trans-sialidase is a T cell-independent B cell mitogen and an inducer of non-specific Ig secretion, Int Immunol, 14 (2002), pp. 299–308.
32. R. Hoff, R. S. Teixeira, J. S. Carvalho, and K. E. Mott, Trypanosoma cruzi in the cerebrospinal fluid during the acute stage of Chagas’ disease, N Engl J Med, 298 (1978), pp. 604–606.
33. E. J. Huang and L. F. Reichardt, Trk receptors: roles in neuronal signal transduction, Annu Rev Biochem, 72 (2003), pp. 609–642.
34. T. N. James, M. A. Rossi, and S. Yamamoto, Postmortem studies of the intertruncal plexus and cardiac conduction system from patients with Chagas disease who died suddenly, Prog Cardiovasc Dis, 47 (2005), pp. 258–275.
35. F. Koberle, The causation and importance of nervous lesions in American trypanosomiasis, Bull World Health Organ, 42 (1970), pp. 739–743.
36. F. Koeberle, Enteromegaly and cardiomegaly in Chagas disease, Gut, 4 (1963), pp. 399–405.
37. J. S. Leon and D. M. Engman, The significance of autoimmunity in the pathogenesis of Chagas heart disease, Front Biosci, 8 (2003), pp. 315–322.
38. M. S. Lo Presti, H. W. Rivarola, A. R. Fern´andez, J. E. Enders, G. Levin, and R. Fretes, et al., Involvement of the beta-adrenergic system in the cardiac chronic form of experimental Trypanosoma cruzi infection, Parasitology, 136 (2009), pp. 905–918.
39. B. Lu, J. Alroy, A. O. Luquetti, and M. PereiraPerrin, Human autoantibodies specific for neurotrophin receptors TrkA, TrkB, and TrkC protect against lethal Trypanosoma cruzi infection in mice, Am J Pathol, 173 (2008), pp. 1406–1414.
40. B. Lu, Z. Petrola, A. O. Luquetti, and M. PereiraPerrin, Autoantibodies to receptor tyrosine kinases TrkA, TrkB and TrkC in patients with chronic Chagas’ disease, Scand J Immunol, 67 (2008), pp. 603–609.
41. J. A. Luther and S. J. Birren, Neurotrophins and target interactions in the development and regulation of sympathetic neuron electrical and synaptic properties, Auton Neurosci, 151 (2009), pp. 46–60.
42. A. B. Machado, C. R. Machado, and M. V. Gomez, Trypanosoma cruzi: acetylcholine content and cholinergic innervation of the heart in rats, Exp Parasitol, 47 (1979), pp. 107–115.
43. C. R. Machado, M. V. Caliari, M. de Lana, andW. L. Tafuri, Heart autonomic innervation during the acute phase of experimental American trypanosomiasis in the dog, Am J Trop Med Hyg, 59 (1998), pp. 492–496.
44. C. R. Machado, M. V. Gomez, and A. B. Machado, Changes in choline acetyltransferase activity of rat tissues during Chagas’ disease, Braz J Med Biol Res, 20 (1987), pp. 697–702.
45. C. R. Machado and A. L. Ribeiro, Experimental American trypanomiasis in rats: sympathetic denervation, parasitism and inflammatory process, Mem Inst Oswaldo Cruz, 84 (1989), pp. 549–556.
46. E. M. Machado, D. J. Camilo J´unior, S. W. Pinheiro, E. R. Lopes, A. J. Fernandes, J. C. Dias, and S. J. Adad, Morphometry of submucous and myenteric esophagic plexus of dogs experimentally reinfected with Trypanosoma cruzi, Mem Inst Oswaldo Cruz, 96 (2001), pp. 545–548.
47. J. A. Marin-Neto, E. Cunha-Neto, B. C. Maciel, and M. V. Sim¨oes, Pathogenesis of chronic Chagas heart disease, Circulation, 115 (2007), pp. 1109–1123.
48. N. M. Matsuda, S. M. Miller, and P. R. Evora, The chronic gastrointestinal manifestations of Chagas disease, Clinics (Sao Paulo), 64 (2009), pp. 1219–1224.
49. U. G. Meneghelli, Chagasic enteropathy, Rev Soc Bras Med Trop, 37 (2004), pp. 252–260.
50. M. Ming, M. Chuenkova, E. Ortega-Barria, and M. E. Pereira, Mediation of Trypanosoma cruzi invasion by sialic acid on the host cell and trans-sialidase on the trypanosome, Mol Biochem Parasitol, 59 (1993), pp. 243–252.
51. J. Mucci, M. G. Risso, M. S. Leguizam´on, A. C. Frasch, and O. Campetella, The trans-sialidase from Trypanosoma cruzi triggers apoptosis by target cell sialylation, Cell Microbiol, 8 (2006), pp. 1086–1095.
52. S. Mukherjee, H. Huang, S. B. Petkova, C. Albanese, R. G. Pestell, and V. L. Braunstein, et al., Trypanosoma cruzi infection activates extracellular signal-regulated kinase in cultured endothelial and smooth muscle cells, Infect Immun, 72 (2004), pp. 5274–5282.
53. R. D. Nascimento, A. de Souza Lisboa, R. T. Fujiwara, M. A. de Freitas, S. J. Adad, and R. C. Oliveira, et al., Characterization of enteroglial cells and denervation process in chagasic patients with and without megaesophagus, Hum Pathol, 41 (2009), pp. 528–534.
54. M. E. Pereira, A developmentally regulated neuraminidase activity in Trypanosoma cruzi, Science, 219 (1983), pp. 1444–1446.
