Is It Possible to Intervene in the Capacity of Trypanosoma cruzi to Elicit and Evade the Complement System?

Chagas' disease is a zoonotic parasitic ailment now affecting more than 6 million people, mainly in Latin America. Its agent, the protozoan Trypanosoma cruzi, is primarily transmitted by endemic hematophagous triatomine insects. Transplacental transmission is also important and a main source for the emerging global expansion of this disease. In the host, the parasite undergoes intra (amastigotes) and extracellular infective (trypomastigotes) stages, both eliciting complex immune responses that, in about 70% of the cases, culminate in permanent immunity, concomitant with the asymptomatic presence of the parasite. The remaining 30% of those infected individuals will develop a syndrome, with variable pathological effects on the circulatory, nervous, and digestive systems. Herein, we review an important number of T. cruzi molecules, mainly located on its surface, that have been characterized as immunogenic and protective in various experimental setups. We also discuss a variety of parasite strategies to evade the complement system - mediated immune responses. Within this context, we also discuss the capacity of the T. cruzi infective trypomastigote to translocate the ER-resident chaperone calreticulin to its surface as a key evasive strategy. Herein, it is described that T. cruzi calreticulin inhibits the initial stages of activation of the host complement system, with obvious benefits for the parasite. Finally, we speculate on the possibility to experimentally intervene in the interaction of calreticulin and other T. cruzi molecules that interact with the complement system; thus resulting in significant inhibition of T. cruzi infectivity.

The Interactions of Parasite Calreticulin With Initial Complement Components: Consequences in Immunity and Virulence.

Because of its capacity to increase a physiologic inflammatory response, to stimulate phagocytosis, to promote cell lysis and to enhance pathogen immunogenicity, the complement system is a crucial component of both the innate and adaptive immune responses. However, many infectious agents resist the activation of this system by expressing or secreting proteins with a role as complement regulatory, mainly inhibitory, proteins. Trypanosoma cruzi, the causal agent of Chagas disease, a reemerging microbial ailment, possesses several virulence factors with capacity to inhibit complement at different stages of activation. T. cruzi calreticulin (TcCalr) is a highly-conserved, endoplasmic reticulum-resident chaperone that the parasite translocates to the extracellular environment, where it exerts a variety of functions. Among these functions, TcCalr binds C1, MBL and ficolins, thus inhibiting the classical and lectin pathways of complement at their earliest stages of activation. Moreover, the TcCalr/C1 interaction also mediates infectivity by mimicking a strategy used by apoptotic cells for their removal. More recently, it has been determined that these Calr strategies are also used by a variety of other parasites. In addition, as reviewed elsewhere, TcCalr inhibits angiogenesis, promotes wound healing and reduces tumor growth. Complement C1 is also involved in some of these properties. Knowledge on the role of virulence factors, such as TcCalr, and their interactions with complement components in host-parasite interactions, may lead toward the description of new anti-parasite therapies and prophylaxis.

In vitro Treatment of a Murine Mammary Adenocarcinoma Cell Line with Recombinant Trypanosoma cruzi Calreticulin Promotes Immunogenicity and Phagocytosis.

American Trypanosomiasis, a parasitic disease produced by Trypanosoma cruzi (T. cruzi), endemic in Latin America, infects about 6 million people. During the chronic stage of the infection, approximately 30% of infected people will develop Chagas Disease, the clinical manifestation. Few decades ago it was reported that, during the chronic stage, the parasite interferes with the development of solid tumors. However, the identification of parasite molecules responsible for such effects remained elusive. Years later, we described T.cruzi Calreticulin (TcCalr), an endoplasmic reticulum resident chaperone that infective trypomastigotes translocate to the parasite exterior, where it displays anticomplement activities. Most likely, at least some of these activities are related with the antitumor properties of TcCalr, as shown in in vitro, ex vivo, in ovum, and in vivo models. In this context we, we have seen that in vivo subcutaneous peritumoral inoculation of rTcCalr enhances local infiltration of T cells and slows tumor development. Based on these precedents, we propose that in vitro treatment of a mammary adenocarcinoma (TA3 cell line) with rTcCalr, will enhance tumor immunogenicity. In agreement with this proposal, we have shown that: i). rTcCalr binds to TA3 cells in a concentration-dependent fashion, ii). C1q binds to TA3 cells in an rTcCalr-dependent fashion, confirmed by the reversion attained using anti-TcS (a central TcCalr domain that binds C1) F(ab')2 antibody fragments, iii). incubation of TA3 cells with rTcCalr, promotes cell phagocytosis by murine macrophages and, iv). rTcCalr decreases the membrane expression of MHC class II, m-Dectin-1, Galectin-9 and PD-L1, while increasing the expression of Rae-1γ. In synthesis, herein we show that in vitro treatment of a murine mammary adenocarcinoma with rTcCalr enhances phagocytosis and modulates the expression of a variety of membrane molecules that correlates with increased tumor immunogenicity.

