advances in the study of schistosomiasis: the postgenomic era

Advances in the Study of Schistosomiasis: The Postgenomic Era

Journal of Parasitology Research

Guest Editors: Cristina Toscano Fonseca, Sergio Costa Oliveira, Andrea Teixeira-Carvalho, and Rashika El Ridi

Advances in the Study of Schistosomiasis:The Postgenomic Era

Journal of Parasitology Research

Advances in the Study of Schistosomiasis:The Postgenomic Era

Guest Editors: Cristina Toscano Fonseca, Sergio Costa Oliveira,Andrea Teixeira-Carvalho, and Rashika El Ridi

Copyright © 2013 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in “Journal of Parasitology Research.” All articles are open access articles distributed under the CreativeCommons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the originalwork is properly cited.

Editorial Board

Takeshi Agatsuma, JapanJeffrey Bethony, USADave Chadee, USAKwang Poo Chang, USAWej Choochote, ThailandAlvin A. Gajadhar, CanadaC. Genchi, ItalyBoyko B. Georgiev, Bulgaria

Ana Maria Jansen, BrazilMaria V. Johansen, DenmarkNirbhay Kumar, USAD. S. Lindsay, USABernard Marchand, FranceRenato A. Mortara, BrazilDomenico Otranto, ItalyBarbara Papadopoulou, Canada

A. F. Petavy, FranceBenjamin M. Rosenthal, USAJoseph Schrevel, FranceJose F. Silveira, BrazilM. J. Stear, UKXin-zhuan Su, USAKazuyuki Tanabe, Japan

Contents

Advances in the Study of Schistosomiasis: The Postgenomic Era, Cristina Toscano Fonseca,Sergio Costa Oliveira, Andrea Teixeira-Carvalho, and Rashika El RidiVolume 2013, Article ID 849103, 2 pages

Cytokine Pattern of T Lymphocytes in Acute Schistosomiasis mansoni Patients following TreatedPraziquantel Therapy, Denise Silveira-Lemos, Matheus Fernandes Costa-Silva,Amanda Cardoso de Oliveira Silveira, Mauricio Azevedo Batista, Lucia Alves Oliveira-Fraga,Alda Maria Soares Silveira, Maria Carolina Barbosa Alvarez, Olindo Assis Martins-Filho,Giovanni Gazzinelli, Rodrigo Correa-Oliveira, and Andrea Teixeira-CarvalhoVolume 2013, Article ID 909134, 13 pages

Cytokine and Chemokine Profile in Individuals with Different Degrees of Periportal Fibrosis due toSchistosoma mansoni Infection, Robson Da Paixao De Souza, Luciana Santos Cardoso,Giuseppe Tittoni Varela Lopes, Maria Cecılia F. Almeida, Ricardo Riccio Oliveira, Leda Maria Alcantara,Edgar M. Carvalho, and Maria Ilma AraujoVolume 2012, Article ID 394981, 10 pages

New Frontiers in Schistosoma Genomics and Transcriptomics, Laila A. Nahum, Marina M. Mourao,and Guilherme OliveiraVolume 2012, Article ID 849132, 11 pages

Changes in T-Cell and Monocyte Phenotypes In Vitro by Schistosoma mansoni Antigens in CutaneousLeishmaniasis Patients, Aline Michelle Barbosa Bafica, Luciana Santos Cardoso, Sergio Costa Oliveira,Alex Loukas, Alfredo Goes, Ricardo Riccio Oliveira, Edgar M. Carvalho, and Maria Ilma AraujoVolume 2012, Article ID 520308, 10 pages

Transcriptional Profile and Structural Conservation of SUMO-Specific Proteases in Schistosomamansoni, Roberta Verciano Pereira, Fernanda Janku Cabral, Matheus de Souza Gomes,Liana Konovaloff Jannotti-Passos, William Castro-Borges, and Renata Guerra-SaVolume 2012, Article ID 480824, 7 pages

Schistosoma Tegument Proteins in Vaccine and Diagnosis Development: An Update,Cristina Toscano Fonseca, Gardenia Braz Figueiredo Carvalho, Clarice Carvalho Alves,and Tatiane Teixeira de MeloVolume 2012, Article ID 541268, 8 pages

Pathogenicity of Trichobilharzia spp. for Vertebrates, Lichtenbergova Lucie and Horak PetrVolume 2012, Article ID 761968, 9 pages

Hindawi Publishing CorporationJournal of Parasitology ResearchVolume 2013, Article ID 849103, 2 pageshttp://dx.doi.org/10.1155/2013/849103

EditorialAdvances in the Study of Schistosomiasis: The Postgenomic Era

Cristina Toscano Fonseca,1 Sergio Costa Oliveira,2

Andrea Teixeira-Carvalho,1 and Rashika El Ridi3

1 Centro de Pesquisas Rene Rachou, Fundacao Oswaldo Cruz, 30190-002 Belo Horizonte, MG, Brazil2 Department of Biochemistry and Immunology, Federal University of Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil3 Zoology Department, Faculty of Science, Cairo University, Giza 12613, Egypt

Correspondence should be addressed to Cristina Toscano Fonseca; [email protected]

Received 3 March 2013; Accepted 3 March 2013

Copyright © 2013 Cristina Toscano Fonseca et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Schistosomiasis remains a serious public health problem in76 countries and territories of the world, with more than200million individuals infected, causing annual losses of 10.4million in DALYs (disability adjusted to life years). Althougheffective drugs against schistosomiasis are used in diseasecontrol programs, the prevalence of the disease remainsunaltered, and new diseases foci and acute outbreaks areobserved worldwide. Also, drug resistance by the parasites isa concern, and new drugs and alternative control measuresneed to be developed.

An effective vaccine against schistosomiasis is an alterna-tive and desirable tool to help in the disease control; however,the complex schistosome life cycle and the complexity ofits interaction with the host immune system turn vaccinedevelopment into a difficult task. Neither drug developmentnor vaccine development is sufficient to ensure the successof schistosomiasis control programs. As a matter of fact, thecombination of an effective vaccine together with chemother-apy is the strategy of choice. Many studies also demonstratethat without a more sensitive diagnosis test, able to detectindividuals with low parasite burden, no control strategywill achieve the desirable result which nowadays is diseaseselimination.

The access to the genome from Schistosoma man-soni, Schistosoma japonicum, and Schistosoma haematobiumrecently published is expected to increase rapidly our under-standing of the parasite biology and also accelerate thedevelopment of new drugs, vaccine, and diagnosis methodsby helping in the identification of new targets.

In this special issue, a review by C. T. Fonseca et al.gives an update on how parasite genomes together withbioinformatics tools have helped in vaccine and diagnosisdevelopment in the last few years. Since the parasite surfacerepresents an interesting target for the host immune system,this review was focused on parasite tegument as a source ofantigens.

Although the genome from the three most socioeconom-ically important schistosome species had already been pub-lished,many questions continue to challenge researchers.Thereview by L. A. Nahum and coworkers discusses the advanceson schistosome genomics and transcriptomics studies andpoint out the gaps and future directions in this research field.

An example on how parasite genome database helpsin understanding the parasite biology is presented in thepaper by R. V. Pereira and colleagues. In their article,using in silico analysis, they identified in the S. mansonigenome orthologs of themammalian Sentrin/SUMO-specificproteases (SENPs) 1 and 7, which are involved inmany cellularprocesses. The S. mansoni SENPs transcriptional profile aswell as its structural conservation was analyzed.

Just as important as understanding the biology of theparasite is deciphering how the host immune system dealswith schistosome infection and its relation to the pathogene-sis of the disease. Clinically schistosomiasis is characterizedby two distinct phases: the acute and the chronic phase.The acute phase is commonly observed in individuals livingin nonendemic area, while chronic phase is observed inendemic-area residents.

2 Journal of Parasitology Research

D. Silveira-Lemos et al. evaluated in their article immuno-logical parameters in patients with acute schistosomiasisbefore and after praziquantel treatment. Interesting differ-ences are demonstrated by the authors between noninfected,acute, and treated group, and a role for CD8+ T cells as asource of cytokines in acute patients is pointed by this study.

The cytokine and chemokine profile observed in PBMCsfrom individuals infected with S. mansoni in response tospecific stimulation and its correlation with different degreesof periportal fibrosis is described by R. P. de Souza andcolleagues in their research article. Their results indicatepotential biomarkers for the progression of liver pathologydue to schistosomiasis.

Many schistosome antigens have been described tomodulate inflammatory diseases such as asthma. A. M. B.Bafica et al. evaluated in their article the impact of theS. mansoni antigens: Sm29, TSP-2, and PIII in PBMCsculture from cutaneous leishmaniasis patients stimulatedwith L. braziliensis antigen. Their results demonstrated thatschistosome antigens induce a modulatory phenotype withdecreased monocyte activation and increased expression ofT-lymphocyte modulatory molecules.

Trichobilharzia spp. infection results in bird schistoso-miases, which causes severe pathogenic impact in birds andfrequently results in cercarial dermatitis in humans. In theirarticle, L. Lucie and H. Petr review the pathogenesis ofbird schistosomiasis and the immune response triggered byTrichobilharzia spp. infection with emphasis on the newspecies T. regent.

We hope you enjoy reading this special issue!

Cristina Toscano FonsecaSergio Costa Oliveira

Andrea Teixeira-CarvalhoRashika El Ridi

Hindawi Publishing CorporationJournal of Parasitology ResearchVolume 2013, Article ID 909134, 13 pageshttp://dx.doi.org/10.1155/2013/909134

Research ArticleCytokine Pattern of T Lymphocytes in Acute Schistosomiasismansoni Patients following Treated Praziquantel Therapy

Denise Silveira-Lemos,1, 2, 3, 4 Matheus Fernandes Costa-Silva,1, 2

Amanda Cardoso de Oliveira Silveira,1 Mauricio Azevedo Batista,4

Lúcia Alves Oliveira-Fraga,5 AldaMaria Soares Silveira,5

Maria Carolina Barbosa Alvarez,6 Olindo Assis Martins-Filho,1 Giovanni Gazzinelli,2

Rodrigo Corrêa-Oliveira,2 and Andréa Teixeira-Carvalho1

1 Laboratório de Biomarcadores de Diagnóstico e Monitoração, Centro de Pesquisas René Rachou, FIOCRUZ, Barro Preto,30190-002 Belo Horizonte, MG, Brazil

2 Laboratório de Imunologia Celular eMolecular, Centro de Pesquisas René Rachou, FIOCRUZ, 30190-002 Belo Horizonte, MG, Brazil3 Laboratório de Imunologia e Genômica de Parasitos, Departamento de Parasitologia, Instituto de Ciências Biológicas (ICB),Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

4 Laboratório de Imunoparasitologia, Departamento de Ciências Biológicas, NUPEB, Instituto de Ciências Exatas e Biológicas,Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil

5 Núcleo de Pesquisa em Imunologia, Faculdade de Ciências da Saúde, Universidade Vale do Rio Doce,Governador Valadares, MG, Brazil

6 Santa Casa de Misericórdia de Belo Horizonte, Belo Horizonte, MG, Brazil

Correspondence should be addressed to Denise Silveira-Lemos; [email protected]

Received 12 July 2012; Revised 24 September 2012; Accepted 26 September 2012

Academic Editor: Rashika El Ridi

Copyright © 2013 Denise Silveira-Lemos et al. is is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Acute schistosomiasis is associated with a primary exposure and is more commonly seen in nonimmune individuals travelingthrough endemic regions. In this study, we have focused on the cytokine pro�le of T lymphocytes evaluated in circulating leukocytesof acute Schistosomiasis mansoni-infected patients (ACT group) before and aer praziquantel treatment (ACT-TR group). Our datademonstrated increased values of total leukocytes, eosinophils, and monocytes in both groups. Interestingly, we have observed thatpatients treated with praziquantel showed increased values of lymphocytes as compared with noninfected group (NI) or ACTgroups. Furthermore, a decrease of neutrophils in ACT-TR was observed when compared to ACT group. Analyses of short-termin vitro whole blood stimulation demonstrated that, regardless of the presence of soluble Schistosoma mansoni eggs antigen (SEA),increased synthesis of IFN-𝛾𝛾 and IL-4 by T-cells was observed in the ACT group. Analyses of cytokine pro�le in CD� T cellsdemonstrated higher percentage of IFN-𝛾𝛾 and IL-4 cells in both ACT and ACT-TR groups apart from increased percentage of IL-10 cells only in the ACT group. is study is the �rst one to point out the relevance of CD� T lymphocytes in the immune responseinduced during the acute phase of schistosomiasis.

1. Introduction

Schistosomiasis is a tropical parasitic chronic disease, causedby worms of the genus Schistosoma. e infection is endemicin tropics and subtropics around the world and is presentin Africa, South America, Arabia, and Asia. ere areapproximately 200 million people infected worldwide, and

the number of deaths caused by schistosomiasis is estimatedat around 41.000 annually [1]. Schistosoma mansoni wormsdwell in peri-intestinal venules and cause intestinal, hepa-tointestinal, and hepatosplenic schistosomiasis [2].

