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.

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.

Triatoma infestans Calreticulin: Gene Cloning and Expression of a Main Domain That Interacts with the Host Complement System.

Triatoma infestans is an important hematophagous vector of Chagas disease, a neglected chronic illness affecting approximately 6 million people in Latin America. Hematophagous insects possess several molecules in their saliva that counteract host defensive responses. Calreticulin (CRT), a multifunctional protein secreted in saliva, contributes to the feeding process in some insects. Human CRT (HuCRT) and Trypanosoma cruzi CRT (TcCRT) inhibit the classical pathway of complement activation, mainly by interacting through their central S domain with complement component C1. In previous studies, we have detected CRT in salivary gland extracts from T. infestans We have called this molecule TiCRT. Given that the S domain is responsible for C1 binding, we have tested its role in the classical pathway of complement activation in vertebrate blood. We have cloned and characterized the complete nucleotide sequence of CRT from T. infestans, and expressed its S domain. As expected, this S domain binds to human C1 and, as a consequence, it inhibits the classical pathway of complement, at its earliest stage of activation, namely the generation of C4b. Possibly, the presence of TiCRT in the salivary gland represents an evolutionary adaptation in hematophagous insects to control a potential activation of complement proteins, present in the massive blood meal that they ingest, with deleterious consequences at least on the anterior digestive tract of these insects.

Does native Trypanosoma cruzi calreticulin mediate growth inhibition of a mammary tumor during infection?

BACKGROUND: For several decades now an antagonism between Trypanosoma cruzi infection and tumor development has been detected. The molecular basis of this phenomenon remained basically unknown until our proposal that T. cruzi Calreticulin (TcCRT), an endoplasmic reticulum-resident chaperone, translocated-externalized by the parasite, may mediate at least an important part of this effect. Thus, recombinant TcCRT (rTcCRT) has important in vivo antiangiogenic and antitumor activities. However, the relevant question whether the in vivo antitumor effect of T. cruzi infection is indeed mediated by the native chaperone (nTcCRT), remains open. Herein, by using specific modified anti-rTcCRT antibodies (Abs), we have neutralized the antitumor activity of T. cruzi infection and extracts thereof, thus identifying nTcCRT as a valid mediator of this effect. METHODS: Polyclonal anti-rTcCRT F(ab')2 Ab fragments were used to reverse the capacity of rTcCRT to inhibit EAhy926 endothelial cell (EC) proliferation, as detected by BrdU uptake. Using these F(ab')2 fragments, we also challenged the capacity of nTcCRT, during T. cruzi infection, to inhibit the growth of an aggressive mammary adenocarcinoma cell line (TA3-MTXR) in mice. Moreover, we determined the capacity of anti-rTcCRT Abs to reverse the antitumor effect of an epimastigote extract (EE). Finally, the effects of these treatments on tumor histology were evaluated. RESULTS: The rTcCRT capacity to inhibit ECs proliferation was reversed by anti-rTcCRT F(ab')2 Ab fragments, thus defining them as valid probes to interfere in vivo with this important TcCRT function. Consequently, during infection, these Ab fragments also reversed the in vivo experimental mammary tumor growth. Moreover, anti-rTcCRT Abs also neutralized the antitumor effect of an EE, again identifying the chaperone protein as an important mediator of this anti mammary tumor effect. Finally, as determined by conventional histological parameters, in infected animals and in those treated with EE, less invasive tumors were observed while, as expected, treatment with F(ab')2 Ab fragments increased malignancy. CONCLUSION: We have identified translocated/externalized nTcCRT as responsible for at least an important part of the anti mammary tumor effect of the chaperone observed during experimental infections with T. cruzi.

Comparative effect of human and Trypanosoma cruzi calreticulin in wound healing.

In orthopaedics, the use of factors that enhance granulation tissue formation and prevent or delay new bone regeneration is sometimes desirable. Calreticulin (CRT), a unique endoplasmic reticulum luminal Ca(2+) -binding chaperone widely distributed in eukaryotic cells, is involved in many cellular functions. Among them, CRT has an important influence in cutaneous wound healing and diverse processes associated with cutaneous repair, inhibition of angiogenesis, promotion of cell adhesion and antitumour effect. One of the molecules involved in several aspects of the host-parasite interplay is Trypanosoma cruzi calreticulin (TcCRT), which is highly homologous to human calreticulin (HuCRT). Here, recombinant (r)HuCRT and rTcCRT are compared on their abilities to affect fibroblast behaviour in a scratch plate assay, and wound healing in in vivo skin rat models. In molar terms, rTcCRT is three orders of magnitude more efficient than rHuCRT in increasing proliferation and migration of human fibroblasts in vitro. A similar effect was observed in vivo on rat skin wounds and inhibition of bone gap bridging in rabbit unicortical bone osteotomies.

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.

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.