Trypanosoma cruzi Calreticulin: Immune Evasion, Infectivity, and Tumorigenesis.

To successfully infect, Trypanosoma cruzi evades and modulates the host immune response. T. cruzi calreticulin (TcCalr) is a multifunctional, endoplasmic reticulum (ER)-resident chaperone that, translocated to the external microenvironment, mediates crucial host-parasite interactions. TcCalr binds and inactivates C1 and mannose-binding lectin (MBL)/ficolins, important pattern- recognition receptors (PRRs) of the complement system. Using an apoptotic mimicry strategy, the C1-TcCalr association facilitates the infection of target cells. T. cruzi infection also seems to confer protection against tumorigenesis. Thus, recombinant TcCalr has important antiangiogenic properties, detected in vitro, ex vivo, and in ovum, most likely contributing at least in part, to its antitumor properties. Consequently, TcCalr is useful for investigating key issues of host-parasite interactions and possible new immunological/pharmacological interventions in the areas of Chagas' disease and experimental cancer.

Structural bases that underline Trypanosoma cruzi calreticulin proinfective, antiangiogenic and antitumor properties.

Microbes have developed mechanisms to resist the host immune defenses and some elicit antitumor immune responses. About 6 million people are infected with Trypanosoma cruzi, the protozoan agent of Chagas' disease, the sixth neglected tropical disease worldwide. Eighty years ago, G. Roskin and N. Klyuyeva proposed that T. cruzi infection mediates an anti-cancer activity. This observation has been reproduced by several other laboratories, but no molecular basis has been proposed. We have shown that the highly pleiotropic chaperone calreticulin (TcCalr, formerly known as TcCRT), translocates from the parasite ER to the exterior, where it mediates infection. Similar to its human counterpart HuCALR (formerly known as HuCRT), TcCalr inhibits C1 in its capacity to initiate the classical pathway of complement activation. We have also proposed that TcCalr inhibits angiogenesis and it is a likely mediator of antitumor effects. We have generated several in silico structural TcCalr models to delimit a peptide (VC-TcCalr) at the TcCalr N-domain. Chemically synthesized VC-TcCalr did bind to C1q and was anti-angiogenic in Gallus gallus chorioallantoic membrane assays. These properties were associated with structural features, as determined in silico. VC-TcCalr, a strong dipole, interacts with charged proteins such as collagen-like tails and scavenger receptors. Comparatively, HuCALR has less polarity and spatial stability, probably due to at least substitutions of Gln for Gly, Arg for Lys, Arg for Asp and Ser for Arg that hinder protein-protein interactions. These differences can explain, at least in part, how TcCalr inhibits the complement activation pathway and has higher efficiency as an antiangiogenic and antitumor agent than HuCALR.

Molluskan Hemocyanins Activate the Classical Pathway of the Human Complement System through Natural Antibodies.

