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Acta Tropica 130 (2014) 51–57

Contents lists available at ScienceDirect

Acta Tropica

journa l homepage: www.e lsev ier .com/ locate /ac ta t ropica

ltrastructural alterations in adult Schistosoma mansoni, harbored inon-antihelminthic treated and low-inflammatory mice byransmission electron microscopy (TEM)

urelizia Maria Lemos Xaviera,b,1, Daniel Tavaresa,∗,1, Erick Vaz Guimarãesc,aria de Fátima Sarro-Silvaa, Antonio Carlos Silvaa,

ntonio Henrique Almeida de Moraes Netod

Laboratório de Imunobiologia, Departamento de Genética, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio deaneiro (UERJ), 20550-900 Rio de Janeiro, BrazilDepartamento de Imunobiologia, Universidade Federal Fluminense (UFF), 24000-000 Niterói, Rio de Janeiro, BrazilLaboratório de Biologia Estrutural (LBE), Instituto Oswaldo Cruz (IOC), Fundacão Oswaldo Cruz (FIOCRUZ), 21040-900 Rio de Janeiro, BrazilLaboratório de Inovacões em Terapias, Ensino e Bioprodutos (LITEB), Instituto Oswaldo Cruz (IOC), Fundacão Oswaldo Cruz (FIOCRUZ),1040-900 Rio de Janeiro, Brazil

r t i c l e i n f o

rticle history:eceived 11 February 2013eceived in revised form 31 July 2013ccepted 15 October 2013vailable online 23 October 2013

eywords:chistosoma mansoniegumentltrastructure

a b s t r a c t

This original study suggests that alterations observed on tegumental structure and egg quality of adultSchistosoma mansoni harvested from TS mice are due to their high immune tolerogenic and low-inflammatory capacity. The tegument of worms harvested from genetically selected mice for extremephenotypes of immune oral tolerance, resistance (TR) and susceptibility (TS) were analyzed by transmis-sion electron microscopy (TEM). Parasites recovered from TR mice showed no tegumental morphologicalchanges. However, specimens collected from TS mice exhibited tubercle swelling with blunted andshortened spines in lower density. These tegumental alterations were similar to those described withartemether or praziquantel treatment, but without to affecting the worm surveillance, supporting obser-vations that the host immune system influences the development and function of the tegument of worms

ransmission electron microscopy (TEM)mmune responseral tolerance

harbored in non-antihelminthic treated TS mice. TS mice showed a higher percentage of dead eggs anda lower percentage of immature eggs than TR mice, but had similar quantities of collected eggs. Thissuggests that in TS mice the alterations in adult worm tegument prevented egg development, but not eggproduction or worm survival. These results corroborate our previous scanning electron microscopy (SEM)study indicating the influence of the host immune regulatory profile on the development and function

ve sy

of the worm’s reproducti

. Introduction

Schistosoma mansoni in its different phases of the life cycle has itshysiological and reproductive state strongly influenced by envi-onmental and host factors (Senft et al., 1978; Cioli et al., 1977).he response of the worms at different stages possibly facilitatesurvival under adverse conditions (Davies and McKerrow, 2003),nd the gene regulation of parasites allows adaptation to different

nvironments associated with morphological and biochemicalransitions (Jolly et al., 2007). The parasite uses the signals pro-ided by the host immune system for replication and spread (Amiri

∗ Corresponding author. Tel.: +55 21 2334 0499; fax: +55 21 2334 0309.E-mail addresses: liz@vm.uff.br (A.M.L. Xavier), tavares.uerj@gmail.com

D. Tavares).1 Both authors contributed equally as first authors to this work.

001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.actatropica.2013.10.014

stem and tegument.© 2013 Elsevier B.V. All rights reserved.

