J. Duaso1; G. Rojo1; G. Cabrera1; J.D. Maya1; C. Bosco1; A. Morello1; N. Galanti1; U Kemmerling1,2
Chagas' disease, one of the major public health concerns in Latin America, is caused by the haemophlagelated protozoan Trypanosoma cruzi. In vector related diseases, it is second to malaria in prevalence and mortality. The disease spreads from southern United States through southern Argentina and Chile. At least twenty-eight million people are at risk of exposure to infection, with an estimated total of fifteen million cases.
T. cruzi presents an indirect life cycle, with hematophagous insects (Triatomids) as intermediary hosts, and mammals including human, domestic animals such as dogs and a wide range of wildlife species, as definitive hosts (Kjos et al 2008). Canines in South and Central America are considered an important reservoir in the domestic Chagas' disease transmission cycle (Gürtler et al 1993), and serve as surveillance sentinels for human infection (Estrada-Franco et al 2006). It has been shown that dogs have a higher capacity to infect the triatomine vectors than human due to persistent parasitemia, further supporting their role in the disease cycle in domestic setting (Gürtler et al 1991, Kjos et al 2008). In the past few years congenital transmission of T. cruzi has increasingly become more important, and partly responsible for the "globalization of Chagas' disease" (Schmunis, 2007), constituting a public health problem of increasing relevance (Lescoure et al 2008).
The placenta is the principal site for the exchange of nutrients and gases between mother and fetus. It is composed of a fetal portion, developed from the chorion frondosum, and a maternal portion, or basal decidua, which originates from the endometrium (Benirschke K et al 2006). The placental barrier or "membrane" is composed of extra fetal tissue that separates maternal blood from that of the fetus. In endotheliochorial placenta, the placental barrier is composed of syncytiotrophoblast, cytotrophoblast, connective tissue from the free chorionic villi, fetal and maternal capillary endothelium; the hemochorial placenta lacks maternal capillary vessels (Benirschke K et al 2006).
Diverse pathogens, including parasites, are able to cross the placental barrier and infect both the placenta and fetus. Among these are Toxoplasma gondii (Correa, et al 2007), Toxocara canis and Toxocara cati (Vignau et al 2005) as well as T. cruzi (Sartori et al 2005).
Parasite invasion in cell cultures has been studied in some depth. On the other hand, studies that analyze parasite invasion in tissues and organs are scarce. T. cruzi penetration in host cells occurs through a complex multi-step process that includes both parasite and host cells.
T. cruzi possesses a series of surface molecules that interact in a differential manner with molecules from the host's cells and extracellular matrix (ECM) (Yoshida, 2006). During tissue invasion by the parasite, interaction between T. cruzi and the ECM is fundamental. The parasite should cross the basal membrane located in different epithelia as well as the adjacent connective tissue and mobilize itself within these structures. T. cruzi secretes proteases (cruzipains) capable of degrading ECM components such as collagen type I, IV and fibronectin (Santana, et al 1997, Scharfstein and Morrot 1999).
Induction of cellular alterations and interaction with the ECM during T. cruzi infection has been studied mainly in mammalian cell cultures, corresponding to cell lines and not to primary cultures. Nevertheless, human placental tissue has been used as a possible study model for ex vivo parasitic tissue infection (Sartori, et al. 2005). The possibility to count on a tissue culture for the study of parasitic invasion creates working conditions that are more similar to those found in vivo.
In our laboratory, we have been able to reproduce ex vivo T. cruzi human placental infection in similar conditions to those previously described (Sartori, et al 2005) and in canine placental tissue as well. So we are able to study the invasion and infection of T. cruzi in the hemochorial human placenta and the endotheliochorial canine placenta.
Materials and Methods
Infection of VERO cells with T. cruzi and trypomastigote harvesting: Green Monkey (Cercopithecus aethiops) renal fibroblast like cells (VERO cells) were grown in RPMI medium enriched with 5% fetal calf serum and antibiotics (penicillin-streptomycin) at 37°C in a humid atmosphere at 5% CO2. After confluence, VERO cells were incubated with a culture of epimastigotes from the MCV, DM28c or SN3 strains while in late stationary phase, which increases the percentage of trypomastigotes. Trypomastigotes then invade fibroblasts and replicate intracellularly as amastigotes. After 72 hours, trypomastigotes lyse host cells and are recuperated by low speed centrifugation (500 g) from the supernatant.