Molluskan hemocyanins are enormous oxygen-carrier glycoproteins that show remarkable immunostimulatory properties when inoculated in mammals, such as the generation of high levels of antibodies, a strong cellular reaction, and generation of non-specific antitumor immune responses in some types of cancer, particularly for superficial bladder cancer. These proteins have the ability to bias the immune response toward a Th1 phenotype. However, despite all their current uses with beneficial clinical outcomes, a clear mechanism explaining these properties is not available. Taking into account reports of natural antibodies against the hemocyanin of the gastropod Megathura crenulata [keyhole limpet hemocyanin (KLH)] in humans as well as other vertebrate species, we report here for the first time, the presence, in sera from unimmunized healthy donors, of antibodies recognizing, in addition to KLH, two other hemocyanins from gastropods with documented immunomodulatory capacities: Fisurella latimarginata hemocyanin (FLH) and Concholepas concholepas hemocyanin (CCH). Through an ELISA screening, we found IgM and IgG antibodies reactive with these hemocyanins. When the capacity of these antibodies to bind deglycosylated hemocyanins was studied, no decreased interaction was detected. Moreover, in the case of FLH, deglycosylation increased antibody binding. We evaluated through an in vitro complement deposition assay whether these antibodies activated the classical pathway of the human complement system. The results showed that all three hemocyanins and their deglycosylated counterparts elicited this activation, mediated by C1 binding to immunoglobulins. Thus, this work contributes to the understanding on how the complement system could participate in the immunostimulatory properties of hemocyanins, through natural, complement-activating antibodies reacting with these proteins. Although a role for carbohydrates cannot be completely ruled out, in our experimental setting, glycosylation status had a limited effect. Finally, our data open possibilities for further studies leading to the design of improved hemocyanin-based research tools for diagnosis and immunotherapy.

Exogenous Calreticulin, incorporated onto non-infective Trypanosoma cruzi epimastigotes, promotes their internalization into mammal host cells.

Chagas disease is an endemic pathology in Latin America, now emerging in developed countries, caused by the intracellular protozoan Trypanosoma cruzi, whose life cycle involves three stages: amastigotes, epimastigotes, and trypomastigotes. T. cruzi Calreticulin (TcCRT), an endoplasmic reticulum resident chaperone, translocates to the external cellular membrane, where it captures complement component C1, ficolins and MBL, thus inactivating the classical and lectin pathways. Trypomastigote-bound C1 is detected as an "eat me" signal by macrophages and promotes the infective process. Unlike infective trypomastigotes, non-infective epimastigotes either do not express or express only marginal levels of TcCRT on their external membrane. We show that epimastigotes bind exogenous rTcCRT to their cellular membrane and, in the presence of C1q, this parasite form is internalized into normal fibroblasts. On the other hand, Calreticulin (CRT)-deficient fibroblasts show impaired parasite internalization. In synthesis, CRT from both parasite and host cell origin is important in the establishment of C1q-dependent first contacts between parasites and host cells.

Trypanosoma cruzi Calreticulin Topographical Variations in Parasites Infecting Murine Macrophages.

Trypanosoma cruzi calreticulin (TcCRT), a 47-kDa chaperone, translocates from the endoplasmic reticulum to the area of flagellum emergence. There, it binds to complement components C1 and mannan-binding lectin (MBL), thus acting as a main virulence factor, and inhibits the classical and lectin pathways. The localization and functions of TcCRT, once the parasite is inside the host cell, are unknown. In parasites infecting murine macrophages, polyclonal anti-TcCRT antibodies detected TcCRT mainly in the parasite nucleus and kinetoplast. However, with a monoclonal antibody (E2G7), the resolution and specificity of the label markedly improved, and TcCRT was detected mainly in the parasite kinetoplast. Gold particles, bound to the respective antibodies, were used as probes in electron microscopy. This organelle may represent a stopover and accumulation site for TcCRT, previous its translocation to the area of flagellum emergence. Finally, early during T. cruzi infection and by unknown mechanisms, an important decrease in the number of MHC-I positive host cells was observed.

Deficiency in mannose-binding lectin-associated serine protease-2 does not increase susceptibility to Trypanosoma cruzi infection.