et al., 1992). According to Hernandez et al. (2004), the male schis-tosome appears to play a central role both in transducing signalsfrom the adaptive immune system (Hernandez et al., 2004; Daviesand McKerrow, 2003) and in facilitating female development. Itis also noted that the genetic variability among individual hostsmay contribute differently to the outcome of infection (Davies andMcKerrow, 2003), and the characteristics of the parasite can changeaccording to the genetic profile of the host (Incani et al., 2001). Dam-age to the tegument and subtegumental structures of the worm,as well as reductions of the schistosome worm burden have beenobserved in infected mice treated with drugs such as artemether,artesunate or praziquantel (PZQ) (Shaohong et al., 2006; Shuhuaet al., 2002, 2000). It is reported that the tegument of parasites

derived from patients with resistance to PZQ, is resistant to itseffects in the original patients, as well as in experimental animalsand in vitro studies of this drug (Melman et al., 2009; William et al.,2001). Protection from schistosomiasis infection is achieved by

52 A.M.L. Xavier et al. / Acta Tropica 130 (2014) 51–57

opula

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Fig. 1. Production of the foundation p

odulating cytokine responses to PZQ (Martins-Leite et al., 2008),hus, the efficacy of PZQ would be dependent on the host immuneesponse (Ribeiro et al., 2004; Farah et al., 2000). Specific antibod-es against antigens expressed in S. mansoni tegument have beeneported to cause damage to adult worms recovered from miceTeixeira de Melo et al., 2010), and combined exposure of praz-quantel and antibodies induce more morphological damage andncreased severity than serum or drugs alone (Modha et al., 1990).

To corroborate previous observations of possible immunomod-latory effects of characteristics of the host on the parasite S.ansoni by scanning electron microscopy (SEM) we used mice

enetically selected to extremes for oral tolerance resistance (TR)nd susceptibility (TS) (Silva et al., 1998) on S. mansoni parasite.ral tolerance is a physiological mechanism that induces immuneeripheral tolerance and has been demonstrated by the suppres-ion of specific immune responses by prior administration of thentigen by oral route (Garside et al., 1999; Mowat, 1994).

The TR and TS mice were obtained by bidirectional geneticelection of mice through assortative matings and consanguinityvoidance, during 25 generations, to establish the best phenotypesf susceptibility (TS strain) and resistance (TR strain) to oral tol-rance of the ovalbumin (OVA) humoral response. The selectionas started from a genetically heterogeneous population produced

y the equilibrated intercrossing of eight inbred mouse strainsA/J, DBA/2J, P/J, SWR/J, SJL/J, CBA/J, BALB/cJ and C57BL/6J) (Fig. 1)nd repeated during generations of consecutive breeding (Silvat al., 1998). This strategy favors the selection of coherent sets ofultiple genes participating in the desired oral tolerance pheno-

ype. Unlike isogenic strains, in which each mouse carries a singleenome, our tolerance-resistant (TR) and tolerance-susceptibleTS) mice are genetically homogeneous at the relevant loci forhe selected character and heterogeneous in terms of backgroundenes. These selected mice show differences in infectious diseasesnd inflammatory responses studies (Silva et al., 2001a,b). TS micere a low-inflammatory strain, producers of high levels of immuneegulatory cytokines such as IL-10 and IL-4 and present high per-entages of CD4+, CD25+, Foxp3+, T regulatory cells. In contrast, TRice are shown as a high-inflammatory strain and present diffi-

ulties in developing immune suppressive or inhibitory responsesSilva et al., 2010, 2006, 2004; Kamphorst et al., 2004).

As TR and TS phenotype selection is based on modification of themmune function, these animals demonstrate altered responses tonfectious agents (Tavares et al., 2006; Silva et al., 2001a,b). Con-equently, the diverse immunoregulatory profile of these strainsf mice could present different conditions for adaptation and sur-ival of the parasite. Our previous studies have indicated that whenompared by SEM the tegument of adult worms recovered fromR mice displayed no morphologic alteration, while specimensollected from TS mice presented tubercle swelling with bluntednd shortened spines in lower density, increased sensory organelleumbers, fusion and tegumental ridge peeling (Xavier et al., 2010).

he present study aimed to determine by TEM if the ultrastructuref adult S. mansoni could be modified in the environments of theseon-antihelminthic treated and non-immunized mice presentingifferent immune regulatory capacities.

tion from eight inbred mouse strains.