Brazil is the country most affected by schistosomiasisin the Americas, with 1.5 million people infected and morethan 36 million at risk of acquiring infection [3, 4]. In the


state of Minas Gerais, Schistosomiasis mansoni is prevalent in519 municipalities, with an estimated number of one millionpeople infected [5].

e propagation and maintenance of schistosomiasisin a given region are conditional of many factors such asappropriate climate, socioeconomic conditions, rapid spreadof intermediate hosts, sanitary conditions, and water supply[6]. Tourist activity in endemic areas may be an importantcontributing factor to the propagation of outbreaks/cases ofschistosomiasis [7–10].

In endemic areas for schistosomiasis, people develop thechronic phase of infection (which begins around six monthsaer exposure) and have different clinical manifestations.People who had never had previous contact with the parasitedevelop acute schistosomiasis when contracting the infection[11, 12].

e acute phase starts aer cercarial infection, andlocal urticaria may appear in a few hours. Between oneand four weeks aer infection, the migrating and maturingschistosomula can cause a systemic hypersensitivity reactionwith fever, general weakness, headache, nausea, vomiting,diarrhea, and dry cough [2, 9]. In this phase, individualspresent a mixed cytokine pro�le with predominance of 1type of CD4+ T-cell differentiation [13, 14].

In the context of cytokine milieu triggered by S. man-soni infection, it has been suggested that interleukin (IL)-4upregulates �broblast chemokine, matrix protein expression,and collagen, which implies that IL-4 is a crucial cytokinefor granuloma formation [15] and reduces the cellularproliferative response to soluble egg antigens (SEA) [13].IL-5 in schistosomiasis induces liver �brosis [16]. IL-10has been shown to be a major cytokine during infectionwith downregulatory activity of both 1 and 2 T cellsubpopulations [17]. Interferon gamma (IFN-𝛾𝛾) is related tothe activation of macrophages and plays a key role in theprotectivemechanism against periportal �brosis, whereas theproin�ammatory tumor necrosis factor-alpha (TNF-𝛼𝛼) mayaggravate the disease [18].

As regards the cytokine pro�le aer speci�c chemother-apy, it has been suggested that peripheral blood mononuclearcells (PBMC) from patients with acute infection respondedto SEA and soluble worm antigen preparation (SWAP) byproducing signi�cantly higher amounts of IFN-𝛾𝛾 and IL-10. However, IL-5 was detected only in SEA-stimulatedcultures, and little or no IL-4 was detected in SEA or SWAP-stimulated cells [19]. De Jesus et al. [9] suggested thatmost patients aer speci�c chemotherapy for schistosomiasisspontaneously released high levels of TNF-𝛼𝛼, IL-1, and IL-6. In addition, detectable levels of IFN-𝛾𝛾 were present inthe supernatants of unstimulated PBMC from these patients.Stimulation of PBMC from patients with acute disease onlyinduced higher levels of IFN-𝛾𝛾 upon SEA stimulation [9].

In the present study, we evaluated the cytokine pro�le(IFN-𝛾𝛾, TNF-𝛼𝛼, IL-4, IL-5, and IL-10) of T lymphocytesand their subsets as well as the ultrasonographic features ofpatients with acute schistosomiasis infection before and aerpraziquantel treatment.

2. Population, Material, andMethods

2.1. Study Population. e patients evaluated in this studywere infected accidentally by S. mansoni in the countryvillage of São Geraldo da Piedade, an endemic area forschistosomiasis, located in the east of the state of MinasGerais (MG), Brazil, during the traveling in a holiday. ispeople reported swimming in a stream that probably harborscercariae released by infected snails. In this area 60% ofBiomphalaria glabrata are infected and the chronic diseaseis frequent in the population. ese individuals went to thehospital because they felt very bad, with acute symptoms.epatients did not live in areas of active transmission of S. man-soni. All patients showed clinical symptoms associated withearly S. mansoni infection such as fever, diarrhoea, headache,and nausea. Only patients who had positive results for quan-titative parasitological stool examinations for S. mansoni bythe Kato-Katz method [20] and signed an informed consentwere included in this study. e group of acutely S. man-soni-infected patients (ACT) without praziquantel treatmentconsisted of twenty-one individuals, �ve women and sixteenmen, with ages ranging from 13 to 44 years, with parasite loadranging from 12 to 1812 eggs per gram of feces (epg). Aerblood sample collection, all S. mansoni-infected individualsreceived speci�c therapeutic treatment with praziquantelin the standard Brazilian dose (50–60mg/Kg praziquantel),regardless of whether or not they were going to participatein this study [21]. One month aer praziquantel treatment,blood was collected from a group of patients (ACT-TR) com-posed of three women and four men, aged between 12 and40 years. An additional noninfected group (NI) composedof nineteen individuals, eleven woman and eight men, withage ranging from 19 to 45 years, who were healthy blooddonors contacted at the Hemominas Blood Bank Foundationin Belo Horizonte, MG, Brazil, participated in the study. Allnoninfected volunteers were included aer three consecutivenegative parasitological exams for S. mansoni infection apartfrom negative serology for Chagas disease, leishmaniasis,human immunode�ciency virus infection, and hepatitis.isstudy was approved by the Ethics Committees at the OswaldoCruz Foundation (FIOCRUZ), the School of Medicine at theFederal University of Minas Gerais (UFMG), and the Brazil-ian National Committee on Ethics in Research (CONEP).

2.2. Ultrasonographic Analysis. Ultrasonographic evaluationwas performed in all individuals using a Nemio SSA/550Aultrasound machine (Toshiba) with a 3MHz sector probe.Liver size, portal vein diameter, thickness of central wallsand peripheral portal branches, spleen size, and splenic veindiameter were assessed as described in previous studies [22,23]. Liver span was measured both in the midclavicularline and the midline. e liver was also examined forsurface smoothness. Portal vein diameter was measured at itsentrance into the liver and its bifurcation to the liver. Obliqueand longitudinal scans of the le upper quadrant were usedto evaluate the spleen. e gallbladder was examined forwall thickness and stones. Periportal thickness was evaluatedaccording to established criteria [22–24]. All individuals wereexamined by the same physician.

Journal of Parasitology Research 3

2.3. Evaluation of Hematological Parameters. Hemogramswere performed with an automated blood cell counter (Coul-ter MD18, USA), using whole blood collected in 5mL vacu-tainer tubes containing the anticoagulant ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson Biosciences, SanDiego, CA,USA).e parametersmeasuredwere total leuko-cytes counts and differential analysis of leukocyte subsetsincluding the absolute counts of eosinophils, neutrophils,lymphocytes, and monocytes.

2.4. Preparation of Antigens. S. mansoni eggs were isolatedfrom the livers of infected mice, exposed 8 weeks previouslyto cercariae, homogenized, and ground in cold phosphate-buffered saline (PBS). e clear supernatant �uid result-ing from high-speed centrifugation of the homogenate at50.000×g for 1 h at room temperature was named soluble eggantigens (SEA) and stored at −70∘C until use [25].

2.5. Monoclonal Antibodies. e experiments used mono-clonal antibodies (mAbs) against both human cell surfacemarkers, including CD3 (UCTH-1), CD4 (SK3), and CD8(SK1) labeled with �uorescein isothiocyanate (FITC), andintracytoplasmic cytokines including IL-4 (MP4-25D2), IL-5 (TRFK5), IL-10 (JES3-9D7), IFN-𝛾𝛾 (B27), and TNF-𝛼𝛼(Mab11) labeled with phycoerythrin (PE). Isotypic controlswere also used in all experiments, including mouse IgG1(679.1Mc7) and IgG2a (UCTH-1) labeled with FITC and PE,respectively. All mAbs and controls were purchased fromBecton Dickinson Biosciences Pharmingen (San Diego, CA,USA).

2.6. Short-Term Whole Blood Culture In Vitro and Intracel-lular Cytokine Immunostaining. Short-term cultures in vitroand cytokine immunostaining were performed as describedby Silveira-Lemos et al. [26]. Brie�y, �ve hundred micro-liters of peripheral blood samples collected into Vacutainertubes containing heparin sodium salt (Becton Dickinson,Mountain View, CA, USA) were dispensed into individual17 × 100mm polypropylene tubes (Falcon 2059 - BectonDickinson, Mountain View, CA, USA). Short-term cultureswere performed in the absence (control cultures) or in thepresence of S. mansoni eggs derived antigens (SEA). For thispurposes, 500 𝜇𝜇L of whole blood samples were incubatedin the presence of 500 𝜇𝜇L of RPMI 1640 plus Brefeldin A,BFA (Sigma Chemical Company, St Louis, MO, USA) at a�nal concentration of 10𝜇𝜇g/mL. e blood samples wereincubated for 4 h at 37∘C in a 5% CO2 humidi�ed incubator.ese conditions were chosen considering that the detectionof cytokines, particularly in the absence of exogenous stimuli,may re�ect the events of cytokine production by bloodleukocytes in vivo. Antigen-speci�c stimulation was per-formed by previous in vitro incubation of 500𝜇𝜇Lwhole bloodaliquots in the presence of SEA at a �nal concentration of25 𝜇𝜇g/mL for 1 h at 37∘C in a 5% CO2 humidi�ed incubator.Following the addition of BFA (Sigma Chemical Company,St Louis, MO, USA) at a �nal concentration of 10𝜇𝜇g/mL,blood samples were incubated for an additional 4 h at 37∘Cin a 5% CO2 humidi�ed incubator. Prior to immunostaining

for intracellular cytokine, all cultures, including control andSEA stimulated, were treated with 2mM EDTA (SigmaChemical Company, St. Louis, MO, USA) and kept at roomtemperature for 15min, to stop cell activation process.

Following the short-term in vitro stimulation, culturedwhole blood samples were washed with 6mL of FACS buffercontaining PBS supplemented with 0.5% Bovine SerumAlbumin (BSA) and 0.1% sodium azide (Sigma ChemicalCompany, St. Louis, MO, USA), by centrifugation at 600×gat room temperature for 7min. Aer resuspension in 1mLof FACS buffer, 400 𝜇𝜇L aliquots were dispensed into one 12 ×75mm polystyrene tube (Becton Dickinson, Mountain View,CA, USA) and labeled in the dark for 30min at room temper-ature with the manufacture’s recommended amount of mAbsanti-CD3, anti-CD4, and anti-CD8-FITC. Following theerythrocyte lyses step, the cells were incubated with 3mL ofFACS permeabilizing solution, containing FACS buffer sup-plemented with 0.5% of saponin (Sigma Chemical Company,St. Louis, MO, USA) in the dark for 10min at room temper-ature. Following incubation, the samples were centrifuged at600×g at room temperature for 7min, the supernatant gentlydecanted and 3mL of FACS buffer added to the resuspendedpellet. Aer centrifugation, the pellet was resuspended and30 𝜇𝜇L aliquots of cell suspension distributed in 96wellsU bot-tom microplate (Falcon-Becton Dickinson, Mountain View,CA, USA) and stained with 20𝜇𝜇L of PE-labeled anticytokinemAb (anti-IL-4, anti-IL-5, anti-IL-10, anti-IFN-𝛾𝛾, and anti-TNF-𝛼𝛼,). e cells were incubated in the dark for 30min atroom temperature. Aer incubation, the cells were washedwith 150 𝜇𝜇L of FACS permeabilizing solution followed by200 𝜇𝜇L of FACS buffer. Aer washing, the stained cells were�xed in 200 𝜇𝜇L of FACS �x solution and the samples stored at4∘C in the dark and analyzed by �ow cytometry.

2.7. Data Collection and Analysis. Immunostained sampleswere run in a FACScan three-color detection �ow cytometer(Becton Dickinson, San Jose, CA, USA). Data were collectedand analyzed with CellQuest soware (aer collection of50.000 events/sample). Cytokine-expressing T lymphocytes,including CD3+, CD4+, and CD8+ T cells, were identi�edby dual color immunophenotyping with FL1/FITC-labeledantihuman cell surface markers against CD3, CD4 andCD8mAbs together with FL2/PE-labeled anticytokinemAbs.Lymphocyte gating was initially based on lymphocyte selec-tion by forward scatter (FSC) versus side light scatter (SSC)properties on dot plot distributions, where they are con�nedinto a region of low size and complexity. Further analysisof lymphocytes subpopulations was based on their selectivestaining with FITC-labeled anti-CD3, anti-CD4, and anti-CD8 mAbs. Cytokine-expressing cell subsets were quanti�edusing FL1 cell surface markers versus FL2 anti cytokine-PEdot plots by setting quadrants to segregate FL2 positive andnegative cells based on the negative control immunostaining.e results are expressed as the percentage of lymphocytesubsets expressing a given cytokine.

2.8. Statistical Analysis. e statistical analysis was per-formed with soware GraphPad PRISM 5.00 for Windows(La Jolla, CA,USA). Considering the parametric nature of the


T 1: Ultrasonographic features of the study group.