55. L. Piacenza, M. N. Alvarez, G. Peluffo, and R. Radi, Fighting the oxidative assault: the Trypanosoma cruzi journey to infection, Curr Opin Microbiol, 12 (2009), pp. 415–421.
56. J. E. Pittella, Central nervous system involvement in Chagas’ disease. An updating, Rev Inst Med Trop Sao Paulo, 35 (1993), pp. 111–116.
57. A. Rassi, Jr., A. Rassi, and J. A. Marin-Neto, Chagas disease, Lancet, 375 (2010), pp. 1388–1402.
58. L. C. Ribeiro, A. A. Barbosa, Jr., and Z. A. Andrade, Pathology of intracardiac nerves in experimental Chagas disease, Mem Inst Oswaldo Cruz, 97 (2002), pp. 1019–1025.
59. N. N. Rocha, S. Garcia, L. E. Gim´enez, C. C. Hern´andez, J. F. Senra, and R. S. Lima, et al., Characterization of cardiopulmonary function and cardiac muscarinic and adrenergic receptor density adaptation in C57BL/6 mice with chronic Trypanosoma cruzi infection, Parasitology, 133 (2006), pp. 729–237.
60. S. S. Rubin-de Celis, H. Uemura, N. Yoshida, and S. Schenkman, Expression of trypomastigote trans-sialidase in metacyclic forms of Trypanosoma cruzi increases parasite escape from its parasitophorous vacuole, Cell Microbiol, 8 (2006), pp. 1888–1898.
61. E. Saavedra, M. Herrera, W. Gao, H. Uemura, and M. A. Pereira, The Trypanosoma cruzi trans-sialidase, through its COOHterminal tandem repeat, upregulates interleukin 6 secretion in normal human intestinal microvascular endothelial cells and peripheral blood mononuclear cells, J Exp Med, 190 (1999), pp. 1825–1836.
62. S. Schenkman, L. Pontes de Carvalho, and V. Nussenzweig, Trypanosoma cruzi trans-sialidase and neuraminidase activities can be mediated by the same enzymes, J Exp Med, 175 (1992), pp. 567–575.
63. P. Scudder, J. P. Doom, M. Chuenkova, I. D. Manger, and M. E. Pereira, Enzymatic characterization of beta-D-galactoside alpha 2,3-trans-sialidase from Trypanosoma cruzi, J Biol Chem, 268 (1993), pp. 9886–9891.
64. J. S. Silva, F. S. Machado, and G. A. Martins, The role of nitric oxide in the pathogenesis of Chagas disease, Front Biosci, 8 (2003), pp. 314–325.
65. B. Stark, M. Risling, and T. Carlstedt, Distribution of the neurotrophin receptors p75 and trkB in peripheral mechanoreceptors; observations on changes after injury, Exp Brain Res, 136 (2001), pp. 101–107.
66. L. V. Tanowitz, H. B. Kirchhoff, D. Simon, S. A. Morris, L. M. Weiss, and M. Wittner, Chagas’ disease, Clin Microbiol Rev, 5 (1992), pp. 400–719.
67. R. L. Tarleton, Parasite persistence in the aetiology of Chagas disease, Int J Parasitol, 31 (2001), pp. 550–554.
68. A. Todeschini, W. B. Dias, M. F. Girard, J. M. Wieruszeski, L. Mendonc¸a-Previato, and J. O. Previato, Enzymatically inactive trans-sialidase from Trypanosoma cruzi binds sialyl and betagalactopyranosyl residues in a sequential ordered mechanism, J Biol Chem, 279 (2004), pp. 5323–5328.
69. F. Villalta, Y. Zhang, K. E. Bibb, S. Pratap, J. M. Burns, Jr., and M. F. Lima, Signal transduction in human macrophages by gp83 ligand of Trypanosoma cruzi: trypomastigote gp83 ligand upregulates trypanosome entry through protein kinase C activation, Mol Cell Biol Res Commun, 2 (1999), pp. 64–70.
70. J. C. Villar, H. Leon, and C. A. Morillo, Cardiovascular autonomic function testing in asymptomatic T. cruzi carriers: a sensitive method to identify subclinical Chagas’ disease, Int J Cardiol, 93 (2004), pp. 189–195.
71. C. Weinkauf and M. PereiraPerrin, Trypanosoma cruzi promotes neuronal and glial cell survival through the neurotrophic receptor TrkC, Infect Immun, 77 (2009), pp. 1368–1375.
72. S. R.Wilkinson and J. M. Kelly, Trypanocidal drugs: mechanisms, resistance and new targets, Expert Rev Mol Med, 11 (2009), p. e31.
73. W. C. Wong, C. K. Tan, M. Singh, and T. Y. Yick, Ultrastructure of murine cardiac ganglia in experimental Chagas’ disease, Histol Histopathol, 7 (1992), pp. 371–378.
74. A. Woronowicz, S. R. Amith, V. W. Davis, P. Jayanth, K. De Vusser, and W. Laroy, et al., Trypanosome trans-sialidase mediates neuroprotection against oxidative stress, serum/glucose deprivation, and hypoxia-induced neurite retraction in Trkexpressing PC12 cells, Glycobiology, 17 (2007), pp. 725–734.
75. A. Woronowicz, K. De Vusser, W. Laroy, R. Contreras, S. O. Meakin, G. M. Ross, and M. R. Szewczuk, Trypanosome transsialidase targets TrkA tyrosine kinase receptor and induces receptor internalization and activation, Glycobiology, 14 (2004), pp. 987–998.
76. J. Yamauchi, J. R. Chan, and E. M. Shooter, Neurotrophin 3 activation of TrkC induces Schwann cell migration through the c-Jun N-terminal kinase pathway, Proc Natl Acad Sci USA, 100 (2003), pp. 14421–14426.

--