Ismael Maldonado

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.

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 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 Ca2+‐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. Copyright

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

BackgroundFor 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.MethodsPolyclonal 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.ResultsThe 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.ConclusionWe 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.

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.