Trypanosoma cruzi is the causative agent of Chagas' disease, a chronic illness affecting 10 million people around the world. The complement system plays an important role in fighting microbial infections. The recognition molecules of the lectin pathway of complement activation, mannose-binding lectin (MBL), ficolins, and CL-11, bind to specific carbohydrates on pathogens, triggering complement activation through MBL-associated serine protease-2 (MASP-2). Previous in vitro work showed that human MBL and ficolins contribute to T. cruzi lysis. However, MBL-deficient mice are only moderately compromised in their defense against the parasite, as they may still activate the lectin pathway through ficolins and CL-11. Here, we assessed MASP-2-deficient mice, the only presently available mouse line with total lectin pathway deficiency, for a phenotype in T. cruzi infection. Total absence of lectin pathway functional activity did not confer higher susceptibility to T. cruzi infection, suggesting that it plays a minor role in the immune response against this parasite.

Trypanosoma cruzi calreticulin inhibits the complement lectin pathway activation by direct interaction with L-Ficolin.

Trypanosoma cruzi, the agent of Chagas' disease, the sixth neglected tropical disease worldwide, infects 10-12 million people in Latin America. Differently from T. cruzi epimastigotes, trypomastigotes are complement-resistant and infective. CRPs, T-DAF, sialic acid and lipases explain at least part of this resistance. In vitro, T. cruzi calreticulin (TcCRT), a chaperone molecule that translocates from the ER to the parasite surface: (a) Inhibits the human classical complement activation, by interacting with C1, (b) As a consequence, an increase in infectivity is evident and, (c) It inhibits angiogenesis and tumor growth. We report here that TcCRT also binds to the L-Ficolin collagenous portion, thus inhibiting approximately between 35 and 64% of the human complement lectin pathway activation, initiated by L-Ficolin, a property not shared by H-Ficolin. While L-Ficolin binds to 60% of trypomastigotes and to 24% of epimastigotes, 50% of the former and 4% of the latter display TcCRT on their surfaces. Altogether, these data indicate that TcCRT is a parasite inhibitory receptor for Ficolins. The resulting evasive activities, together with the TcCRT capacity to inhibit C1, with a concomitant increase in infectivity, may represent T. cruzi strategies to inhibit important arms of the innate immune response.

Is it all That Bad When Living with an Intracellular Protozoan? The Role of Trypanosoma cruzi Calreticulin in Angiogenesis and Tumor Growth.

The immune system protects against disease, but may aberrantly silence immunity against "altered self," with consequent development of malignancies. Among the components of the endoplasmic reticulum (ER), important in immunity, is calreticulin (CRT) that, in spite of its residence in the ER, can be translocated to the exterior. Trypanosoma cruzi is the agent of Chagas disease, one of the most important global neglected infections, affecting several hundred thousand people. The syndrome, mainly digestive and circulatory, affects only one-third of those infected. The anti-tumor effects of the infection are known for several decades, but advances in the identification of responsible T. cruzi molecules are scarce. We have shown that T. cruzi CRT (TcCRT) better executes the antiangiogenic and anti-tumor effects of mammal CRT and its N-terminus vasostatin. In this regard, recombinant TcCRT (rTcCRT) and/or its N-terminus inhibit angiogenesis in vitro, ex vivo, and in vivo. TcCRT also inhibits the growth of murine adenocarcinomas and melanomas. Finally, rTcCRT fully reproduces the anti-tumor effect of T. cruzi infection in mice. Thus, we hypothesize that, the long reported anti-tumor effect of T. cruzi infection is mediated at least in part by TcCRT.

The interaction of classical complement component C1 with parasite and host calreticulin mediates Trypanosoma cruzi infection of human placenta.