Site of measurement Reference values CT (𝑁𝑁 𝑁 𝑁𝑁) ACT (𝑁𝑁 𝑁 𝑁𝑁)Longitudinal le lobe of liver 107.0 102.2 ± 16.0 (91.9–112.4) 99.7 ± 20.9 (89.9–109.6)Anteroposterior le lobe of liver 70.0 42.7 ± 4.4 (39.6–45.9) 53.7 ± 7.6∗ (50.2–57.3)Longitudinal right lobe of liver 150.0 134.3 ± 10.5 (127.3–141.4) 147.4 ± 11.5∗ (142.0–152.8)Anteroposterior right lobe of liver 100.0 70.3 ± 10.6 (63.2–77.5) 84.3 ± 9.6∗ (79.7–89.0)Longitudinal spleen 120.0 88.1 ± 7.0 (83.4–92.9) 116.9 ± 15.6∗ (109.2–124.7)Anteroposterior spleen 70.0 33.7 ± 3.9 (31.0–36.3) 46.6 ± 8.5∗ (42.6–50.6)Portal vein <12.0 9.9 ± 1.2 (9.2–10.7) 10.5 ± 1.30 (9.9–11.1)Hilar portal vein wall <3.0 1.5 ± 0.3 (1.3–1.7) 2.0 ± 0.1∗ (1.9–2.1)Splenic vein <9.0 6.4 ± 1.3 (5.5–7.2) 6.5 ± 0.9 (6.1–6.9)Superior mesenteric vein <9.0 6.4 ± 0.8 (5.9–7.1) 6.2 ± 0.8 (5.8–6.6)Values are represented in mm as mean ± standard deviation and 95% con�dence intervals.∗Represents a statistically signi�cant difference (𝑃𝑃 < 𝑃𝑃𝑃𝑃) among groups CT and ACT.

data, all results were analyzed by the ANOVA test followed byTukey�s multiple comparison test. Signi�cance was de�ned at𝑃𝑃 < 𝑃𝑃𝑃𝑃.

3. Results

3.1. Ultrasonographic Features of the Study Group. Table 1shows the most important ultrasonographic features fromACT in comparison with control group. Our data demon-strated that ACT had signi�cant increase in themeasurement(in mm) of the longitudinal right lobe of liver (ACT: 𝑁47𝑃4 ±𝑁𝑁𝑃𝑃; CT: 𝑁34𝑃3±𝑁𝑃𝑃𝑃), anteroposterior le lobe of liver (ACT:𝑃3𝑃7 ± 7𝑃6; CT: 4𝑁𝑃7 ± 4𝑃4), the anteroposterior right lobe ofliver (ACT: 84𝑃3±9𝑃6; CT: 7𝑃𝑃3±𝑁𝑃𝑃6), size of longitudinal andthe anteroposterior spleen (ACT: 𝑁𝑁6𝑃9 ± 𝑁𝑃𝑃6; CT: 88𝑃𝑁 ± 7𝑃𝑃and ACT: 46𝑃6 ± 8𝑃𝑃; CT: 33𝑃7 ± 3𝑃9, resp.), dimension ofHilar portal vein wall (ACT: 𝑁𝑃𝑃 ± 𝑃𝑃𝑁; CT: 𝑁𝑃𝑃 ± 𝑃𝑃3, resp.)as compared with the CT group. No signi�cant differenceswere found according to the sex categories.

3.2. Hematological Parameters during Acute Schistosomiasismansoni Infection before and aer Treatment with Praziquan-tel. e results showed an increase in the values of totalleukocytes from ACT and ACT-TR as compared with NI.A differential analysis of leukocyte subsets provides a moredetailed description of the speci�c differences across celltypes. e data showed increased counts of eosinophils andmonocytes in the ACT and ACT-TR groups as comparedwith NI. Moreover, increased values were detected for lym-phocytes from ACT-TR as compared with NI and ACT.Interestingly, aer speci�c therapy, count of neutrophils inACT-TR was decreased in comparison with NI (Figure 1).

3.3. Cytokine Producing T Lymphocytes following In VitroSEA Stimulation. Aiming to evaluate the impact of SEAstimulation in triggering proin�ammatory and regulatorycytokines by T lymphocytes and the effect of the speci�ctherapy for this pro�le, we have performed a detailed singlecell �ow cytometric analysis to �uantify the fre�uency ofcytokine+ cells for IFN-𝛾𝛾, TNF-𝛼𝛼, IL-4, IL-5, and IL-10

aer in vitro short-term incubation of whole blood samplesfocusing on T lymphocytes and their subsets (CD4+ or CD8+T cells).

Regardless of the addition of SEA, the data revealedincreased synthesis for IFN-𝛾𝛾 and IL-4 by T cells in ACTas compared with NI (Figures 2(a) and 2(c), resp.). AerSEA stimulation, we also observed increased synthesis of IL-4 in ACT-TR as compared with NI (Figure 2(c)). Moreover,the analysis of the impact of SEA addition to the culturesdetected increased synthesis of TNF-𝛼𝛼 in ACT (Figure 2(b)).No signi�cant differences were found according to the sexcategories.

3.4. Cytokine Producing T CD4+ Lymphocytes following InVitro SEA Stimulation. e analysis of the results showed,in the control culture, increased synthesis of IL-4 by CD4+T lymphocytes in ACT and ACT-TR groups as comparedwith NI (Figure 3(c)). Furthermore, aer SEA stimulation,we observed increased synthesis of TNF-𝛼𝛼 by CD4+ Tlymphocytes in ACT as compared with the control culture(Figure 3(b)).

3.5. Cytokine Producing T CD8+ Lymphocytes following InVitro SEA Stimulation. e analysis of the cytokine pro�le inthe CD8+T subset revealed a higher number of differencesthan in the CD4+ T cells. Regardless of SEA stimulation,increased synthesis of IFN-𝛾𝛾 and IL-4 by CD8+ T lympho-cytes was observed in ACT as compared with NI (Figures4(a) and 4(c), resp.). Moreover, in cultures stimulated withSEA, increased synthesis of IL-4 was detected in ACT-TRas well as increased synthesis of IL-10 in ACT as comparedwith NI (Figures 4(c) and 4(e), resp.). Aer SEA stimulation,data analysis also showed increased synthesis of IFN-𝛾𝛾 orIL-5 by CD8+ T lymphocytes as compared with the controlculture (Figures 4(a) and 4(d), resp.). When the impact ofSEA addition in the cultures from ACT-TR patient wasinvestigated, increased synthesis of IFN-𝛾𝛾 or IL-4 by CD8+T lymphocytes were observed as compared with the controlcultures (Figures 4(a) and 4(c), resp.).


Total leukocytes

NI ACT ACT-TR

0

10000

20000

= 0.0201

= 0.0012

Nu

mb

er o

f le

uk

ocy

tes

sub

po

pu

lati

on

s (m

m3)

(a)

Eosinophils

NI ACT ACT-TR

0

5001000

7500

14000

= 0.0002

< 0.0001

Nu

mb

er o

f le

uk

ocy

tes

sub

po

pu

lati

on

s (m

m3)

(b)

Neutrophils

NI ACT ACT-TR

0

3500

7000

= 0.0014

Nu

mb

er o

f le

uk

ocy

tes

sub

po

pu

lati

on

s (m

m3)

(c)

Lymphocytes

NI ACT ACT-TR

0

3000

6000

= 0.0068

= 0.0112

Nu

mb

er o

f le

uk

ocy

tes

sub

po

pu

lati

on

s (m

m3)

(d)

Monocytes

NI ACT ACT-TR

0

750

1500

= 0.0049

= 0.0029

Nu

mb

er o

f le

uk

ocy

tes

sub

po

pu

lati

on

s (m

m3)

(e)

F 1: Analysis of hematological pro�le from patients with acute Schistosomiasis mansoni before (ACT, faint grey square = 21) and aerpraziquantel treatment (ACT-TR, grey square = 07) and noninfected individuals (NI, white square = 19). e results are shown in bar-plotformat highlighting the mean counts of datasets/mm3± standard deviation. �tatistically signi�cant differences (connecting lines) observedbetween groups NI, ACT, and ACT-TR were considered at 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃.

4. Discussion

Acute schistosomiasis is a severe disease and the mechanisminvolved in its clinical manifestations is not completelyunderstood. In the present study, we evaluated patientsexposed to the same place of contaminated water, 40–60 daysaer exposure to cercaria. Different studies concerning thisacute disease refer to groups of tourists, �shermen, or sailorsoriginally from a nonendemic country who has visited atropical zone [27–29]. However, as schistosomiasis is a focallydistributed disease [29–31], the acute form is also diagnosedin inhabitants from endemic countries who do not live inendemic areas. e main clinical �ndings presented in our

study such as headache, fever, diarrhea, and weight loss wereconsistent with those found by others authors [7, 9, 10, 29,32].

�etween two and eight weeks aer a �rst contact withnatural water infested by Schistosoma cercariae, susceptibleinfected patients may present a syndrome comprising of aperiod of 2 to 30 days of fever, diarrhea, toxemia and weak-ness, weight loss, abdominal pain, cough, myalgia, arthralgia,edema, urticaria, nausea/vomiting, and hepatosplenomegaly[33]. us, the diagnosis poses a challenge to physiciansowing to the nonspeci�city symptoms as well as the lack ofpositivity for S. mansoni eggs in feces from acute patients in


NI ACT ACT-TR NI ACT ACT-TR

0

1.3

2.6

Control culture SEA stimulation

IFN-

= 0.0006

= 0.0005

Cyt

ok

ines

+to

tal+

T ly

mp

ho

cyte

s (%

)

(a)


0

1.3

2.6


TNF-

= 0.0395

Cyt

ok

ines

+to

tal+

T ly

mp

ho

cyte

s (%

)

(b)

IL-4


0

1.3

2.6


= 0.0001

= 0.0119

< 0.0001

Cyt

ok

ines

+to

tal+

T ly

mp

ho

cyte

s (%

)

(c)

IL-5


0

1.3

2.6


Cyt

ok

ines

+to

tal+

T ly

mp

ho

cyte

s (%

)

(d)

IL-10


0

1.3

2.6


Cyt

ok

ines

+to

tal+

T ly

mp

ho

cyte

s (%

)

(e)

F 2: Cytokine pattern of circulating T lymphocytes from patients with acute Schistosomiasis mansoni before (ACT, faint grey square =21) and aer praziquantel treatment (ACT-TR, grey square = 07) and noninfected individuals (NI, white square = 19), following short-termin vitro cultivation in the absence (control culture) or presence of SEA stimulation. e results are shown in bar-plot format highlighting themean percentage of datasets ± standard deviation. Statistically signi�cant di�erences (connecting lines) observed between groups NI, ACT,and ACT-TR were considered at 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃.



0

1.1

2.2


IFN-

Cyt

ok

ines

+C

D4+

T ly

mp

ho

cyte

s (%

)

(a)


0

1.1

2.2


= 0.0252

TNF-

Cyt

ok

ines

+C

D4+

T ly

mp

ho

cyte

s (%

)

(b)

IL-4


0

1.1

2.2


= 0.0169

= 0.0002

Cyt

ok

ines

+C

D4+

T ly

mp

ho

cyte

s (%

)

(c)

IL-5


0

1.1

2.2


Cyt

ok

ines

+C

D4+

T ly

mp

ho

cyte

s (%

)

(d)

IL-10


0

1.1

2.2


Cyt

ok

ines

+C

D4+

T ly

mp

ho

cyte

s (%

)

(e)

F 3: Cytokine pattern of circulating T CD4+-lymphocytes from patients with acute Schistosomiasis mansoni before (ACT, faint greysquare = 21) and aer praziquantel treatment (ACT-TR, grey square = 07) and noninfected individuals (NI, white square = 19), followingshort-term in vitro cultivation in the absence (control culture) or presence of SEA stimulation. e results are shown in bar-plot formathighlighting the mean percentage of datasets ± standard deviation. Statistically signi�cant di�erences (connecting lines) observed betweengroups NI, ACT, and ACT-TR were considered at 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃.