Placenta and chorionic villi culture: Human term placentas were obtained from uncomplicated pregnancies with a single, normal fetus delivered by elective caesarean section, in order to ensure asepsis and preservation of the sample. Canine placenta was obtained from an uncomplicated cesarean section after dog owner´s request. The organs were collected in cold sterile saline-buffered solution (PBS) and processed no more than 1 h after delivery. The maternal and fetal surfaces were discarded and villous tissue was obtained from the central part of the cotyledons. The isolated chorionic villi were washed with PBS in order to remove blood, and cut in 0.5 cm3 pieces. Placental villi were co-cultured with 1x106 trypomastigotes of MCV, DM28c or SN3 strains for 24 hours in culture media RPMI supplemented with inactivated FBS and antibiotics. Control villi were maintained in the same conditions without parasites.
Histological techniques: The placental villi were fixed in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.3) for 24 h, then dehydrated in alcohol, clarified in xylene, embedded in paraffin, and sectioned at 5μm. Paraffin histological sections were stained with haematoxylineosin and Picrosirius-hematoxylin.
Immunohistochemistry: Standard immunoperoxidase techniques were used to show the distribution of different antigens in tissue sections of the chorionic placental villi incubated in presence or absence of the parasite. The following antibodies were used: 1) anti-T. cruzi cruzipain as T. cruzi marker (from the laboratory of Dr. Juan José Cazzulo, Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, Buenos Aires, Argentina), dilution 1:1000 v/v. 2) antihuman placental lactogen as syncyciotiotrophoblast marker (Novocastra NCL-PLp), dilution 1:250 v/v. Immunostaining was performed by using a horseradish peroxidase-labelled streptavidin biotin kit (ScyTek, ACA, Kit RTU Vectastain) following the manufacturer's directions, using diaminobenzidine as chromogen. Sections were counterstained with Mayer's hematoxylin (ScyTek) and mounted with Entellan (Merck). Immunohistochemical controls were carried out by replacing the primary antibodies with saline phosphate buffer. All sections were examined by light microscopy (Zeiss Axioplan 2).
Parasite DNA detection by the polymerase chain reaction: Genomic DNA was extracted from the placental tissue according to Lahiry and Nürnberger (Lahiry and Nürnberger, 1991). The DNA solution was suspended in endonuclease free sterile water and stored at -20°C until further use. A 450 base pair fragment of the T. cruzi GAPDH gene (T. cruzi GAPDH CONTIG AAHK01000012) was amplified. The sequence of the oligonucleotides is the following: forward: 5´-CAGAGC-CTCAGTGTTGGTG-3´ and backward: 5´-TCAATACT-CACTCTTGTTTGG-3'. The PCR product was subjected to electrophoresis in 1.6% agarose gels and stained with ethidium bromide. PCR markers from Promega were used.
The infection of canine and human placental chorionic villi was confirmed by immunohistochemistry and semiquantitative PCR. Chorionic villi incubated with trypomastigotes showed presence of the parasite inside the chorionic villi and in the haematic chamber that surrounds them in the hemochorial placenta and inside the maternal capillaries in the endotheliochorial placenta. In all samples incubated with trypomastigotes, from different parasite strains, important tissue damage with respect to control villi was observed. The syncytiotrophoblast separates itself from the cytotrophoblast, in some areas the syncytiotrophoblast is disaggregated, and the immunoreactivity of the placental lactogen, a syncytiotrophoblast marker, is clearly altered. The ECM of the fetal connective tissue in the central part of the chorionic villi presents also an important damage. The normal distribution of collagen I fibers is disorganized and the relation collagen I versus collagen III fibers is altered as demonstrated by Picrosirius stain.
Discussion and Conclusions
The placental tissue has been widely used in biomedical studies, specifically the human placental tissue. This is the first report of the use of canine placental tissue as an ex vivo model for the study of the mechanism of infection and invasion of T. cruzi. The use of placental tissue of both origins allows compare the infection and invasion mechanism of the parasite in a hemochorial and in an endotheliochorial placenta. The placental barrier in an endotheliochorial placenta is supposed to be more efficient than a hemochorial placenta because of the presence of maternal capillaries, which are absent in the hemochorial placenta. One limitation of this ex vivo infection model is that the parasite supply occurs via the culture media and not via maternal circulation. In this way, the barrier formed by the maternal capillary and their basal lamina is not functional. Nevertheless we were able to infect the canine chorionic villi with the same efficiency as the human chorionic villi, observing similar damage in both tissues. These results validate the use of canine placental explants as a model for study the infection of T. cruzi in canine tissue. This is of major importance considering that canines participate in the domestic transmission cycle of Chagas' disease. Additionally, the use of placental tissue explants to study the mechanisms of T. cruzi infectivity and invasion will allow advancement in the knowledge of the pathophysiology of congenital Chagas' disease.
Supported by "Bicentennial Program in Science and Technology Act-29; Fondecyt 11080166".
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