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From the Laboratorios de Biologia Celular* and Ultraestrutura Celular,¶ Departamento de Ultra-estrutura e Biologia Celular and Laboratorio de Genomica Funcionale Bioinformatica, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; the Laboratorio de Processamento Digital de Imagens, Pontifica Universidade Catolica, Rio de Janeiro, Brazil; and the Institut National de la Santé et de la Recherche Médicale Equipe Mixte 01-05, Département Réponse et Dynamique Cellulaire, Commissariat à l’Énergie Atomique, Grenoble, France
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Abstract |
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15 million people in Latin America.1
Infective nonreplicative trypomastigote forms of the parasites circulate periodically in the blood of chronic patients whereas proliferative intracellular amastigotes persist in tissues.2
Heart damage and dysfunction are important features in patients with chronic Chagas’ disease and numerous studies are conducted to elucidate the physiopathology of this disease.3
A role for parasite antigens has been proposed to explain the development of extensive fibrosis that is characteristic of the cardiac form of Chagas’ disease.4
We previously reported that circulating levels of transforming growth factor-ß1 (TGF-ß1) are increased in patients with the cardiac form of Chagas’ disease.5
In addition, we observed a contrasting pattern of fibronectin and phosphorylated Smad 2 (an intracellular signal-transducing protein phosphorylated by activated TGF-ß receptors) immunoreactivity in the hearts of patients with chagasic cardiopathy,5
indicating that the TGF-ß signaling pathway is highly active in these patients. All these observations point to a functional link between TGF-ß1 and the parasite T. cruzi in the etiology of chagasic myocardiopathy. TGF-ß1 is the prototypic member of a family of polypeptidic growth and differentiation factors that play a great variety of biological functions in such diverse processes as inflammation, fibrosis, immunosuppression, cell proliferation, cell differentiation, and cell death.6-8 Virtually all cells synthesize and secrete TGF-ß as a biologically inactive protein complex termed latent TGF-ß, which is stored in the pericellular environment. Latent TGF-ß activation results from different enzymatic and nonenzymatic mechanisms9 and only the active form of TGF-ß can interact with the specific transmembrane TGF-ß receptors at the cell surface, inducing cell-signaling and biological responses. TGF-ß1 has already been implicated in three important processes associated with Chagas’ disease: 1) stimulation of fibrosis,5,10 2) parasitic cell invasion,11,12 3) down-regulation of cellular and immune mechanisms of parasite control.13,14 During the course of our studies on the regulation of fibrosis during T. cruzi infection,10 an interesting observation was made: immunolabeling of infected cardiomyocytes using a polyclonal antiserum against human TGF-ß1 revealed immunoreactivity in the intracellular amastigote forms of T. cruzi. In the present study, we further documented this observation and addressed the question of the origin of this intraparasitic TGF-ß. Did it result from synthesis by the parasite or was it taken up from the host cell cytoplasm? Our results indicated that the parasite is able to internalize host cell TGF-ß, to accumulate it during its intracellular proliferation phase and suggested that it may use it as a signaling mediator to trigger differentiation into trypomastigote. So, like for various other species, TGF-ß appears as a regulator of the developmental events driving T. cruzi life cycle.
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Materials and Methods |
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Paraffin-embedded myocardial sections (5 µm) were obtained from T. cruzi-infected mice as described elsewhere.10 Sections were incubated in 10 mmol/L citrate buffer and microwaved for 2 x 10 minutes, followed by saturation for 1 hour at room temperature with 5% normal goat serum in Tris-buffered saline/1% bovine serum albumin. Sections were double stained with anti-human TGF-ß antibody (AB-100-NA; R&D Systems, Oxon, UK) 1:50 and 4,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO) 1:5000. After three washes with Tris-buffered saline/1% bovine serum albumin, secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgGs (Jackson Laboratories, West Grove, PA) were added at 1:100 for 1 hour at room temperature.
In Vitro T. cruzi-Heart Cell Infection
Mouse embryo cardiomyocytes were obtained and grown in primary culture as previously described.15 Briefly, cells were seeded in 24-well plates, incubated for 24 hours at 37°C in a 5% CO2 atmosphere, and cultured in Eagle’s medium supplemented with 0.1% fetal calf serum, 1 mmol/L glutamine, and 2.5 mmol/L CaCl2. To analyze T. cruzi proliferation and differentiation in cardiomyocytes, subconfluent monolayers were incubated at 37°C with T. cruzi trypomastigotes (Y strain) in a parasite/host cell ratio of 10:1, washed out after 24 hours, and monitored for different periods of time (24 to 96 hours). At each time point, the cultures were washed twice in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 20 minutes at 4°C, and processed for immunocytochemistry.