0

1.1

2.2


IFN-

Cyt

ok

ines

+C

D8+

T ly

mp

ho

cyte

s (%

)

= 0.0381

= 0.0258

= 0.0112< 0.0001

(a)


0

1.1

2.2


TNF-

Cyt

ok

ines

+C

D8+

T ly

mp

ho

cyte

s (%

)

(b)

IL-4


0

1.1

2.2


Cyt

ok

ines

+C

D8+

T ly

mp

ho

cyte

s (%

)

= 0.0008

= 0.0173

< 0.0001< 0.0001

(c)

IL-5


0

1.1

2.2


Cyt

ok

ines

+C

D8+

T ly

mp

ho

cyte

s (%

)

= 0.0299

(d)

IL-10


0

1.1

2.2


Cyt

ok

ines

+C

D8+

T ly

mp

ho

cyte

s (%

)

= 0.0156

(e)

F 4: Cytokine pattern of circulating T CD8+-lymphocytes from patients with acute Schistosomiasis mansoni before (ACT, faint greysquare = 21) and aer praziquantel treatment (ACT-TR, grey square = 07) and noninfected individuals (NI, white square = 19), followingshort-term in vitro cultivation in the absence (control culture) or presence of SEA stimulation. e results are shown in bar-plot formathighlighting the mean percentage of datasets ± standard deviation. Statistically signi�cant di�erences (connecting lines) observed betweengroups NI, ACT, and ACT-TR were considered at 𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃.


the earlier stage of the infection. In this context, abdominalultrasound imaging can be a complementary tool to assist thediagnosis of S. mansoni infection and its clinical monitoring.e ultrasonographic �ndings presented in this study wereconsistent with those described by Barata et al. [34] andCosta-Silva [32], which showed nonspeci�c increase in thesize of liver and spleen (Table 1). Costa-Silva [32] showed thatthe ACT group had increased measurement of longitudinalle/right lobe of liver, size of longitudinal spleen as well asdimension of Hilar portal vein wall, and incipient periportalechogenic thickening, namely, grade I �brosis. us, the datapresented here suggest that alterations identi�ed by analysesof ultrasonographic features, although not speci�c, are com-patible with the acute phase of Schistosomiasis mansoni andprovide a reliable complementary tool for the diagnosis andclinical followup of the disease.

Analyses of hematological parameters showed that ACTand ACT-TR groups presented increased values for totalleukocytes, eosinophils, or monocytes. On the other hand,increased values for lymphocytes in addition to decreasedvalues for neutrophils were observed only in the ACT-TRgroup (Figure 1). ese results suggest the existence of a sys-temic activation of various hematopoietic lineages, antigen-induced secretion of parasite eggs [35–37]. Smithers et al.[38] demonstrated in experimental models that the cellularin�ltrate observed in hepatic granulomas is predominantlyformed by eosinophils and monocytes. Different studiesshowed that acute schistosomiasis patients have increasedlevels of circulating eosinophils [26, 32, 39]. Previous studieshave also demonstrated that eosinophilia occurs between the5th and 7th weeks aer exposure to the parasite and maybe induced by 2 cytokines, such as IL-4, IL-10, IL-13, IL-9, and especially IL-5 [40]. Increased count of circulatingmonocytes was also observed by different studies [26, 41].Moreover, Borojevic et al. [41] showed a delayed differen-tiation of bone marrow neutrophil granulocytes and bloodmonocytosis of S. mansoni-infected animals. ese changesare compatible with modi�cations in the differentiation ofbone marrow myeloid precursors, favoring the production ofa monomacrophage cell lineage. Indeed, some previous stud-ies show that during acute Schistosomiasis mansoni infectiona higher percentage of neutrophil apoptosis is found, com-pared to patients with chronic illness or the control subjects[42]. Also, aer the praziquantel treatment, we have a higherexposure of cryptic antigens which could be contributing toincrease lymphocyte rather than neutrophil counts. Anotherimportant point to be addressed is that, aer treatment, wehave lesser levels of in�ammatory cytokines such as IL-1-betaandTNF-𝛼𝛼, important inducers of neutrophilmobilization tothe peripheral blood during the acute phase of the disease.

Although our results do not show increased valuesfor lymphocytes in the ACT group, in contrast with dataobtained by Costa-Silva [32], a special interest in this studywas the analysis of cytokines producing T lymphocytes,considering the importance of the speci�c immune responseto solve the infection. Analyses of proin�ammatory cytokines(IFN-𝛾𝛾 and TNF-𝛼𝛼) and regulatory cytokines (IL-4, IL-5,and IL-10) demonstrated higher percentage of TNF-𝛼𝛼+ TCD4+ lymphocytes in the ACT group aer SEA stimulation

in comparison with the control culture. Furthermore, in thecontrol cultures, the results showed increased percentage ofIL-4+T CD4+ lymphocytes in both ACT and ACT-TR groupin comparison with NI (Figure 3).

e 1-2 model of CD4+ T-cell differentiation isa well-established paradigm for understanding the basis ofprotective versus pathogenic immune responses for a hostof important human pathogens [43]. 1 cells secrete IFN-𝛾𝛾 and IL-2 and promote cell-mediated immunity, while 2cells secrete IL-4, IL-5, and IL-10, among others. In thiscontext, an important concept that evolved from the studyof 1 and 2 cell responses is the fact that both responsescross regulate each other. us, IFN-𝛾𝛾 downregulates 2cell development, while IL-4 and IL-10 antagonize 1 celldifferentiation [44]. In humans with acute schistosomiasis,higher levels of IFN-𝛾𝛾 and lower levels of IL-5 are found insupernatants from PBMC compared to patients with chronicdisease [19]. However, the results by de Jesus et al. [9] showedonly high production of the proin�ammatory cytokines IL-1, IL-6, and TNF-𝛼𝛼 in cultures of unstimulated PBMC frompatients with acute schistosomiasis. us, our results suggesta mixed cytokine pro�le produced by CD4+ T cells. iscytokine milieu might be preventing tissue damage. In addi-tion, development of a severe in�ammatory disease duringacute schistosomiasis has been documented in knockoutmice for IL-4 and IL-10 genes [45, 46]. Interestingly, ourresults demonstrated predominant increased percentage ofIFN-𝛾𝛾+ and IL-4+ T CD8+ lymphocytes, following short-term incubation of whole blood with SEA in both ACT andACT-TR groups and increase of IL-10+ cells only in theACT group (Figure 4). is study is the �rst one to pointout the relevance of CD8+ T lymphocytes in the immuneresponse of acute phase of schistosomiasis. CD8+ T cellshave been implicated in several immunopathological eventsduring helminthic infection including schistosomiasis [47].e presence of CD8+ T cells was observed in both earlyand chronic murine granulomas with an increased ratio ofCD8+ cells during the chronic phase of the disease [48].Studies developed by Pancré et al. [49] in murine modeldemonstrated that SEA was able to stimulate IFN-𝛾𝛾 or IL-2 producing CD8+ T cells, suggesting a type 1 responseinduced by SEA. In humans,Oliveira-Prado et al. [50] showeda smaller percent of CD4+ and a higher percent of CD8+cells in peripheral blood from patients with chronic schis-tosomiasis. Moreover, our group has previously describedthe putative role of CD8+ T subsets in controlling morbidityduring human schistosomiasis, which was evidenced by theincreased levels of activated HLA-DR+ CD8+ T lymphocytesin patients presenting intestinal clinical form (INT) of thedisease and low levels of CD28+ CD8+ cells in hepatosplenicpatients [51–53]. In addition, Teixeira-Carvalho et al. [54]observed increased percentage of IL-4+, IL-5+, and IL-10+CD8+ lymphocytes following short-term SEA stimulation ofwhole blood samples in vitro in the INT group. Althoughthe recruitment of CD8+ T cells by exogenous antigens (suchas those derived from S. mansoni) primarily seems to beunexpected, recent studies have demonstrated that CD8+T cells are prone to respond to extracellular antigens ininfectious diseases [55, 56] via bystander activation triggered


by persistent antigenic stimulation, cytokines milieu [52,57, 58], or interaction with antigen-presenting cells (APC)that acquire exogenous antigens by phagocytosis and presentthem throughout MHC class I molecules [59–62]. Similarly,data of Montenegro et al. [19] showed that PBMC fromacute schistosomiasis patients responded to SEA and SWAPby producing signi�cantly higher amounts of IFN-𝛾𝛾 and IL-10. A study carried out by de Jesus et al. [9] demonstratedthe occurrence of higher levels of IFN-𝛾𝛾 production inpatients with acute schistosomiasis than those who havechronic infection. However, fewer patients with acute diseaseproduced IL-10 in response to SEA. ese authors alsoshowed a positive correlation between IL-10 levels in SEA-stimulated PBMC and time aer water exposure. On theother hand, Falcão et al. [63] showed that hepatosplenicschistosomiasis patients produced high levels of IFN-𝛾𝛾 andlow levels of IL-10 as compared to INT, associating this lattercytokine with the establishment/maintenance of the severeclinical forms. Interestingly, studies by Pedras-Vasconcelosand Pearce [64] demonstrated that mice infected with schis-tosomes present a regulatory pathway in which type 1 CD8+cells, under the control of IL-4, dampen immunopathologictype 2 responses. Corrêa-Oliveira et al. [13] showed thatthe blockage of IL-4 and IL-5 using anti-IL-4 and anti-IL-5 antibodies signi�cantly reduced the PBMC proliferativeresponse to SEA antigens in acute, chronic intestinal, andhepatosplenic patients.

Another interesting data found was the high IL-10 pro-ducing cells in response to SEA in uninfected individuals.Like SEA is a complex mix of proteins, glycoproteins, andpolysaccharides, this composition inducing could be induc-ing cross-reactivity sharing some epitopeswith other antigensthat previously sensitized the uninfected individuals.

Studies show that praziquantel is an effective drug incontrolling infection with S. mansoni, promoting a reductionin egg excretion and regression of �brosis in mice andhumans [65–70]. We observed that one month aer speci�cchemotherapy with praziquantel, the patients in this studyshowed similar changes in the number of eosinophils andmonocytes, but had a speci�c decrease in the number ofboth neutrophils and lymphocytes as compared with NIand ACT or only NI, respectively (Figure 1). Some authorshave demonstrated that chemotherapy-induced immuneresponses are heterogeneous, depending on the type ofantigen used and time of analysis aer treatment [70, 71].In this context, we observed a higher number of IFN-𝛾𝛾+ andIL-4+ T CD8+ lymphocytes in response to SEA in the ACT-TR group (Figure 4). De Souza et al. [72] demonstrated—sixmonths aer speci�c treatment with oxamniquine—that theIFN-𝛾𝛾 levels increased signi�cantly in response to SEA.Similarly, it was reported that patients with chronic diseaseliving in endemic areas showed increased levels of IFN-𝛾𝛾 aertreatment in response to parasite antigens [73]. Probably,the increase in T-cell reactivity aer chemotherapy can beexplained by exposure of released antigens to the immunesystem following the destruction of worms by chemotherapy[43, 73–76]. In contrast to our study, De Souza et al. [72]have not found increased levels of IL-4 in patients with acuteschistosomiasis aer treatment with oxamniquine.

In conclusion, the data showed that a mixed cytokinepro�le is present during the acute phase of schistosomiasismansoni. One month aer treatment with praziquantel thereis a increase in the production of IL-4 and IL-10 cytokinesfollowing SEA stimulation in circulating lymphocytes frominfected patients. is �nding is associated with a reductionin their morbidity.

Acknowledgments

e authors would like to thank the technical staff of theLaboratório de Imunologia Celular e Molecular, FundaçãoOswaldo Cruz and Núcleo de Pesquisa em Imunologia, UNI-VALE, Brazil, for their invaluable assistance during this study.e authors also wish to thank the Program for Techno-logical Development in Public Health—PDTIS—FIOCRUZfor use of its facilities. is study was supported byFIOCRUZ, Conselho Nacional de Desenvolvimento Cien-t��co e Tecnológico (CNPq), the Fundação de Amparo �Pesquisa de Minas Gerais (FAPEMIG), the National Insti-tutes of Health NIH-ICIDR/Grant AI45451-01, and theUNDP/World Bank/WHO Special Program for Researchand Training in Tropical Diseases. O. A. Martins-Filho, A.Teixeira-Carvalho and R. Corrêa-Oliveira are thankful toCNPq for the fellowships received (PQ). ATC is thankful tothe US Food and Drug Administration for the fellowshipgranted.