BACKGROUND: 9 million people are infected with Trypanosoma cruzi in Latin America, plus more than 300,000 in the United States, Canada, Europe, Australia, and Japan. Approximately 30% of infected individuals develop circulatory or digestive pathology. While in underdeveloped countries transmission is mainly through hematophagous arthropods, transplacental infection prevails in developed ones. METHODOLOGY/PRINCIPAL FINDINGS: During infection, T. cruzi calreticulin (TcCRT) translocates from the endoplasmic reticulum to the area of flagellum emergence. There, TcCRT acts as virulence factor since it binds maternal classical complement component C1q that recognizes human calreticulin (HuCRT) in placenta, with increased parasite infectivity. As measured ex vivo by quantitative PCR in human placenta chorionic villi explants (HPCVE) (the closest available correlate of human congenital T. cruzi infection), C1q mediated up to a 3-5-fold increase in parasite load. Because anti-TcCRT and anti-HuCRT F(ab')2 antibody fragments are devoid of their Fc-dependent capacity to recruit C1q, they reverted the C1q-mediated increase in parasite load by respectively preventing its interaction with cell-bound CRTs from both parasite and HPCVE origins. The use of competing fluid-phase recombinant HuCRT and F(ab')2 antibody fragments anti-TcCRT corroborated this. These results are consistent with a high expression of fetal CRT on placental free chorionic villi. Increased C1q-mediated infection is paralleled by placental tissue damage, as evidenced by histopathology, a damage that is ameliorated by anti-TcCRT F(ab')2 antibody fragments or fluid-phase HuCRT. CONCLUSIONS/SIGNIFICANCE: T. cruzi infection of HPCVE is importantly mediated by human and parasite CRTs and C1q. Most likely, C1q bridges CRT on the parasite surface with its receptor orthologue on human placental cells, thus facilitating the first encounter between the parasite and the fetal derived placental tissue. The results presented here have several potential translational medicine aspects, specifically related with the capacity of antibody fragments to inhibit the C1q/CRT interactions and thus T. cruzi infectivity.

Roles of Trypanosoma cruzi calreticulin in parasite-host interactions and in tumor growth.

In Latin America, there are about 10-12 million people infected with Trypanosoma cruzi, the agent of Chagas' disease, one of the most important neglected tropical parasitism. Identification of molecular targets, specific for the aggressor or host cells or both, may be useful in the development of pharmacological and/or immunological therapeutic tools. Classic efforts in Chagas' disease explore those strategies. Although the immune system frequently controls parasite aggressions, sterile immunity is seldom achieved and chronic interactions are thus established. However, laboratory-modified immunologic probes aimed at selected parasite targets, may be more effective than their unmodified counterparts. Calreticulin (CRT) from vertebrates is a calcium binding protein, present mainly in the endoplasmic reticulum (ER), where it directs the conformation of proteins and controls calcium levels. We have isolated, gene-cloned, expressed and characterized T. cruzi calreticulin (TcCRT). Upon infection, the parasite can translocate this molecule from the ER to the surface, where it inhibits both the classical and lectin complement pathways. Moreover, by virtue of its capacity to bind and inactivate first complement component C1, it promotes parasite infectivity. These two related properties reside in the central domain of this molecule. A different domain, amino terminal, binds to endothelial cells, thus inhibiting their angiogenic capacity. Since tumor growth depends, to a large extent on angiogenesis, their growth is also inhibited.

Extracellular Trypanosoma cruzi calreticulin in the host-parasite interplay.

Calreticulin (CRT) from vertebrates is a calcium-binding protein present mainly in the endoplasmic reticulum (ER). There, it directs the conformation of proteins and controls calcium levels. This review will focus on several extracellular roles of Trypanosoma cruzi CRT (TcCRT) in relation to its capacity to inhibit the complement system, mediate parasite infectivity, interfere with angiogenesis and, as a possible consequence, with tumor growth. The TcCRT antiangiogenic effect parallels with the capacity of T. cruzi infection to inhibit tumor development in vivo. Thus, the TcCRT, complement, and endothelial cell interactions seem to be an evolutionary adaptation to promote prolonged parasite-host relationships.

Trypanosoma cruzi calreticulin: a novel virulence factor that binds complement C1 on the parasite surface and promotes infectivity.