Immunocytochemical Staining
Cell monolayers were incubated with PBS-bovine serum albumin 2% for 3 x 10 minutes and then incubated overnight at 4°C with rabbit anti-human TGF-ß antibodies (AB-100-NA, R&D Systems) or with nonimmune rabbit serum diluted 1:100 in PBS. The monolayers were further incubated for 1 hour at room temperature with the secondary antibody (goat anti-rabbit IgG-FITC diluted 1:100; Jackson Laboratories), incubated for 30 minutes at room temperature with phalloidin-tetramethyl-rhodamine isothiocyanate (TRITC) (1:500) to stain actin fibers and then with DAPI (1:5000) to stain DNA. The slides were then mounted in CytoFluor AF1 (Agar Scientific, Stansted, UK) and observed under a confocal laser microscope (Leica Microsystems, Wetzlar, Germany). Image processing was performed using Zeiss KS-400 software.
Electron Microscopy Analysis
Cells were fixed for 60 minutes at 4°C in a solution containing 0.2% glutaraldehyde, 4% freshly prepared formaldehyde, 0.8% picric acid in 0.1 mol/L cacodylate buffer, pH 7.2. After a postfixation in 1% OsO4 containing 1.5% potassium ferrocyanide for 30 minutes at 4°C, the samples were dehydrated in graded ethanol series, embedded in lowicryl, and collected on nickel grids coated with formvar and carbon. For immunolabeling, sections were washed in phosphate-buffered saline-3% albumin, quenched in 50 mmol/L NH4Cl for 30 minutes, incubated for 1hour at 37°C in the presence of rabbit anti-TGF-ß antibodies (diluted 1:50, R&D Systems), washed three times, and incubated with 5-nm gold particles linked to goat anti-rabbit IgGs (1:100 dilution) for 1 hour. Sections were thinly embedded in a 9:1 mixture of 3% polyvinyl alcohol and uranyl acetate and observed with a transmission electron microscope (EM10C; Zeiss, Oberkochen, Germany) operated at 80 kV. Controls were performed using normal rabbit IgGs or omitting the primary antibody.
Amastigogenesis in Vitro
The infective trypomastigote forms of T. cruzi Y strain were obtained from the blood of infected mice at the peak of parasitemia. In all assays, the living parasites were incubated in serum-free medium. The multiplicative amastigote forms were obtained 24 and 48 hours after acid induction as previously described,16 and counted in a Neubauer chamber.
In Vitro Proliferation of Amastigotes
After 4 hours of acid induction, amastigotes (106/ml) were incubated with 10 ng/ml of recombinant TGF-ß1 (Promega, Madison, WI) and/or anti-TGFß antibodies (AB-100-NA; R&D Systems). 24 hours and 48 hours later, the live parasites were counted in each sample in a Neubauer hematometer.
TGF-ß Binding to Amastigotes in Vitro
The multiplicative amastigote forms were obtained 48 hours after acid induction as previously described.16 Axenic amastigotes (0.5 x 106) were incubated for 60 minutes at 4°C with 20 ng of biotinylated human TGF-ß1 (R&D Systems) in 45 µl of PBS. Ten µl of streptavidin-FITC (10 µg/ml) were then added and incubation was pursued in the dark for 30 minutes at 4°C. The parasites were washed twice, suspended in 0.2 ml of washing buffer (RDF1, R&D Systems), and analyzed as living organisms in a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA). For confocal fluorescence microscopy observation, the parasites were sequentially incubated with biotinylated TGF-ß and streptavidin-FITC as described above and eventually further incubated for 2 hours at 37°C to allow internalization of the TGF-ß biotin-avidin-FITC complexes. The labeled parasites were then fixed with 4% paraformaldehyde and seeded onto polylysine-coated slides. Negative controls were incubated with biotin instead of TGF-ß-biotin or with streptavidin-FITC alone.