2. Material and methods

2.1. Animals

TR and TS strains of mice from the F25 generation, obtainedby two-way genetic selection according to susceptibility (TS) orresistance (TR) to ovalbumin (OVA) oral tolerance, were adopted.The original foundation population, from which the TR and TSstrains were derived, was achieved by the balanced intercrossingof eight inbred mouse strains (Jackson Laboratory, Bar Harbor, ME)of distant origins, (A/J, DBA/2J, P/J, SWR/J, SJL/J, CBA/J, BALB/cJ, andC57BL/6J) (Silva et al., 1998) (Fig. 1). The selective breeding thatgave rise to these strains was carried out in the Genetics Depart-ment of the Rio de Janeiro State University. The mice were lodgedtogether in our animal facilities. The Committee for the Care andUse of Laboratory Animals of the Universidade do Estado do Rio deJaneiro, Brazil, approved the protocols of the experiments describedin this paper (029/2010/UERJ).

2.2. Parasites and infection

Eight week old mice of TR and TS strains were exposed to50 transcutaneous cercariae obtained from Biomphalaria glabrata,infected with S. mansoni (BH strain, Brazil) from a life cycle main-tained in the Reference Laboratory in Malacology of InstitutoOswaldo Cruz (IOC), FIOCRUZ, RJ, Brazil.

2.3. Helminths

Ten adult worms of each sex, obtained by perfusion from 4 TSand TR mice each, 8 weeks after infection were collected from theportal and mesenteric veins in PBS pH 7.2 during necropsies of theabdominal cavity (Xavier et al., 2010). Each specimen was dividedin three regions: anterior, midbody and posterior.

2.4. Transmission electron microscopy (TEM)

For TEM, the previously fixed helminths in Karnovsky moisturefor 2 h at room temperature (2.5% glutaraldehyde, with 4% freshlyprepared paraformaldehyde and 5 mM calcium chloride in 0.1 Msodium cacodylate buffer, pH 7.2). Post-fixation was conductedwith 1.0% osmium tetroxide in cacodylate buffer pH 7.2 + 0.8%potassium ferricyanide + 5 mM CaCl2 and washed in 0.1 M cacody-late buffer; pH 7.2. The worms were subsequently dehydrated inacetone series; embedded in Spurr’s resin; thin sections (60–70 nm)were collected on 300 mesh copper grids, counterstained withuranyl acetate and lead citrate, and finally, observed with a JEOL EM1011 Transmission Electron Microscope of Microscopy Platform ofthe Instituto Oswaldo Cruz, FIOCRUZ.

2.5. Tegument measurements

The ImageJ program was used to obtain the tegument mea-surements. Three to five measurements of each micrograph wereacquired from each worm body region and observed at the same

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utting plane and magnification. At least 10 micrographs wereerformed from each body region of the helminth specimen corre-ponding to a total of 30–50 measurements to obtain the arithmeticean of each region. The measurements are given in micrometers

�m) unless otherwise stated. Mean values and standard devia-ions of the measurements of the structures are followed by rangesnclosed in parentheses.

.6. Oogram

The eggs were quantified by digestion of the ileum, secondheever (1968). Fragments of tissues were weighed and kept insolution of potassium hydroxide (KOH) at 4%, at room tempera-

ure (26 ◦C), for 5 h and incubated at 37 ◦C for 1 h. The digested eggsere resuspended in 10 mL of 4% KOH and counted at least 100 �Ler sample in triplicate in optical microscope (an increase of 100×).

The average count of aliquots of each sample were extrapo-ated to the total sample volume and weight related to the digestedample.

.7. Statistical analysis

The comparisons of the tegument measurements, the recoveryf adult worms and the egg numbers harvested from TS and TRice utilized Student’s t test and ANOVA. Groups were considered

tatistically different if p ≤ 0.05.

. Results

The TEM analysis of adult male worms harvested from TRice showed the tegument intact in the anterior region (Fig. 2A).

pines with a regular morphology (Fig. 2B) near the basal lam-na were observed as well as a large number of vacuoles withntact membranes. At the midbody region of the tegument and inhe subtegument the presence of fat cells and cells that metab-lize nutrients were observed (Fig. 2A and B). The muscularayer exhibited circular fibers with preserved membranes (Fig. 2A)hroughout the body. The subtegumental cytons showed well-efined membranes. The tubercles showed spines with unchangedegular structure ranging in size from 2.0 to 4.0 �m. Vacuoles werebserved with intact membranes inside the tubercles. At the pos-erior region of adult male worms from TR mice, the tegumenthowed tubercles with spines and regular structures, as well as anntact internal structure (Fig. 2C). In contrast, TS tegument showedubercles without spines (Fig. 2F).