In Trypanosoma cruzi, calreticulin (TcCRT) translocates from the endoplasmic reticulum (ER) to the area of flagellum emergence. We propose herein that the parasite uses this molecule to capture complement C1, in an infective apoptotic mimicry strategy. Thus, TcCRT/C1 interactions, besides inhibiting the classical pathway of complement activation as previously shown in our laboratories, will also promote infectivity. This fact correlates with significant increases in TcCRT mRNA levels during early infection stages of a VERO cell line. In vitro, the collagenous and globular C1q domains simultaneously bind TcCRT and antigen aggregated Igs, respectively. Accordingly, mouse immunizations with TcCRT induced humoral responses that, after challenge, correlated with increased parasitemia. Thus, on the parasite surface, whole Igs anti-TcCRT promote C1 deposits on trypomastigotes while, as expected, F(ab')2 fragments decrease it. Likewise, pretreatment of the parasites with whole anti-TcCRT antibodies augmented parasitemia and mortality in mice. In contrast, pretreatment with F(ab')2 fragments anti-TcCRT, devoid of their capacity to provide additional C1q binding sites, was protective. Most important, while pretreatment of trypomastigotes with C1q increased infectivity in the RAW murine cell line, as well as mice mortality and parasitemia, the F(ab')2 fragments significantly interfered with the C1q-dependent infectivity. Differently from other surface molecules involved in infectivity, TcCRT uses C1 as an adaptor molecule to recognize host cells. As expected, since TcCRT is one of several cell surface parasite molecules participating in infectivity, attempts to interfere with the C1/TcCRT interactions with F(ab')2 fragments, were moderately but significantly effective, both in vitro and in vivo.

Antiangiogenic and antitumor effects of Trypanosoma cruzi Calreticulin.

BACKGROUND: In Latin America, 18 million people are infected with Trypanosoma cruzi, the agent of Chagas' disease, with the greatest economic burden. Vertebrate calreticulins (CRT) are multifunctional, intra- and extracellular proteins. In the endoplasmic reticulum (ER) they bind calcium and act as chaperones. Since human CRT (HuCRT) is antiangiogenic and suppresses tumor growth, the presence of these functions in the parasite orthologue may have consequences in the host/parasite interaction. Previously, we have cloned and expressed T. cruzi calreticulin (TcCRT) and shown that TcCRT, translocated from the ER to the area of trypomastigote flagellum emergence, promotes infectivity, inactivates the complement system and inhibits angiogenesis in the chorioallantoid chicken egg membrane. Most likely, derived from these properties, TcCRT displays in vivo inhibitory effects against an experimental mammary tumor. METHODOLOGY AND PRINCIPAL FINDINGS: TcCRT (or its N-terminal vasostatin-like domain, N-TcCRT) a) Abrogates capillary growth in the ex vivo rat aortic ring assay, b) Inhibits capillary morphogenesis in a human umbilical vein endothelial cell (HUVEC) assay, c) Inhibits migration and proliferation of HUVECs and the human endothelial cell line Eahy926. In these assays TcCRT was more effective, in molar terms, than HuCRT: d) In confocal microscopy, live HUVECs and EAhy926 cells, are recognized by FITC-TcCRT, followed by its internalization and accumulation around the host cell nuclei, a phenomenon that is abrogated by Fucoidin, a specific scavenger receptor ligand and, e) Inhibits in vivo the growth of the murine mammary TA3 MTXR tumor cell line. CONCLUSIONS/SIGNIFICANCE: We describe herein antiangiogenic and antitumor properties of a parasite chaperone molecule, specifically TcCRT. Perhaps, by virtue of its capacity to inhibit angiogenesis (and the complement system), TcCRT is anti-inflammatory, thus impairing the antiparasite immune response. The TcCRT antiangiogenic effect could also explain, at least partially, the in vivo antitumor effects reported herein and the reports proposing antitumor properties for T. cruzi infection.

Molecular mechanisms involved in the inactivation of the first component of human complement by Trypanosoma cruzi calreticulin.

Trypanosoma cruzi (T. cruzi), the agent of Chagas' disease, the sixth most important neglected tropical disease worldwide, causes 50,000 deaths per year in Latin America. T. cruzi calreticulin (TcCRT), a highly pleiotropic chaperone molecule, plays important roles in several host/parasite interactions. Among other functions, we have previously shown that TcCRT, translocated from the endoplasmic reticulum to the area of flagellar emergence, binds human C1q and inhibits activation of the classical pathway in vitro. Based on a series of in vitro experiments, we propose here two mechanisms to explain how TcCRT inhibits the classical pathway at the initial stages of C1 (q, r, s) activation. First, TcCRT interacts in vitro with both solid phase bound active C1s and C1, but impairment of C4 activating capacity is evident only when the serine proteases are within the structural context of the macromolecular first component. Although C1s activity, in this context, is inhibited by TcCRT, the serine protease is not displaced from the C1 complex. Second, TcCRT prevents C1 formation, by interfering with the ability of the (C1r-C1s)(2) tetramer to bind C1q. These complement inhibitory effects are better explained by direct interaction of the parasite protein with C1, rather than by the TcCRT capacity to bind calcium, an essential element for the functional integrity of C1.