Search for TGF-ß Genes in T. cruzi Genome
Genomic DNA was prepared from the T. cruzi II CL-Brenner and T. cruzi I Dm28c strains using standard procedures. Search for TGF-ß-like sequences in the T. cruzi genome were performed by BLAST alignments of the TGF-ß sequences from various species including Homo sapiens, Mus musculus, Drosophila melanogaster, Brugia malayi, and Caennorhabditis elegans on the T. cruzi genome resource (TcruziDB, release 2.2, http://tcruzidb.org/). The same sequences were aligned between them using Mac Vector Align software and several sets of degenerate oligonucleotide primers were selected from the most conserved protein sequence domains using the CODEHOP software on the Infobiogen website (http://www.infobiogen.fr/). These were used for polymerase chain reaction (PCR) amplification of T. cruzi genomic DNA. The sequences of the obtained amplicons were determined by Genome Express (Meylan, France). Control reverse transcriptase (RT)-PCR amplification of mammalian TGF-ß genes was performed using RNAs from human placenta or mouse adrenal glands. Control of T. cruzi DNA quality was achieved by amplifying the parasite actin gene.
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Results |
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We previously reported that the TGF-ß signaling pathway is activated in the infected hearts of human patients with chagasic cardiomyopathy.5
To better understand this mechanism, we wondered whether TGF-ß was detectable in the hearts of T. cruzi-infected mice. The analysis was performed on day 22 after infection, when the parasites have infected various tissues including the heart.10
To our surprise, using a pan-specific polyclonal anti-TGF-ß antibody that recognizes mammalian TGF-ß1 and TGF-ß2 as well as Xenopus TGF-ß5, we observed that TGF-ß immunoreactivity was more intense on intracellular parasites (essentially amastigotes) than in the cytoplasm of cardiomyocytes (Figure 1B)
. To confirm this observation, we performed an in vitro infection of cultured mouse embryo cardiomyocytes with T. cruzi trypomastigotes and stained the infected cells on day 2 after infection with the same anti-TGF-ß antibody. The immunostaining was similar to that observed in infected heart with a stronger staining of the intracellular forms of the parasite (again amastigotes) than of the host cell cytoplasm (Figure 1, F and G)
. Control staining with nonimmune IgGs only displayed a weak background reactivity (Figure 1D)
in regions where DAPI staining (Figure 1C)
showed the presence of the parasites. Careful analysis of the pictures clearly indicated that TGF-ß immunoreactivity (Figure 1, B and F)
was present in every parasite as assessed by DAPI staining of their nuclei and kinetoplast (Figure 1, A and E)
. Using a monoclonal anti-TGF-ß1 antibody (Genzyme), we obtained a similar although slightly less intense immunostaining (data not shown).
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, but absent in the nucleus and kinetoplast because DAPI staining of these DNA-containing structures (Figure 1E
, arrows) did not overlap with TGF-ß immunoreactivity. An enlarged view of amastigotes disclosed a patchy staining (Figure 1G)
and serial confocal sections of amastigotes revealed the presence of large fluorescent spots in the parasite cytoplasm that could correspond to internalization or externalization vesicles (Figure 2; A to C)
. In these latter images, which corresponded to three of nine inner sections in parasites that measure 3 to 5 µm in diameter, the labeling was localized in granules (Figure 2, B and C
; arrows) and in the area of the flagellar pocket, as seen in longitudinal (Figure 2, B and C
; filled arrowhead) or sagittal (Figure 2, B and C
; open arrowhead) section planes. To further characterize the intraparasitic TGF-ß immunostaining, we deconvoluted the confocal images taken on infected cardiomyocytes along the z axis (Figure 2D)
. This allowed us to confirm that the stained vesicular structures were inside the parasite rather than on its surface.
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. In all 38 electron microscopic images that were generated, gold particles could be seen on the surface, in the flagellar pocket, and in granules of amastigotes. TGF-ß thus appeared to be localized in the endocytic/exocytic parasite machinery.
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These intriguing observations suggested two possibilities concerning the origin of TGF-ß immunoreactivity. Either a TGF-ß-like molecule is synthesized by the parasite and its sequence is sufficiently conserved to be recognized by both polyclonal and monoclonal anti-vertebrate TGF-ß antibodies or mammalian TGF-ß is taken up by the intracellular parasites from the host cells.