The tegument and subtegumental structures of adult S. mansoniorms harvested from TS mice showed ultrastructural damages.t the anterior region of adult male worms from TS mice, the tegu-ent was thinner (1.56 ± 0.74; 0.55–3.27) when compared to the

ame region from TR mice (2.18 ± 0.47; 1.33–3.32). At this regionFig. 2D) and the midbody of male adult worms collected fromS mice, the tegument showed swelling with the occurrence ofarge vacuoles, with a tendency to detach from the surface of the

orm and lysis of subtegumental cytons. The spines were internal-zed (Fig. 2E). In the tubercles, internal disorganization with loss of

atrix integrity, lysis in the internal structures, vacuoles of variousizes with residual bodies and no spines were observed (Fig. 2F).

Tegumental thickness at the midbody region did not showifferences between the strains. It was 2.33 ± 0.94 (0.92–4.89)nd 2.45 ± 0.91 (1.11–4.55) in worms harvested from TS and TRtrains, respectively. In contrast, at the posterior region, the tegu-ent thickness of worms harvested from TS strain was thinner,

.36 ± 0.60 (1.24–3.86), when compared to TR worms, 3.74 ± 0.882.38–5.54). The gut epithelial cells had not been affected.

The anterior and midbody regions of adult female wormsrom TR mice presented well-organized vitelline cells, organized

ica 130 (2014) 51–57 53

membranes, vacuoles and integral tegument without apparentchanges (Fig. 3A). The tegument thickness in these regions was4.29 ± 1.20 (2.11–6.99) and 3.15 ± 1.01 (1.45–5.43), respectively.At the posterior region, the tegument showed spines and intactuterine cells (Fig. 3B).

The midbody and posterior regions of adult female worms fromTS mice showed a disorganized intensely vacuolated tegument(Fig. 3 C), which were thinner than in worms from TR mice. Thetegument thickness of TS worms at these regions, respectively,were: 1.25 ± 0.32 (0.57–1.84), 3.01 ± 0.66 (1.91–4.30); and in theTR worms: 3.15 ± 1.01 (1.45–5.43), 4.30 ± 0.84 (2.15–5.75). Femaleworms from TS mice presented vitelline cells with vitelline ballsdivided into small pieces and fragmented endoplasmic reticulum(Fig. 3D and E).

TR and TS mice showed a high percentage (TR: 46%, TS: 41%) ofadult worms recovered after percutaneous infection (Fig. 4A). Therewas/were no differences in gender of recovered worms (Fig. 4B).

There was no difference in oviposition of female worms in bothmice strains. However, the percentage of dead eggs was signifi-cantly higher in TS, while the percentage of immature eggs presentin the ileum at the 8th week of infection was significantly higher inTR (Fig. 4C).

4. Discussion

It is known that schistosomes are highly dependent on the hostmetabolism. The tegument of schistosomula, being the interfacebetween the parasite and host, is an important target for the hostimmune system. Mice are permissive to infection with S. mansoni(Senft et al., 1978), and, as expected, the normal development of theintegument of the parasite occurs. Thus, changes in the tegument ofschistosomes collected from mice not treated with anti-helminthicdrugs would not be expected.

TEM analysis is consistent with information from our previouswork with SEM (Xavier et al., 2010), which showed that the TS miceenvironment promotes changes in the worm’s tegument (Table 1).Helminths parasitizing TS mice showed the tegument disorganized,swollen and intensely vacuolated with a tendency to detach fromthe surface of the worm, unlike the TR strain that was unable tocause changes in the worm.

Ultrastructural alterations in the tegument of S. mansoni havebeen studied by others authors with animal models treated withartemether (Xiao et al., 2002; Shuhua et al., 2002). This is an orig-inal TEM study using a non-antihelminthic treated murine modelwhich allowed observation of the ultrastructural alterations in thetegument of S. mansoni.