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Hindawi Publishing CorporationJournal of Parasitology ResearchVolume 2012, Article ID 394981, 10 pagesdoi:10.1155/2012/394981

Research Article

Cytokine and Chemokine Profile in Individualswith Different Degrees of Periportal Fibrosis due toSchistosoma mansoni Infection

Robson Da Paixao De Souza,1 Luciana Santos Cardoso,1, 2, 3

Giuseppe Tittoni Varela Lopes,1 Maria Cecılia F. Almeida,1

Ricardo Riccio Oliveira,1, 3, 4 Leda Maria Alcantara,1

Edgar M. Carvalho,1, 3, 4, 5 and Maria Ilma Araujo1, 3, 4, 5

1 Servico de Imunologia, Complexo Hospitalar Universitario Professor Edgard Santos, Universidade Federal da Bahia,Rua Joao das Botas s/n, Canela, 40.110-160 Salvador, BA, Brazil

2 Departamento de Ciencias da Vida, Universidade do Estado da Bahia, 2555 Rua Silveira Martins,Cabula, 41.150-000 Salvador, BA, Brazil

3 Instituto Nacional de Ciencia e Tecnologia em Doencas Tropicais (INCT-DT-CNPQ/MCT), Rua Joao das Botas s/n,Canela, 40.110-160 Salvador, BA, Brazil

4 Departamento de Analises Clınicas e Toxicologicas, Faculdade de Farmacia, UFBA, Avenida Adhemar de Barros s/n,Ondina, 40.170-970 Salvador, BA, Brazil

5 Escola Bahiana de Medicina e Saude Publica, Rua Frei Henrique No. 08, Nazare, 40.050-420 Salvador, BA, Brazil

Correspondence should be addressed to Robson Da Paixao De Souza, [email protected]

Received 13 July 2012; Accepted 7 November 2012

Academic Editor: Sergio Costa Oliveira

Copyright © 2012 Robson Da Paixao De Souza et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Periportal fibrosis in schistosomiasis has been associated to the host immune response to parasite antigens. We evaluated theimmune response in S. mansoni infected individuals with different degrees of periportal fibrosis. Cytokine and chemokineswere measured in serum and in supernatants of PBMC cultures stimulated with the soluble adult worm (SWAP) or egg (SEA)antigens, using a sandwich ELISA. The levels of IL-5 in response to SEA were higher in individuals with moderate to severe fibrosis(310.9 pg/mL) compared to individuals without fibrosis (36.8 pg/mL; P = 0.0418). There was also a higher production of TNF-α incultures stimulated with SWAP in patients with insipient fibrosis (1446 pg/mL) compared to those without fibrosis (756.1 pg/mL;P = 0.0319). The serum levels of IL-13 and MIP-1α were higher in subjects without fibrosis than in those with moderate to severefibrosis. However a positive association between serum levels of IL-13, TNF-α, MIP-1α, and RANTES and S. mansoni parasiteburden was found. From these data we conclude that IL-5 and TNF-α may participate in liver pathology in schistosomiasis. Thepositive association between IL-13, TNF-α, MIP-1α, and RANTES with parasite burden, however, might predict the developmentof liver pathology.

1. Introduction

Schistosomiasis is a chronic parasitic infection that affects200 million people in Africa, South America, and Asia andit is estimated that roughly 700 million people in the worldlive at risk of infection [1, 2]. In Brazil, the only speciesdescribed is Schistosoma mansoni, and it is estimated thatseven million people are infected by the parasite and about 25

million are at risk of infection [3]. The liver pathology resultsfrom the host immune response to parasite antigens from theeggs that become trapped in the portal venous system, andit is associated to the morbidity and mortality described inschistosomiasis [4].

The granuloma formation around S. mansoni eggs iscomplex and represents an interaction between productssecreted by the miracidia, which are released from the egg


and the host immune response [5]. Consequently, the gran-ulomas formed act as barriers that prevent the dispersion ofegg antigens of S. mansoni, and the consequent damage tothe liver parenchyma. However, when caused by depositionof large numbers of eggs, the inflammatory process mayprogress to severe fibrosis, which leads to interruption ofnormal blood flow in the venous system to the sinusoidsresulting in portal hypertension, hepatosplenomegaly, andformation of gastric and esophageal varices that can leadto bleeding and even death. This severe form of the diseaseoccurs in five to ten percent of infected subjects living inendemic areas [6–8].

Some studies have evaluated the immune responseassociated with granuloma formation and development ofperiportal fibrosis due to schistosomiasis, both in experi-mental models and in vitro models of granuloma or tissuebiopsies [9–11].

Recently described, the cytokine interleukin-17 (IL-17)is considered as an inflammatory mediator and may beassociated to the pathology of several chronic illnesses [12].IL-17 stimulates the production of IL-6, nitric oxide, andprostaglandin E2 (PGE2) and acts in synergy with othercytokines, such as IL-1, TNF-α, and IFN-γ. Furthermore,this cytokine promotes the proliferation and recruitment ofmonocytes and neutrophils to inflammatory sites. Th17 cellsmay have been involved in the pathogenesis of schistosomia-sis in experimental models [4, 13–15].

There are few studies evaluating the serum levels ofcytokines and chemokines in schistosomiasis patients withdifferent degrees of periportal fibrosis and the participationof IL-17 in the formation of granuloma and progressionto fibrosis in human schistosomiasis remains unclear. Thus,in this study apart from assessment of Th1, Th2, and Tregulatory immune response we also evaluate the levels of IL-17 in human schistosomiasis.

2. Materials and Methods

2.1. Study Design and the Endemic Area. This study wascarried out in an endemic area from schistosomiasis namedAgua Preta, in the state of Bahia, Brazil. Agua Preta is located280 km south of Salvador, the capital of the State Bahia. Itis composed of a residential area in the center of the villageand some surrounding farms. A total of approximately 800people live in the community. They live in poor sanitaryconditions and agriculture is the predominant occupation.There is one river in this region that is used for bathing,washing clothes and utensils, and leisure, exposing theresidents to high risk of Schistosoma infection.

A cross-sectional parasitological surveys using Kato-Katz[16] and sedimentation techniques were conducted in threedifferent stool samples collected on different days. The cur-rent degree of exposure to S. mansoni infection was assessedby a previously developed questionnaire, which providesfour categories of reported level of exposure to infestedwater: no exposure, low exposure (<1 h/week), mediumexposure (1–3 h/week), or high exposure (1–3 h/day) [17–19].

The inclusion criteria for this study were individuals fromendemic areas who have at least one positive parasitologicalexam for S. mansoni. From the 537 individuals who agreedto participate in this study 334 were infected with S. mansoni(62.5%). The frequency of other helminthic infections was43.4% for Trichuris trichiura, 37.4% for Ascaris lumbricoides,33.7% for Hookworms, and 3.5% for Strongyloides stercoralis.From 334 individuals who were infected with S. mansoni,220 agreed to perform abdominal ultrasound, in order todetermine the degree of periportal fibrosis. They also agreedto donate blood for the study of the immunological response.For this particular aim, we did not include individuals underfive or above 60 years old. We also did not include alcoholicindividuals and those with positive serology for HIV, HTLV-1, or hepatitis virus types B and C, all of which are conditionsthat could interfere with the immunological response.

2.2. Ultrasound Examination. Abdominal ultrasound (USG)were performed using the Quantum 2000 Siemens andElegra Siemens ultrasound with a convex transductor of 3.5–5.0 Mhz. Liver span was measured in the midclavicular lineand midline. The liver was also examined for smoothness ofsurface, echogenicity and posterior attenuation of the soundbean, and portal vein diameter outside the liver midwaybetween its entrance into the portal hepatic and its firstbifurcation in the liver. Periportal fibrosis was observedas multiple diffuse echogenic areas. Grading of periportalfibrosis was determined by the mean total thickness of fourportal tracts after the first division from the right and leftbranches of portal vein (PT1) as follow: degree 0, meanthickness <3 mm; degree I, mean thickness 3 to 5 mm; degreeII, mean thickness >5 to 7 mm; and degree III mean thickness>7 mm [20–22]. Of the 220 individuals evaluated 62 (28.2%)had some degree of periportal fibrosis as shown in Table 1.The scores of periportal fibrosis were grouped accordingto the severity, being degree 0 without periportal fibrosis.Incipient periportal fibrosis was considered to individualswith degree I and moderate to severe periportal fibrosis tothose with degrees II and III [23].

To perform the immune response we included 30 indi-viduals with degree 0 and 25 individuals with degree I, whileall individuals with severe forms of the disease characterizedby the grade II (n = 13) or III (n = 04) were included.

2.3. Schistosoma mansoni Antigens. The S. mansoni antigensused in this study were the soluble extract of the adult wormof S. mansoni (SWAP) and the soluble egg antigen (SEA),prepared as previously described [24].

2.4. Cell Culture and Cytokine Measurements. Peripheralblood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque gradient sedimentation and adjusted to a concen-tration of 3 × 106/mL in RPMI 1640 medium containing10% normal human serum (AB positive and heat inacti-vated), 100 U/mL of penicillin, 100 mg/mL of streptomycin,2 mmol/L of L-glutamine, and 30 mmol/L of HEPES (allfrom Life Technologies GIBCO, BRL, Gaithersburg, MS).Cells were cultured without any stimulation or stimulated


Table 1: Demographic characteristics of the individuals enrolled in the study (n = 220).

Characteristic Without fibrosis (n = 158) Incipient fibrosis (n = 45) Moderate to severe fibrosis (n = 17) P

Age (median/range)∗

19 (5–60) 30 (9–58) 40 (21–59) <0.001a,b

Gender n (%)∗∗

Male 83 (52.5) 23 (51.1) 8 (47.1)ns

Female 75 (47.5) 22 (48.9) 9 (52.9)

Burden parasite for Schistosoma mansoni (EPG/range)∗

72 (24–4752) 42 (24–1380) 60 (24–200) 0.0451a

Coinfection (Schistosoma mansoni and other helminth infections) (%)∗∗

Yes 74.7 68.3 66.7ns

No 25.3 31.7 33.3∗Kruskal-Wallis; ∗∗Chi-squared Test; awithout fibrosis versus incipient fibrosis; bwithout fibrosis versus moderate to severe fibrosis.EPG: eggs per gram of feces; ns: not significant (P > 0.05).

with 10 mg/mL of SWAP, SEA, or Phytohemagglutinin(PHA). Plates were incubated at 37◦C in an atmospherecontaining 5% CO2. Supernatants were collected after 72hours of incubation and maintained at −20◦C for measure-ment of cytokines by Enzyme-linked immunosorbent assay(ELISA). Levels of IL-5, IL-13, IL-17, IL-10, IFN-γ, and TNF-α were determined (R&D Systems, Inc, Minneapolis, MN),and results were expressed as pg/mL on the basis of standardcurves.

2.5. Serum Cytokine and Chemokine Measurement. Levels ofthe cytokines IL-5, IL-13, IFN-γ, TNF-α, IL-17, IL-10, TGF-β(R&D systems Inc., Minneapolis) and the chemokines MIP-1α/CCL3 and RANTES/CCL5 were measured in serum usingsandwich ELISA (eBioscience). The results are expressed aspg/mL, based in standard curves.

2.6. Statistical Analysis. Statistical analyses were performedusing the Statistical Package for the Social Sciences software(version 9.0 for Windows, SPSS). Statistical differencesbetween groups were assessed using the Kruskal-Wallisanalysis of variance test. Fisher’s exact test was used tocompare proportions. To assess the association between theserum levels of cytokines and chemokines and the parasiteburden of S. mansoni the Linear Regression tests usingGraphpad PRISM 3.03 software (La Jolla, CA, USA) wereused. All statistical tests were two-tailed and the statisticalsignificance was established at the 95 percent confidenceinterval and significance was defined to P < 0.05.

The Ethical Committee of Climerio de Oliveira Mater-nity, Federal University of Bahia, approved the present study.Informed consent was obtained from all study participantsor their legal guardians (License number 240/2008).

3. Results

3.1. Features of the Studied Subject. The demographiccharacteristics, parasite burden, and the ultrasonographyevaluation for periportal fibrosis measurement are shown inTable 1. It was observed that the mean age of individuals

with incipient and moderate to severe periportal fibrosis washigher than in individuals without fibrosis (P < 0.05).

There was no significant difference in gender distributionamong groups. There was also no significant differencein the levels of exposure to the contaminated water, withmedium to high contact to the water being found in60.5%, 55.3%, and 80.0% of individuals without periportalfibrosis, incipient fibrosis, and moderate to severe fibrosis,respectively (P > 0.05).

The S. mansoni parasite burden of individuals withoutperiportal fibrosis was higher than in patients with incipientfibrosis (P < 0.05). There was no statistically significantdifference of being coinfected with other helminths in allgroups of patients (Table 1).

3.2. Cytokine Responses Induced by S. mansoni Antigens.Levels of IL-5, IL-13, IL-17, IFN-γ, TNF-α, and IL-10 weremeasured in supernatant of PBMCs cultures unstimulatedand stimulated with SWAP and SEA as shown in Table 2. Asignificantly higher levels of IL-5 in cultures stimulated withSEA was observed in the group of patients with moderateto severe fibrosis (median levels = 310.9 pg/mL) comparedto those without fibrosis (median levels = 36.8 pg/mL; P =0.0418).

Levels of TNF-α were significantly higher in supernatantsfrom SWAP-stimulated PBMC of subjects with incipientperiportal fibrosis (median levels = 1446 pg/mL) than inindividuals without fibroses (median levels = 756.1 pg/mL;P = 0.0319). Moreover, the levels of TNF-α in nonstimulatedcultures were higher in individuals with moderate to severefibrosis (median levels = 488.7 pg/mL) compared to subjectswithout periportal fibrosis (median levels = 61.9 pg/mL; P =0.0182).

The levels of IL-13, IL-17, IFN-γ, and IL-10 did not differsignificantly between individuals of the different groups(Table 2).

3.3. Serum Levels of Cytokines. The levels of serum cytokinesin patients with different degrees of periportal fibrosis areshown in Figure 1. With the exception of IFN-γ and IL-17,whose levels were below the detection limit of 15.6 pg/mL


Table 2: Levels of cytokines produced by peripheral blood mononuclear cells (PBMC) of studied individuals.