We first tried to address the question of the possible existence of a TGF-ß gene in the T. cruzi genome. Members of the TGF-ß superfamily of growth and differentiation factors have been identified in a wide variety of organisms, ranging from invertebrates to mammals,6,17-19
and the existence of molecular mimicry between T. cruzi and mammalian hosts20
suggested that a TGF-ß ortholog might exist in the T. cruzi genome. Conserved peptide motifs throughout TGF-ß proteins, spanning species from nematodes to human, were identified. We chose to study four such motifs from the N-terminal part, and four motifs from the C-terminal part, corresponding to the least amount of degenerate codon possibilities. Next, using a T. cruzi-specific codon table, nondegenerate primers were designed corresponding to the selected sequences. In this way, four forward primers and four reverse primers were synthesized and used in different combinations so as to amplify potential TGF-ß gene fragments by PCR on T. cruzi genomic DNA because the T. cruzi genes are intronless (Table 1)
. Under stringent PCR conditions, no amplification was obtained whereas, under less stringent conditions, several bands could be obtained with some combinations of primers. However, after sequencing of the amplification products, none of these fragments proved to have any homology with TGF-ß. We then designed conserved and/or degenerate primers from the alignment of human, bovine, murine, and C. elegans TGF-ß and performed RT-PCR analyses on RNAs from human placenta and murine adrenal glands and PCR analyses on T. cruzi DNA (Table 1)
. Although correct amplification could be obtained with mammalian tissues, no amplification of a TGF-ß-related gene was obtained from T. cruzi cDNA or genomic DNA.
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T. cruzi Can Take Up Exogenous TGF-ß
We then tried to check whether T. cruzi amastigotes could bind and internalize exogenous TGF-ß. An experimental model allowing to produce T. cruzi amastigotes under host cell-free conditions has been described: acidic pH treatment of trypomastigotes collected either from the supernatant of infected mammalian cells or from the blood of infected mice induces amastigogenesis and yields viable and proliferating amastigotes.16,22
Blood trypomastigotes were acid-induced in vitro to differentiate into amastigotes and these were incubated with biotinylated TGF-ß for 1 hour at 4°C. After extensive washes, the parasites were incubated with streptavidin-FITC and analyzed by flow cytometry. In parallel, some preparations were eventually further incubated at 37°C to allow internalization, then fixed, spread on a glass slide, and observed under a confocal microscope. Flow cytometry analysis of amastigotes permitted the design of an unambiguous window containing parasites (polygon in Figure 4A
), and the fluorescence analysis inside this window revealed that 25% of the parasites had bound biotinylated TGF-ß (Figure 4B)
. Images of the parasites incubated at 4°C with biotinylated TGF-ß revealed patches of fluorescence in the region of the cytostome (Figure 4, C and D)
. If the parasites that had bound biotinylated TGF-ß at 4°C were then submitted to a further incubation for 120 minutes at 37°C, microscopic observation of the parasites revealed homogeneous fluorescent labeling of the whole cytoplasm (Figure 4E)
. Confocal deconvolution of the images confirmed that the fluorescence was intracellular but, due to the extreme flatness of the fixed axenic amastigotes, it was impossible to discern intraparasitic structures. No staining was observed when the parasites were incubated with biotin instead of biotinylated TGF-ß (Figure 4F)
or with streptavidin-FITC alone (Figure 4G)
. This prompted us to conclude that axenic amastigotes are able to bind and internalize exogenous TGF-ß.
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We then wondered whether T. cruzi parasites were constantly immunoreactive for TGF-ß during the intracellular parasitic cycle. Cultures of infected cardiomyocytes were fixed at various periods of time after infection and TGF-ß immunoreactivity (stained with anti-TGF-ß-FITC) was imaged by piling up confocal microscopy images. The cells were stained for actin using phalloidin-TRITC to visualize the host cell architecture in red. Image processing using a threshold value for eliminating background FITC fluorescence confirmed the localization in the parasite cytoplasm and the specificity of the TGF-ß staining (shown in light blue Figure 5; C, F, and I
). At 24 hours and 48 hours, the parasites were mainly amastigotes as characterized by DAPI staining (Figure 5, A and D)
and were intensely stained for TGF-ß (Figure 5; B, C, E, and F)
. As shown in the inset of Figure 5E
, the pattern of staining appeared cytoplasmic with the location of the nucleus appearing as a black hole (as already noted in Figure 1, F and G
). At 72 hours, TGF-ß staining was still strong, but more heterogeneous (Figure 5, H and I)
, indicating a progressive decrease of TGF-ß immunoreactivity. This decrease was much more pronounced at 96 hours, when differentiation to trypomastigote was complete as shown by DAPI staining (Figure 5J
, arrows) and only a faint staining remained detectable (Figure 5, K and L
; arrows). Specificity of the TGF-ß labeling was confirmed by absence of staining in negative controls that were treated with nonimmune serum and FITC-labeled secondary antibodies (data not shown). Image processing of the original immunofluorescence micrographies allowed a better view of the changes in TGF-ß reactivity in the parasites during the intracellular cycle (Figure 5; C, F, I, and L)
and confirmed the dramatic decrease in TGF-ß immunoreactivity during the transition of amastigotes toward trypomastigotes.