The tegument in both genders of helminths parasitizing TS micewere equally damaged, differing from murine models treated withartemether or mefloquine where the tegument of female wormswas more affected than that of male worms (Keiser et al., 2009;Xiao et al., 2000) and differing from mice using praziquantel orvaccinated rabbit sera, where the tegument of male worms wasmore affected than in females (Abdeen et al., 2012; Cioli and Pica-Mattoccia, 2003).

The spines in S. mansoni parasitizing TS mice, apparently short-ened as reported in our previous work by SEM (Xavier et al., 2010),are in reality internalized by the swollen tegument (like suggestedby Dr. Vannier dos Santos, MA, in personal communication) with atendency to become detached from the worm surface as observedby TEM in the present work.

The ultrastructural changes in S. mansoni parasitizing TS mice

were not restricted only to the tegument. The changes in the germcells of female worms were evidenced by the rupture of the mem-brane of these cells, compromising the quality of the eggs. Irie et al.(1987) proposed the correlation of a degenerative alteration of the

54 A.M.L. Xavier et al. / Acta Tropica 130 (2014) 51–57

Fig. 2. Ultrastructural micrographs of adult male S. mansoni tegument recovered from genetically selected mice. (A–C) Representative figures of male worms recovered fromTR mice. (D–F) Representative figures of male worms recovered from TS mice. (A) Tegument (T) and sub-tegumental cytons (STC) intacts in the anterior region of wormsharvested from TR mice. Circular fibers with preserved membranes (small arrow) and fat cells (FC) were observed. (B) Spines (small arrow) in the tegument (T) of wormsharvested from TR mice with a regular morphology, vacuoles with intact membranes (large arrow) and fat cells (FC) were observed. (C) Tubercles from tegument posteriorregion of worms from TR mice showed spines (small arrow) and regular internal structure. (D) At anterior region of worms from TS mice the thin tegument (T) showedswelling with the occurrence of large vacuoles (V), with a tendency to detach from the surface of the worm (arrows) and lysis of sub-tegumental cytons (STC). (E) Internalizedspines (small arrow) of worms harvested from TS mice. (F) Tubercles from tegument posterior region of worms from TS mice without spines and internal disorganizationwith loss of matrix integrity, lysis in the internal structures (L) and vacuoles of various sizes with residual bodies (asterisk). Bars: A and B: 5 �m; C–E: 2 �m; F: 1 �m.

Table 1Major differences in tegument, subtegument and vitelline cells from the worms obtained from TR and TS mice strains.

Major alterations Worms recovered from TS mice Worms recovered from TR mice

Tegument The tegument was disorganized, intensely vacuolated with a tendencyto detach from the surface of the worm. The spines were internalized

Intact with a regular morphology and vacuoleswith intact membranes

The tubercles present total internal disorganization with loss ofintegrity of the matrix, extensive lysis in the internal structures,vacuoles of various sizes with residual bodies, and few spines

Tubercles with spines

Subtegument Ultrastructural damage with lysis of subtegumental cytons,n of th

Intact with a regular morphology

mall p

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disorganization of syncytium, and vacuolizatioVitelline cells Vitelline cells with vitelline balls divided into s

fragmented endoplasmic reticulum

eproductive organs of female schistosomes with their change ingg-laying rate. These modifications of vitelline cells did not causeeduction in the number of eggs produced by worms (there waso significant difference in the amount of eggs between TR and TS

e tissueieces and Healthy vitelline cells

mice). Although there were no interstrain differences in the num-ber of eggs in the liver (unpublished results) and intestinal tissue,a change in the quality of eggs was observed. The ileum of TS miceshowed a high percentage of unviable eggs (dead), representing

A.M.L. Xavier et al. / Acta Tropica 130 (2014) 51–57 55

Fig. 3. Ultrastructural micrographs of adult female S. mansoni tegument recovered from genetically selected mice. (A and B) Representative figures of female worms recoveredfrom TR mice. (C and D) Representative figures of female worms recovered from TS mice. (A) Midbody region of adult female worms from TR mice showing healthy vitellinec arentT anizet reticu

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ells, organized membranes (small arrow), tegument (T) and vacuoles without appR mice. (C) Midbody region of adult female worms from TS mice presented disorghe vitelline balls divided into small pieces (large arrow), fragmented endoplasmic

4.5% of eggs found in tissues, suggesting that these eggs wereroduced with low viability.