Cytocines (pg/mL) Without fibrosis Incipient fibrosis Moderate to severeP

median (min–max) (n = 30) (n = 25) Fibrosis (n = 17)

IL-5

Without antigen 31.2 (31.2–112.2) 31.2 (31.2–75.5) 31.2 (31.2–127.8) ns

SWAP 374.4 (31.2–5153.0) 524 (31.2–5905.0) 1988.0 (31.2–5135.0) ns

SEA 36.8 (31.2–3820.0) 188.4 (31.2–4232.0) 310.9 (31.2–4256.0) 0.0418a

IL-13


SWAP 246.6 (93.8–1518.0) 190.3 (93.8 –1290.0) 213.7 (93.8–2091.0) ns

SEA 171.2 (93.8–1497.0) 93.8 (93.8–1442.0) 93.8 (93.8–1857.0) ns

IL-17


SWAP 21.4 (15.6–790.5) 57.1 (15.6–974.8) 21.4 (15.6–345.2) ns

SEA 18.9 (15.6–291.1) 15.6 (15.6–615.3) 27.2 (15.6–557.5) ns

IFN-γ


SWAP 73.1 (31.2–3038.0) 1205.0 (31.2–5507.0) 215.8 (31.2–5249.0) ns

SEA 83.1 (31.2–288.0) 203.8 (31.2–2905.0) 130.1 (31.2–2714.0) ns

TNF-α

Without antigen 61.9 (31.2–633.3) 346.6 (31.2–2421.0) 488.7 (31.2–3229.0) 0.0182a

SWAP 756.1 (80.6–3366.0) 1446.0 (236.4–3057.0) 1002.0 (35.7–2351.0) 0.0319b

SEA 389.8 (102.7–2521.0) 827.3 (31.2–3551.0) 927.3 (89.1–2517.0) ns

IL-10


SWAP 393.8 (22.1–1976.0) 399.6 (15.6–1779.0) 318.1 (15.6–1213.0) ns

SEA 585.7 (175.7–2075.0) 1342.0 (15.6–2472.0) 745.8 (35.5–1543.0) ns∗Kruskal-Wallis; awithout fibrosis versus moderate to severe fibrosis; bwithout fibrosis versus incipient fibrosis; ns: not significant (P > 0.05).SWAP: soluble worm antigen preparation of adult Schistosoma mansoni; SEA: Schistosoma mansoni egg antigen.

and 31.2 pg/mL, respectively, in the three groups of patients(data not shown), high levels of serum cytokines wereobserved in the different groups of individuals (Figure 1).

While the median levels of IL-13 were higher in indi-viduals without periportal fibrosis compared to those withmoderate to severe fibrosis (113.3 pg/mL and 97.7 pg/mL,respectively; P = 0.0006), there was no significant differencein the median levels of IL-5, IL-10, TNF-α and TGF-βbetween groups (P > 0.05; Figure 1).

3.4. Serum Level of the Chemokines . The median levels of thechemokines MIP-1α and RANTES in serum of individualswith different degrees of periportal fibrosis are shown inFigure 2. It was observed that the median level of MIP-1α was higher in individuals without periportal fibrosis(70.9 pg/mL) compared to those with moderate to severefibrosis (7.8 pg/mL; P = 0.0232). There was no significantdifference in serum levels of RANTES in individuals withdifferent degrees of periportal fibrosis.

3.5. Association between Serum Levels of Cytokines andChemokines and S. mansoni Parasite Burden. We testedthe possible association between serum levels of cytokinesand chemokines and S. mansoni parasite burden in thestudied population. A positive association between serum

levels of IL-13, TNF-α, MIP-1α, and RANTES and parasiteburden was observed (Figure 3). There were no significantassociations between serum levels of IL-5 (R2 = 0.003413;P > 0.05), IFN-γ (R2 = 0.005831; P > 0.05), IL-10 (R2 =0.004651; P > 0.05), and TGF-β (R2 = 0.003391; P > 0.05)and the parasite burden of S. mansoni.

4. Discussion

The development of periportal fibrosis can occur as a resultof chronic schistosomiasis infection and accounts for thesevere forms of the disease [6]. This fact characterizesschistosomiasis as a serious public health problem.

The present study aimed to evaluate cytokine andchemokine profile in individuals with different degrees ofperiportal fibrosis living in the schistosomiasis endemicarea of Agua Preta, Brazil. Additionally, possible personaland environmental features which could interfere with thedevelopment of periportal fibrosis were also evaluated. Weshowed that despite the high prevalence of S. mansoniinfection in Agua Preta, Bahia, the frequency of severeforms of the disease determined by ultrasonography waslow. This could be due to intrinsic characteristics of thispopulation, since there is no report of previous treatmentfor schistosomiasis in this region. Other possible explanation


IL-5

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Without fibrosis Incipient Moderate to severe

(a)

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3 (p

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(pg/

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)

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Figure 1: Serum cytokine levels in schistosomiasis individuals with different degrees of periportal fibrosis: (first box) individuals withoutfibrosis (n = 30), (second box) incipient fibrosis (n = 25), and (third box) moderate to severe periportal fibrosis (n = 17). Levels of IL-5, IL-13, TNF-α, TGF-β, and IL-10 were determined by sandwich ELISA technique. Horizontal lines represent the median values, boxes representthe 25th to the 75th percentiles, and vertical lines represent the 10th to 90th percentiles. Asterisks indicate statistically significant differences(P < 0.05; ANOVA).

would be the Cairo’s methodology to classify the periportalfibrosis which considers only degrees II and III as severe.We opted to use the Cairo’s classification because we haveperformed previous studies using these parameters andbecause the physicians who have performed the USG in ourstudies are very well familiar with this classification.

The majority of individuals who had the most severedegree of periportal fibrosis were over 40 years of age, andthere was no influence of gender on periportal fibrosis devel-opment. This is in agreement with other authors who havedemonstrated that most individuals who develop periportalfibrosis are over 50 years of age [22]. This could be explained


0

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-1α

/CC

L3

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L5


RANTES

(b)

Figure 2: Serum chemokine levels in schistosomiasis individuals with different degrees of periportal fibrosis: (first box) individuals withoutfibrosis (n = 30), (second box) incipient fibrosis (n = 25), and (third box) moderate to severe periportal fibrosis (n = 17). Levels of MIP-1αand RANTES were determined by sandwich ELISA technique. Horizontal lines represent the median values, boxes represent the 25th tothe 75th percentiles, and vertical lines represent the 10th to 90th percentiles. Asterisks indicate statistically significant differences (P < 0.05;ANOVA).

P < 0.05

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-1α

MIP-1α

(pg

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900900

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RA

NT

ES

(pg/

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RANTES∗R2 = 0.0713

P < 0.05

(d)

Figure 3: Correlation between serum levels of IL-13, TNF-α, MIP-1α, and RANTES (n = 65) and S. mansoni parasite burden. P < 0.05indicates statistically significant differences (Linear Regression).


by the immune response induced by constant reexposure tothe parasite over lifetime, or by the slow process of fibrosisformation. Therefore, younger individuals probably havenot been exposed long enough to the cumulative effects ofcollagen deposition in the periportal tract [25].

Many variables may influence the magnitude of theimmune response in human schistosomiasis which includesgender and age [26], intensity of infection [27–30], andgenetic characteristics of the population [31]. However, thereason why severe fibrosis develops only in a fraction ofthe population, which is under the same environmentalconditions as others who do not develop fibrosis, remainsnot well understood. In this study, we found no associationbetween level of exposure to contaminated water and degreeof periportal fibrosis. Moreover, individuals without peripor-tal fibrosis had a higher parasite burden than individuals withincipient periportal fibrosis. A possible explanation for thisobservation is that chronic infection can lead to intestinalfibrosis that impairs the migration of eggs to the intestinallumen and thereby decreases eggs count in parasitologicalexams [32].

It has been described that the specific type 2 cytokinepattern is a marker of S. mansoni chronic infection inmice and humans, and that this response is involved in thedevelopment of periportal fibrosis due to schistosomiasis[21, 33–35]. In this study we evaluated cytokine productionby PBMC stimulated with the S. mansoni antigens SWAP andSEA as well as its serum levels and correlation with parasiteload. We observed a greater variability in cytokines levels,which shows heterogeneity of response to the S. mansoniantigens.

Levels of IL-5 in supernatants of SEA-stimulated PBMCswere higher in cultures of individuals with moderate tosevere periportal fibrosis, when compared to the groupwithout fibrosis. These data are similar to what was observedin a study conducted in schistosomiasis patient’s residents inother endemic areas of Bahia [36, 37]. These data suggestthat the Th2 immune response is developedand directedto antigens from the eggs. IL-5 has been associated withgranuloma formation around S. mansoni eggs in the liver,since this cytokine participates in eosinophil growth andactivation contributing to a significant source of profibroticmediators such as IL-13 [38]. Likewise, IL-5 may participatedirectly in tissue remodeling and fibrosis in schistosomiasis[38]. In contrast to our data that showed no significantdifference or correlation between serum levels of IL-5 and S.mansoni parasite burden in individuals with different degreesof periportal fibrosis, it has been described that serum levelsof IL-5 is higher in patients with severe degrees of periportalfibrosis than in those without fibrosis [39].

Many studies in experimental models have shown thatIL-13 plays an important role in the development of liverfibrosis. The blockage of IL-13 and IL-4 receptors preventedgranuloma development and liver fibrosis in mice [33].Human studies suggest a correlation between productionof high levels of IL-13 and development of more advanceddegree of liver fibrosis [36, 37]. However, in this study wedid not observe differences in IL-13 levels in supernatants ofSWAP or SEA stimulated PBMCs in all groups evaluated. We

found, however, higher levels of serum IL-13 in the group ofindividuals without periportal fibrosis compared to patientswith severe fibrosis. It has been suggested that levels of IL-13 tend to be higher during the prefibrotic phase, acting asan inducer of the lesion [35, 39, 40]. The parasite burden isone of many other variables that can influence the magnitudeof the immune response in human schistosomiasis [32, 41]and a high parasite burden has been associated with thedevelopment of fibrosis [5, 6]. Corroborating with theseobservation, we also found a positive association betweenparasite burden and serum levels of IL-13.

There are no published data about the role of IL-17 indevelopment of hepatic fibrosis due to schistosomiasis inhumans. As reviewed byTallima et al. (2009), this cytokineis involved in the development of severe disease by recruit-ment of neutrophils and macrophages in inflammatorysites, demonstrated only in experimental models [42]. Weobserved production of IL-17 in cultures stimulated by allthe antigens tested without, however, differences amongthe groups. It is suggested that induction of severe liverdisease associated with expression of IL-17 in experimentalmodels, with polarized immune response to a Th1 profile, isdependent on the presence of IL-23, another Th17 cytokine[12, 13]. In this study, we evaluated the IL-17 productionin individuals chronically infected by S. mansoni, whohave a predominantly Th2 immune response, which mayexplain the low and not detectable levels of IL-17. Whenevaluating serum levels of this cytokine we also found levelsof IL-17 below the detection limit in most individuals,which corroborates the results observed when measuring thesupernatants of PBMCs cultures. Moreover, there was nocorrelation between serum levels of IL-17 and S. mansoniparasite load.

Some authors have suggested that IFN-γ has certainantifibrotic activities, since this cytokine acts by inhibitingproduction of extracellular matrix proteins, enhances theactivity of collagenase in liver tissue, and downmodulatesthe Th2 response [10, 43]. In this study, we found nosignificant differences in levels of IFN-γ between groups,and no significant association between parasite burden andserum levels of this cytokine in individuals with differentlevels of periportal fibrosis.

Although controversy, TNF-α is another cytokine thatmay participate in the granuloma formation and evolution offibrotic tissue process.Hoffmann and colleagues (1998) havedemonstrated in experimental models that TNF-α exertsa protective effect, whereas other authors attribute to theTNF-α proinflammatory and profibrogenic effects [7, 44].In the present study it was observed that cells of individualswith incipient or moderate to severe fibrosis, even withoutantigenic stimuli, produced higher levels of TNF-α whencompared to those without fibrosis. Additionally, therewas a positive association between serum levels of TNF-αand S. mansoni parasite burden, which suggests that thiscytokine may contribute to the liver pathology observed inschistosomiasis [45]. Supporting the role of TNF-α in thedevelopment of liver pathology due to schistosomiasis, astudy conducted in a schistosomiasis endemic area in Brazilhas demonstrated that individuals with moderate to severe


periportal fibrosis have higher serum levels of TNF-α thanthose without fibrosis [39].