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(shown in higher magnification in Figure 6; C, E, G, and I
) contained parasites that were heterogeneously immunoreactive for TGF-ß. DAPI staining of parasite DNA (Figure 6B
, shown at higher magnification in Figure 6; D, F, H, and J
) allowed to recognize amastigotes (yellow circles in Figure 6; C, D, E, and F
) from transitional forms (orange ellipses in Figure 6; C, D, G, and H
) and trypomastigotes (purple ellipses in Figure 6; C, D, I, and J
). TGF-ß immunoreactivity was strong in all amastigotes, weak in all trypomastigotes, and either strong, mild, or weak in the transitional forms. Because all these forms co-existed in the same cardiomyocyte, it can be concluded that the loss of TGF-ß immunoreactivity is not dependent on changes in the host cell cytoplasm but is rather an intrinsic response of the parasite associated with cycle progression from amastigote to trypomastigote.
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The progressive decrease of TGF-ß immunoreactivity observed during the amastigote-trypomastigote transition is concomitant with the arrest of intracellular parasite proliferation. Because TGF-ß is a well established growth inhibitor for a number of mammalian cell types,23
we wondered whether it could have a similar effect on T. cruzi amastigotes. We measured the proliferation of axenic amastigotes for 24 and 48 hours, in the presence or absence of recombinant TGF-ß1 (Table 2)
. The results from three different experiments showed that, under control conditions, the parasite population doubled between 24 hours and 48 hours (ratio 48:24 hours = 1.9 ± 0.2), whereas in the presence of 10 ng/ml TGF-ß1, the growth was markedly reduced (ratio 48:24 hours = 1.3 ± 0.1). This difference was statistically significant (P = 0.05) and was emphasized (P = 0.007) by the effect of neutralizing the cytokine with anti-TGF-ß. This halt in proliferation was not due to parasite cell death because parasite motility was sustained and vital labeling with propidium iodide did not show any important modification (data not shown).
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Discussion |
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How can the parasite pick up TGF-ß inside its host cell? This is an intriguing question because TGF-ß, being a secreted protein, possesses a signal peptide and is therefore synthesized in the lumen of the endoplasmic reticulum, glycosylated in the lumen of the Golgi apparatus, and constitutively secreted without being released in the cytoplasm. Also, TGF-ß is synthesized under a latent form consisting of a noncovalent association between the dimeric precursor part of the TGF-ß gene product (LAP: latency-associated peptide) and the dimeric mature protein (C-terminal peptide). This maturation occurs along the secretion pathway. The antibody that we used for TGF-ß immunolocalization has been raised against the mature isoforms TGF-ß1, TGF-ß2, and TGF-ß5 and does not recognize the C-terminal peptide when it is engaged in a latent complex. However, it has been widely shown that the fixation steps necessary for immunofluorescence analyses can activate latent TGF-ß and render the C-terminal peptide accessible to the antibody. In other terms, under our experimental conditions, the antibody recognizes both mature and latent TGF-ß, and the observed immunoreactivity may correspond to either of these two forms. Three properties of the parasite may explain how it can gain access to cellular TGF-ß. First, the parasite has been shown to be in close contact with endoplasmic reticulum membranes of the infected cell (M.N.L. Meirelles, personal communication); second, it has the capacity to engulf membrane vesicles;24
third, as shown in this study, it has the capacity to bind and internalize recombinant TGF-ß. Our hypothesis is that amastigotes could take up TGF-ß-rich secretory vesicles through their flagellar pocket. In agreement with this hypothesis, electron microscopy showed that TGF-ß was present in the flagellar pocket, a major exchange vesicle, as well as in other intracellular granules. Multifunctional endocytic receptors that interact with TGF-ß or with other proteins associated with TGF-ß could be potential candidates, eg, the LRP (LDL-receptor related protein)/
2M-R(2-macroglobulin receptor) that we previously described in T. cruzi.25
It was recently shown that the type-V TGF-ß receptor, which plays an important role in growth inhibition by TGF-ß in responsive cells, is identical to the LRP-1/2-M receptor.26
The absence of a TGF-ß-like gene in the genome of T. cruzi was somehow unexpected because such orthologs (homologous genes in different organisms) have been found in the genomes of nematodes, ascidians, and insects. However, T. cruzi belongs together with other kinetoplastids and with euglenoids to the phylum of Euglenozoa.27 It must be noted that this phylum is more ancestral than those of Craniates, Arthropods, and Nematods in which TGF-ß orthologs have been characterized.