S. mansoni infection is characterized by the permanent pres-nce of antigens derived from the worms and eggs, released inhe mesenteric venous system, liver and intestinal mucosa. Thegg’s antigens are immunogenic glycoproteins that are activelyecreted or released after death and degradation of the egg. So, newmmunogenic epitopes originating from the tegument and disinte-

rated eggs could induce the Th1 cytokines (Asahi and Stadecker,003) in TS, consistent with the observed severe morbidity in othernimal models (Stadecker et al., 2001; Hernandez et al., 1998; Cait al., 1996; Mahanty and Nutman, 1995). Nevertheless, the TS mice

ig. 4. Worms and eggs of S. mansoni recovered from TS and TR strain. (A) Percentage odult male and female worms of S. mansoni was not different between the two strains. (C)nd TS mice. TS and TR strain eight weeks after infection with 50 S. mansoni cercariae (m

changes (large arrow). (B) Healthy vitelline cells of female worms recovered fromd tegument (T). (D and E) Vitelline cells of female worms recovered from TS mice:lum (small arrow). Bars: A and B: 2 �m; C: 1 �m; D and E: 0.5 �m.

infected with S. mansoni produce higher levels of cytokines than TRmice, not only IFN-g but also the IL-10 and IL-4 immunomodula-tory cytokines (unpublished results). Thus, we cannot rule out thepossibility that growth and fecundity of the worm can be depend-ent on signals provided by modulating T CD4+ cells (Davies et al.,2001), and that the suppressor activity of IL-10 and IL-4 producedby T CD4+ cells are associated with reduction of these signals. So,it is quite feasible that worm evasive strategies might be impaired

in the TS mice.

The repair process of induced mucocutaneous lesions may occurrelatively quickly (Shaw and Erasmus, 1987; Popiel et al., 1985),however, the tegumental damage might lead to the disappearance

f adult worms recovery was not different between the two strains. (B) Number ofS. mansoni eggs in different stages of development harvested from the ileum of TR

ean ± SD, n = 4–6).

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f the immunological ‘disguise’ of the worm, which could exposearge amounts of immunogens and possibly further immunogenicpitopes.

Differently from models treated with drugs (artemether, praz-quantel and mefloquine) (Abdeen et al., 2012; Xiao et al., 2002;haw and Erasmus, 1983), the gut epithelial cells of the worm fromS strain had not been affected and these worms did not block theay to provide the nutrients necessary to maintain life. In spite of

imilarities in the tegument damage between worms from animalodels treated with drugs and that ones from TS strain; the dam-

ge to the worm from TS strain is not extensive and the worms mayurvive in the TS mice environment.

All focal damage to tegument, subtegument and vitelline cellsevealed in worms from TS mice does not confer a milder disease. Inact, the opposite has occurred; the TS strain has higher weakness,pathy, prostration and mortality, probably due to anemia and aore intense hepatosplenomegaly than the TR strain (unpublished

esults). The less deleterious disease in TR reinforces the idea fromur previous work (Xavier et al., 2010), as well as the recent workf Abdeen et al. (2012), that the host immune system influenceshe tegument development, and its function, in non-antihelminthicreated mice models, depending upon the host immune regulatoryapacity.

cknowledgments

We are grateful to Dr. Lygia dos Reis Corrêa, Mr. Paulo Cesaros Santos and Ms. Heloisa from Laboratorio de Malacologia, IOC,iocruz/RJ, for providing the cercariae of Schistosoma mansoni, andr. Cid Couto and Dr. Marcia Giesta for caring for the animal colonies

Laboratório de Imunobiologia). English review by Caroline Fer-az Ignacio Valente. This work was supported by Plataforma deicroscopia Eletrônica do Instituto Oswaldo Cruz (IOC), Fundacãoswaldo Cruz (FIOCRUZ); Conselho Nacional de Desenvolvimentoientífico e Tecnológico (MCT-CNPq); Fundacão Carlos Chagas Filhoe Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) – Grant-26/171.423/04.

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