Some studies have pointed out TGF-β as an importantfactor in periportal fibrosis development during chronicschistosomiasis. This cytokine is considered a multifunc-tional cytokine that regulates biological processes such asinflammation, development, and differentiation of many celltypes, tissue repair, and tumor genesis. It is also associatedwith pro-inflammatory responses and immunosuppressiveactivities [46, 47] and participates in the process of Th17 cellsdifferentiation [42]. In this study, we found no significantdifferences in serum levels of TGF-β between groups withdifferent degrees of periportal fibrosis, nor did we find anassociation between parasite burden and levels of TGF-β.Recent studies in experimental models have shown a negativeassociation between serum levels of TGF-β and S. mansoniparasite burden in chronically infected animals, suggestingthe involvement of this cytokine in controlling the parasiteload [48].

Other molecule assessed in this study was the regulatorycytokine IL-10, which is associated with the switch from theTh1 immune response observed during the acute phase toTh2 responses and consequently preventing the developmentof severe disease. In this study we found no significantdifference in the serum levels of this cytokine,nor in thelevels measured in supernatant of stimulated cultures amongindividuals with different degrees of periportal fibrosis.This is in agreement with Silva-Teixeira and coworkers(2004) who demonstrated no association between levelsof IL-10 and parasite burden, suggesting that IL-10 maynot be involved in periportal fibrosis development duringschistosomiasis [39].

Levels of cytokines in patients with schistosomiasis havebeen evaluated in serum or supernatants of cell cultures. Inthis study we decided to evaluate cytokine in both, serumand supernatants. We believe that serum cytokine levelsrepresent the in vivo profile status. However, as paracrinecytokines, such as IL-5, IL-13, IFN-γ, and IL-10, are not welldetected in serum using convenient technique such as ELISA,we decided to evaluate these molecules in supernatantsof PBMC restimulated in vitro with parasite antigens. Webelieve that these two measurements are complementary andlead to a better knowledge of the cytokine profile in humanschistosomiasis.

The role of chemokines in periportal fibrosis due toschistosomiasis is also not completely understood [49]. Inthis study, we found higher levels of serum MIP-1α inindividuals without periportal fibrosis than in patients withincipient or moderate to severe fibrosis, which is in contrastto what has been found by Souza and colleagues [50, 51]. Weshowed, however, a positive association between serum levelsof MIP-1α and S. mansoni parasite burden. This finding issimilar to what was obtained for IL-13 and reinforces thethought that individuals without fibrosis enrolled in thisstudy may have been in a prefibrotic stage. Interestingly, ithas been also demonstrated that MIP-1α induces productionof Th2-pattern cytokines, including IL-13 [52, 53].

Many authors have suggested that high levels of IFN-γ and TNF-α are associated with increased expression of

RANTES, whereas Th2 cytokines, such as IL-4 and IL-13, areassociated with decreased expression [54–56]. In agreementwith these studies, it has shown that RANTES deficient miceshowed a significant increase granulomatous response whencompared to the control group [57]. These studies reinforcethe idea that RANTES regulates negatively the developmentof granuloma and fibrosis. On the other hand, there are nodata in the literature on the possible role of RANTES in thedevelopment of periportal fibrosis in human schistosomiasis.

The present study showed high levels of IL-5 and TNF-αin individuals with periportal fibrosis. Higher levels of IL-13and MIP-1α in individuals without periportal fibrosis werealso documented. Considering that these former moleculeswere positively associated with S. mansoni parasite burden, aswere TNF-α and RANTES, they may represent biomarker forthe progression of liver pathology in schistosomiasis. Largercasuistic further studies are needed however to confirm therole of these cytokines and chemokines in the developmentof periportal fibrosis in human schistosomiasis.

Acknowledgments

The authors would like to thanks to Dr. Marta Leite and Dr.Antonio Carlos M. Lemos for their support in the selectionof patients, and Dr. Irisma Souza and Dr. Delfin Gonzalez forperforming the ultrasound assessment. they are very gratefulto all volunteers from the community of Agua Preta, Gandu,who agreed to partipate in this study, to the local health agentIrene Jesus for her support, and to Michael Andrew Sundbergfor the corrections and suggestions made in the text. Theyalso thank the Fundacao de Amparo a Pesquisa do Estadoda (Bahia FAPESB) for the financial support. E. Carvalhoand M. I. Araujo are investigators supported by The Con-selho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq). This work was supported by the Brazilian NationalResearch Council (CNPq) Universal (482113/2010-3).

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Review Article

New Frontiers in Schistosoma Genomics and Transcriptomics

Laila A. Nahum,1, 2, 3 Marina M. Mourao,1 and Guilherme Oliveira1, 2

1 Genomics and Computational Biology Group, Centro de Pesquisas Rene Rachou (CPqRR), Instituto Nacional de Ciencia e Tecnologiaem Doencas Tropicais, Fundacao Oswaldo Cruz (FIOCRUZ), 30190-002 Belo Horizonte, MG, Brazil

2 Centro de Excelencia em Bioinformatica (CEBio), Fundacao Oswaldo Cruz (FIOCRUZ), 30190-110 Belo Horizonte, MG, Brazil3 Faculdade Inforium de Tecnologia, 30130-180 Belo Horizonte, MG, Brazil

Correspondence should be addressed to Guilherme Oliveira, [email protected]

Received 10 August 2012; Accepted 16 October 2012

Academic Editor: Sergio Costa Oliveira

Copyright © 2012 Laila A. Nahum et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Schistosomes are digenean blood flukes of aves and mammals comprising 23 species. Some species are causative agents of humanschistosomiasis, the second major neglected disease affecting over 230 million people worldwide. Modern technologies includingthe sequencing and characterization of nucleic acids and proteins have allowed large-scale analyses of parasites and hosts, openingnew frontiers in biological research with potential biomedical and biotechnological applications. Nuclear genomes of the threemost socioeconomically important species (S. haematobium, S. japonicum, and S. mansoni) have been sequenced and are underintense investigation. Mitochondrial genomes of six Schistosoma species have also been completely sequenced and analysed froman evolutionary perspective. Furthermore, DNA barcoding of mitochondrial sequences is used for biodiversity assessment ofschistosomes. Despite the efforts in the characterization of Schistosoma genomes and transcriptomes, many questions regarding thebiology and evolution of this important taxon remain unanswered. This paper aims to discuss some advances in the schistosomeresearch with emphasis on genomics and transcriptomics. It also aims to discuss the main challenges of the current research andto point out some future directions in schistosome studies.

1. Introduction

Schistosomatidae (Platyhelminthes: Digenea) includes sev-eral digenetic endoparasites with complex life cycles, whosedevelopmental stages alternate between intermediate (fresh-water gastropods) and definitive hosts (birds, reptiles, fishes,and mammals) [1]. These parasites differ from other bloodflukes in having separate sexes. Another important featureis the increased longevity (over 5 years) of the Schistosomaspecies in the human host [1].

Schistosoma, the avian and mammalian blood flukes,is the best studied genus of Schistosomatidae [1]. Severalspecies are described, six of which infect humans causingschistosomiasis: S. haematobium, S. intercalatum, S. japon-icum, S. malayensis, S. mansoni, and S. mekongi. Otherspecies are known to infect a broad range of mammalssuch as hippopotamus, rodents, carnivores, and ruminantanimals like buffalo, cattle, goat, lechwe, and sheep. Somehybrid species are reported [1–4]. For example, S. mattheei,

more commonly found in cattle, is believed to form hybridsbetween S. mattheei and S. haematobium thus increasing itssnail and definitive host range [3].

Schistosomiasis, a chronic and debilitating disease, isconsidered by the World Health Organization as one ofthe most serious public health issues and the second mostprevalent tropical disease with high morbidity in the world[5]. Schistosomiasis is endemic in 77 countries [6] and itstransmission is dependent on the existence and distributionof intermediate hosts. It is estimated that 237 million peoplerequire treatment worldwide and that 600 to 779 millionpeople live in endemic areas, under infection risk [6]. Yet,exacerbating this scenario, this disease is responsible for theloss of 1.7 to 4.5 millions of years of life in the world,measured by disability-adjusted life years (DALY) [7], one ofthe highest indexes among all the neglected tropical diseases.Control of schistosomiasis represents a great challenge andit is based on drug treatment (Praziquantel), snail control,improved sanitation, and health education [5].


The development of powerful and scalable methodsto analyse nucleic acids and proteins has changed theway biological data is surveyed. The application of suchtechnologies, together with the development of powerfulcomputational tools and methods, have expanded ourperspective of schistosome biology and allowed a betterunderstanding of processes such as host-parasite interaction[8–10].

This paper aims to discuss some advances in schistosomeresearch with emphasis on genomics and transcriptomics.First, we summarize the current status of sequencing projectsof nuclear and mitochondrial genomes. We also discusssome aspects of evolutionary genomics and biodiversity ofschistosomes. Then, we present key findings in transcrip-tomic analyses. Finally, we point out the main challenges ofthe current research and suggest some future directions inSchistosoma genomic and transcriptomic studies.

2. Schistosoma Genomics

In order to strengthen traditional methods and providenew information to improve success towards schistosomiasiscontrol, the scientific community joined efforts to assessthe Schistosoma genomic information, starting in 1994, asan initiative of WHO/TDR (UNICEF-UNDP-World Bank-WHO Special Programme for Research and Training inTropical Diseases) [11, 12]. At that time, only a few hundredexpressed sequence tags (ESTs) from S. mansoni were avail-able [13]. The WHO/TDR support opened the possibilityto generate data that could be translated into new tools forschistosomiasis diagnostics and treatment, representing the“kickoff” of Schistosoma nuclear genome studies.

2.1. Nuclear Genomes. The karyotype of known Schistosomaspecies comprises seven pairs of autosomes and one pair ofsex chromosomes (female = ZW, male = ZZ), ranging in sizefrom 18 to 73 megabases (Mb) [14, 15].

The nuclear genome of three Schistosoma species wassequenced (Table 1). The S. japonicum (Anhui isolate) andS. mansoni (Puerto Rico isolate) genomes were decoded andsimultaneously published as an initiative of the WellcomeTrust Sanger Center Institute in collaboration with theInstitute for Genomic Research (TIGR) and the Schistosomajaponicum Genome Sequencing and Functional AnalysisConsortium [16, 17]. Recently, the S. haematobium (Egyp-tian isolate) genome was sequenced opening new possibilitiesin comparative genomics of schistosomes [18].

S. haematobium, S. japonicum, and S. mansoni carry anuclear genome of 385, 397, and 363 Mb composed byapproximately, 13,073, 13,469, and 10,852 protein-codinggenes, respectively [16–19]. It is worth to mention that thenumber of predicted protein-coding genes does not reflectthe genome structure per se, but the actual state of analysesof the different draft genome data.

The genetic linkage map of S. mansoni was obtainedand allowed the refinement of the genome sequencing dataproviding a means for gene discovery and gene functionanalysis [20, 21]. More importantly, it has allowed the

identification of genetic loci that determine important traitssuch as host specificity, virulence, and drug resistance.

In fact, although the quality and annotation of the S.mansoni genome sequence data have been greatly improved(see below); the corresponding data of S. haematobiumand S. japonicum are still considered drafts [16–19]. Theirgenome sequences are distributed in a large number ofcontigs; therefore extensive work is required. Constant datarefinement is necessary to provide a reliable comprehensionof the genome and gene models and to provide a curatedannotation.

The S. mansoni genome annotation has been improvedby combining different sequencing strategies [19]. Suchstrategies include new assembly of existing capillary readssupplemented with an additional ∼90,000 fosmid and BACend sequences, deep sequencing of clonal DNA (NMRIstrain, Puerto Rican origin) using 108-based paired reads onIllumina Genome Analyzer IIx platform, and RNA-seq dataof different parasite life stages.

Progress on the knowledge of S. mansoni genome featuresis still expanding, especially with the application of the next-generation sequencing platforms and single nucleotide poly-morphism typing methods. For instance, 14 new microsatel-lite markers were recently identified by de novo assemblyof massive sequencing reads; these new features can beemployed for pedigree studies [22]. Yet, newer sequencingtechnologies will allow for the development of populationgenomics studies of field, laboratory, and clinical isolates.

As a result of the availability of the Schistosoma genomesequence data, comparative analyses across different speciesbecame possible bringing together different areas of research[21, 23, 24]. Comparative genomics of three Schistosomaspecies revealed several conserved features such as theoverall GC content, sequence identity, presence of repetitiveelements, and synteny [18]. This genome-wide analysiscorroborates previous genetic studies and supports theevolutionary hypothesis of S. haematobium and S. mansonias the most related species, followed by S. japonicum, to theexclusion of other Trematoda such as Clonorchis, Fasciola,and Schmidtea.

2.2. Mitochondrial Genomes. Mitochondrial genes have beenused as molecular markers for species and strain identifica-tion, which is key to a variety of studies such as phylogenetics,population genetics, biogeography, and molecular ecology[25–29]. Besides helping to elucidate the evolutionary rela-tionships among taxa, mitochondrial genes have been suc-cessfully applied into epidemiological studies, monitoring,and control of microbes, parasites, and vectors of medicaland socioeconomic importance. The analysis of the wholemitochondrial genome of diverse taxa has changed theperspective of such studies.