The rapid decrease of TGF-ß immunoreactivity during the transition from amastigote to trypomastigote also opens a number of questions. This decrease may result either from secretion into the host cell cytoplasm of TGF-ß that was accumulated inside the parasite, or from a degradation or modification of the stored TGF-ß in a way that masks its immunoreactivity. If this is the result from secretion, then TGF-ß would flow into the cell cytoplasm and probably induce parasite proliferation arrest, which is what occurs at this specific stage of the parasite cycle. Then, active TGF-ß released during host cell disruption could directly induce extracellular matrix protein synthesis by other infected and/or noninfected cardiomyocytes, thus promoting heart fibrosis by itself, as shown previously.4,5,10,13
Moreover, the present results also suggest the presence of TGF-ß receptor(s) on T. cruzi cell surface, as well as the existence of a downstream signaling pathway that would trigger the anti-proliferative effect of TGF-ß. Orthologs of the canonical serine-threonine kinase TGF-ß receptors (TßRI and TßRII) and Smad proteins have been identified in the helminth parasite Schistosoma mansoni28-30
but could not be found after in silico analysis of the T. cruzi genome. However, several non-Smad signaling pathways are now known to be activated or modulated by TGF-ß in eucaryotic cells. These include the Jun-kinase, p38MAP-kinase, Ras/MEK/ERK, Rho-A/p160ROCK, and PP2A/S6kinase.31
Interestingly, homologs of Ras,32
Rho,33
and ERK34,35
have been characterized in T. cruzi and Trypanosma brucei, suggesting that at least some of these alternative TGF-ß pathways might be functional in these parasites. A more detailed molecular analysis of T. cruzi TGF-ß binding proteins and downstream signaling molecules is under current investigation in our laboratories. It should be remarked that other mammalian growth factors (namely epidermal growth factor and TGF-) have been shown to induce signal transduction events and cellular proliferation in T. cruzi amastigotes through binding to specific receptors.36,37
The novel role of host cell TGF-ß described herein, adds complexity to T. cruzi biology and discloses additional functions for this cytokine in Chagas’ disease. TGF-ß thus appears: 1) to be generated at the host cell surface via parasite-mediated activation of latent TGF-ß,38 2) to induce downstream signaling along the host cell TGF-ß receptor pathway thereby favoring cell invasion,11,12 3) to be taken up intracellularly by the parasites and to control differentiation from amastigotes into trypomastigotes (present study), and 4) to trigger fibrosis in Chagas’ cardiomyopathy.4,5,10,13,14
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Acknowledgements |
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Footnotes |
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Supported by an Institut National de la Santé et de la Recherche Médicale-Fundação Instituto Oswaldo Cruz (FIOCRUZ) collaborative research program, by grants from Programa de Apoio à Pesquisa Estratégica em Saúde/FIOCRUZ, Instituto Oswaldo Cruz, Conselho Nacional de Desenvolvimento Científico e Techológico, Fundação de Amparo a Perquisa do Estado do Rio de Janeiro to the Brazilian laboratories, and by recurrent funding from Commissariat à l’Energie Atomique and INSERM to the French laboratory.
This work was part of the Ph.D. thesis of M.C.W.
M.C.W. and M.K. equally contributed to this work and should be considered as first co-authors.
T.C.A.-J. and J.-J.F. equally contributed to the scientific direction of this work and should be considered as last co-authors.
Accepted for publication June 30, 2005.
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