Comparative in vivo antiangiogenic effects of calreticulin from Trypanosoma cruzi and Homo sapiens sapiens.

Angiogenesis is a complex multi-step process of neovascularization arising from preexisting blood vessels whose generation is regulated by pro- and anti-angiogenic factors. Both Trypanosoma cruzi calreticulin (TcCRT) and its human counterpart (HuCRT) are antiangiogenic. This is the first report where the TcCRT and HuCRT anti-angiogenic properties are compared in vivo. In the chick embryonic chorioallantoid membrane assay (CAM) and at equimolar concentrations, TcCRT displayed significantly higher antiangiogenic activities than its human counterpart. LPS had marginal effects at the concentrations present in the recombinant protein preparations and the TcCRT antiangiogenic effects were largely inhibited by specific polyclonal antibodies, thus, reinforcing the fact that the observed TcCRT effects can be attributed to the parasite-derived molecule and not to the endotoxin. The antiangiogenic TcCRT effects correlate with its anti-tumor in vivo effects, as recently shown in our laboratory.

Comparative in vivo antiangiogenic effects of calreticulin from Trypanosoma cruzi and Homo sapiens sapiens

Angiogenesis is a complex multi-step process of neovascularization arising from preexisting blood vessels whose generation is regulated by pro- and anti-angiogenic factors. Both Trypanosoma cruzi calreticulin (TcCRT) and its human counterpart (HuCRT) are antiangiogenic. This is the first report where the TcCRT and HuCRT anti-angiogenic properties are compared in vivo. In the chick embryonic chorioallantoid membrane assay (CAM) and at equimolar concentrations, TcCRT displayed significantly higher antiangiogenic activities than its human counterpart. LPS had marginal effects at the concentrations present in the recombinant protein preparations and the TcCRT antiangiogenic effects were largely inhibited by specific polyclonal antibodies, thus, reinforcing the fact that the observed TcCRT effects can be attributed to the parasite-derived molecule and not to the endotoxin. The antiangiogenic TcCRT effects correlate with its anti-tumor in vivo effects, as recently shown in our laboratory.

Trypanosoma cruzi calreticulin: a possible role in Chagas' disease autoimmunity.

Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas' disease, an endemic and chronic illness that affects 18 million people in Latin America. The mechanisms underlying its pathogenesis are controversial. There is a growing body of evidence supporting the view that T. cruzi infection elicits severe autoimmune responses in the host, which significantly contribute to the pathogenesis of Chagas' disease, and several recent studies have reported the presence of autoantibodies and effector T lymphocytes against parasite and self antigens in infected patients and experimentally infected animals. T. cruzi calreticulin (TcCRT) is a 45kDa protein, immunogenic in humans, rabbits and mice. It has a high degree of homology with human (HuCRT) and mouse calreticulin (MoCRT), which would explain why an immune response to TcCRT could contribute to autoimmune reactions in Chagas' disease. Anti-TcCRT antibodies generated in A/J mice immunized with recombinant TcCRT (rTcCRT) reacted with rHuCRT and bound to neonatal and adult isogenic cardiomyocytes cultured in vitro. Interestingly, histological alterations, such as edema formation and cell infiltrates, which include CD3(+) cells, were detected in heart sections from immunized animals. Therefore, in rTcCRT-immunized mice, an autoimmune reaction against host CRT, paralleled by histological cardiac alterations, suggests a role of the parasite molecule in the induction of immunologically mediated heart tissue damage. The data presented here propose that TcCRT participates in the induction of cardiac autoimmunity in Chagas' disease.