The mitochondrial genome of six Schistosoma specieswas sequenced (Table 1) including S. haematobium(NC 008074), S. japonicum (NC 002544), S. mansoni(NC 002545), S. malayensis, S. mekongi (NC 002529),and S. spindale (NC 008067) [27, 30–32]. These genomesrange in size from 13,503 to 16,901 bp and encode 36 genes


Table 1: Availability of genomic and transcriptomic data for Schistosoma species.

Taxid Taxon ncDNA mtDNA ESTs Barcoding

6184 Schistosoma bovis No No No Yes

6186 Schistosoma curassoni No No No Yes

230327 Schistosoma edwardiense No No No Yes

393876 Schistosoma guineensis No No No Yes

6185 Schistosoma haematobium Yes Yes Yes Yes

157462 Schistosoma hippopotami No No No Yes

198245 Schistosoma incognitum No No No Yes

216970 Schistosoma indicum No No No Yes

6187 Schistosoma intercalatum No No No Yes

6182 Schistosoma japonicum Yes Yes Yes Yes

646316 Schistosoma kisumuensis (∗) No No No Yes

216972 Schistosoma leiperi No No No Yes

53353 Schistosoma malayensis No Yes No Yes

6183 Schistosoma mansoni Yes Yes Yes Yes

48269 Schistosoma margrebowiei No No No Yes

31246 Schistosoma mattheei No No No Yes

38744 Schistosoma mekongi No Yes No Yes

216971 Schistosoma nasale No No No Yes

6188 Schistosoma rodhaini No No No Yes

191505 Schistosoma sinensium No No No Yes

230328 Schistosoma sp. Uganda-JATM-2003 No No No Yes

6189 Schistosoma spindale No Yes No Yes

Taxid: NCBI taxonomy identifier. ncDNA: nuclear DNA genome (SchistoDB.org). mtDNA: mitochondrial DNA genome (NCBI Organelle GenomeResources). ESTs: data from NCBI dbEST (release 120701, July 1, 2012). Barcoding: DNA barcoding used on cox1 sequences (BOLD Systems). ∗Schistosomasp. BH-2009.

comprising two ribosomal genes (large and small subunitrRNA genes), and 22 transfer RNA (tRNA) genes, as well as12 protein-encoding genes (atp6, cob, cox1–3, nad1–6, andnad4L). The atp8 gene, commonly found in other phyla, isabsent from Platyhelminthes, whose mitochondrial genomeshave been analysed [31]. Moreover, each Schistosomamitochondrial genome contains a long noncoding regionthat is divided into two parts by one or more tRNA genes,which is found in other Platyhelminthes and vary in lengthaccording to each taxon.

Comparative mitogenomics have revealed a highly con-served gene order among distinct Platyhelminthes withthe exception of some Schistosoma species [25, 30–33].The mitochondrial genome of the Asian schistosomes, S.japonicum and S. mekongi, displays the same gene order asother Digenea and Cestoda [25, 32].

In contrast, the gene order in the mitochondrial genomeof S. haematobium, S. mansoni, and S. spindale is strik-ingly different from other taxa [25, 30]. The unique geneorder rearrangements identified in these species constitutevaluable information to the reconstruction of the phyloge-netic relationships of Schistosoma and other Platyhelminthes(Section 3.1).

3. Evolutionary Genomics and Biodiversity

3.1. Evolutionary Genomics of Schistosoma. The use ofgenomic data is valuable to the understanding of the

evolutionary history of Schistosoma especially due to theabsence of fossil records. Molecular markers have been usedto test hypotheses in a variety of Schistosoma phylogenetic,phylogeographic and epidemiological studies [4, 25, 34–36].Traditionally, these markers include the nuclear ribosomalRNA genes (18S, 5.8S, and 28S) and the internally tran-scribed spacer (ITS) region besides a number of mitochon-drial genes (cob, cox1–3, nad1–6, etc.).

Based on mitochondrial data from different studies, astandard phylogeny of 23 Schistosoma species was proposed[25]. Six clades were identified that correlate with thegeographic distribution of the species analysed. These cladesinclude (1) the S. japonicum complex (S. japonicum, S.malayensis, S. mekongi, S. ovuncatum, and S. sinensium)found in Central and South Eastern Asia; (2) the S.hippopotami clade (S. edwardiense and S. hippopotami)distributed in Africa; (3) the proto-S. mansoni clade (S.incognitum and S. turkestanicum) in Central Asia, Near East,and Eastern Europe; (4) the S. mansoni clade (S. mansoni andS. rodhaini) found in Africa and South America; (5) the S.haematobium clade (S. bovis, S. curassoni, S. guineensis, S.haematobium, S. intercalatum, S. kisumuensis, S. leiperi, S.margrebowiei, and S. mattheei) occurring in different partsof Africa; (6) the S. indicum clade (S. indicum, S. nasale, andS. spindale) found in India and South Asia.

There are divergent opinions concerning the origin of thegenus Schistosoma and its intermediate snail host [25, 37–39]. The “out of Asia” hypothesis is the most generally


accepted, indicating a migration followed by dispersion ofthese parasites from Asia to Africa [25]. In this regard, studiesof molecular phylogeny suggested that the genus Schistosomahad its origin in Asia and (at least two descendants) colonizedthe African continent independently, where they easilyradiated, becoming exclusive parasites of planorbid snails.Returning to Asia, the parasites diversified into groups ofspecies, characterized by the position of the spike in the eggs[38]. The molecular phylogeny of seven Schistosoma speciessuggested that schistosomes were endoparasites of rodentsand ruminants and were transmitted to the first hominidsin Africa as a migration to savanna areas occurred [40].However, in order to better define the Schistosoma origin,there is a need to sample a broader range of species.

Most of the widely accepted Schistosoma phylogenies areprimarily based on sequence alignments of mitochondrialgenes such as cox1–3, and nad1–6, [25, 30]. Other studiesuse changes in the mitochondrial gene order as phylogeneticmarkers. Indeed, these gene rearrangements, consideredto be rare evolutionary events, have been applied to anincreasing number of studies (cf. [41]).

As mentioned before, major changes in the mitochon-drial gene order were identified in some Schistosoma species(Section 2.2), more specifically in the African clades (S.mansoni and S. haematobium) and the S. indicum group(S. spindale). This provides further evidence for the Asianancestry of schistosomes and corroborates the concept of thereinvasion of Asia by members of the S. indicum group fromAfrica [27, 30].

Besides reconstructing the phylogenetic relationships ofdiverse taxa, the evolutionary framework has been applied toimprove functional annotation of genes and gene products,as well as to study gene/protein family evolution [41].Phylogenomics has allowed the identification and character-ization of all eukaryotic protein kinases encoded in the S.mansoni genome and improved the functional annotationof over 40% of them, highlighting the molecular diversity ofthese enzymes [42, 43].

A phylogenetic analysis was also employed in the clas-sification of S. mansoni histone proteins, which play akey role in epigenetic modifications that might reflect theparasite complex lifestyle [44]. By applying an evolutionaryframework to analyse a large sequence dataset from a broadrange of organisms, functional annotation of the S. mansonihistone proteins was improved.

Moreover, phylogenomics has unraveled the distinctevolutionary histories of three expanded endopeptidasefamilies in S. mansoni [45]. This analysis included membersof the metallopeptidase M8, serine peptidase S1, and asparticpeptidase A1 families, which were originated from successiveevents of gene duplication followed by divergence in the par-asite lineage after its diversification from other metazoans.

Other protein families such as nuclear receptors andantioxidants have been characterized in S. mansoni byapplying evolutionary and functional approaches [46, 47].The identification of SmNR4A, a member of the nuclearreceptor subfamily 4, and its relatedness to the humanhomologs were corroborated by phylogenetic analysis [46].

This study also demonstrated that SmNR4A expression wasregulated throughout parasite development.

Thioredoxin and glutathione systems, involved in avariety of cellular processes including antioxidant defense,differ in parasitic and free-living Platyhelminthes [47]. Byapplying different phylogenetic methods and experimentaltesting, it has been shown that the canonical enzymes of suchsystems were lost in the parasitic lineages.

3.2. DNA Barcoding for Biodiversity Assessment. The avail-ability of sequencing data from mitochondrial genomes fromclosely related taxa to Schistosoma has provided a basis forstudies ranging from phylogenetic systematics to molecularecology and so forth [27, 29, 48, 49]. Importantly, it allowedthe assessment of molecular markers prior to broad scalesampling by comparing data from individual genes versuscompletely sequenced mitochondrial genomes.

Data from African and Asian schistosomes were com-pared to an S. mansoni intraspecific dataset showing apositive correlation between polymorphism and speciesdivergence [27]. A positive correlation rather than randomdistribution was identified for all mitochondrial genes withthe exception of cox1 and nad1. Furthermore, partialsequences of cox1 have been shown not to be the idealmarker for either species identification or population studiesof Schistosoma species. Instead, authors have suggested theuse of cox3 and nad5 sequences for both phylogenetic andpopulation studies of such species [27].

DNA barcoding was performed for samples of S. haema-tobium using cox1 and nad1 as molecular markers [49].The results supported the identification of group 1 and 2haplotypes based on phylogenetic analysis. Moreover, nochange in genetic diversity was detected across samplescollected over different time points. This approach allowedthe development of new assays based on group-specific PCRprimers and SNaPshot probes to assist genetic screening ofschistosome isolates.

Another study employing mitochondrial sequences wasperformed in Schistosoma cercariae harvested from Biom-phalaria choanomphala as part of parasitological and mala-cological surveys for S. mansoni across Lake Victoria [48].DNA barcoding of cercarial samples revealed the presenceof hybrid species between S. mansoni and S. rodhaini,which is typically found in small mammals. Moreover, thephylogenetic analysis identified a new sublineage within S.rodhaini.

DNA barcoding studies are underway for an increas-ing number of Schistosoma species (Table 1), whose datais made available through the Barcode of Life Database(BOLD) system (http://www.boldsystems.org/). This initia-tive includes contributors from the Biodiversity Institute ofOntario, Canada, and the University of New Mexico, USA,among others. Other Schistosomatidae genera are subjectto DNA barcoding analysis such as Allobilharzia, Austro-bilharzia, Bilharziella, Bivitellobilharzia, Dendritobilharzia,Gigantobilharzia, Griphobilharzia, Heterobilharzia, Macrobil-harzia, Orientobilharzia, Ornithobilharzia, Schistosomatium,and Trichobilharzia.


Advances in DNA barcoding of a broader range ofSchistosomatidae will create a framework for species iden-tification from environmental and clinical samples as wellas specimens deposited in biological collections. Moreover,DNA barcoding analysis will help reconstructing the evo-lutionary relationships across different Platyhelminthes andultimately improve our understanding of parasite biologyand evolution.

4. Schistosoma Transcriptomics

Transcriptomic analyses are valuable not only for studies ofdifferential regulation in gene expression, but also for theidentification of splicing variants. These approaches haveproved to be essential in the identification of gene-codingregions during the process of genome annotation.

In this section, we aim to review the achievementsin Schistosoma transcriptomic studies that range from thewhole organism and tissue analyses to those performed atthe cellular level. Transcriptomics in physiological conditionsand in response to a variety of treatments are investigatedwith respect to the interaction with the host as well as otherenvironments. This section summarizes the accomplish-ments of gene function studies and the diverse techniquesthat are being employed.

4.1. Schistosoma Transcriptomics. A fine tuning in gene ex-pression of parasites must occur to enable them to survivein such a complex and extremely diverse milieu, adaptingto diverse environments as evading the immune system and,at the same time, exploiting different hosts. Transcriptomicstudies came along allowing a temporal and quantitativeanalyses of gene expression, which are crucial to completelyexploit different datasets and assess parasite biology at themolecular level.

Aiming to elucidate the differential regulation on theSchistosoma transcriptomics, a large number of projectsemployed diverse strategies, such as the ones using expressedsequence tags (ESTs) [50], open reading frame expressedsequence tags (ORESTEs) [51], and serial analysis of geneexpression (SAGE) [52, 53].

The increasing number of projects resulted in the pro-duction of a vast amount of sequence data and theiravailability in public databases. It is noteworthy that onlyS. japonicum, S. mansoni and, not until very recently, S.haematobium had some of their transcriptome explored(Table 1).

4.2. Exploring the Transcriptome. The large number of tran-scriptome projects accelerated the studies in this area byproviding means to the use of complementary approaches,especially microarray analysis. Together, these approacheshave been used to answer a wide range of questions. In theearly years, microarray datasets conceived the profile of geneexpression between genders of Schistosoma species such asS. japonicum and S. mansoni [54–56]. This greatly extendedthe number of known sex-associated genes and also providednew insights into changes in gene expression promoted

by parasite pairing. In addition, gender biased alternativesplicing patterns were observed, which are perhaps involvedin the generation and maintenance of the sexual dimorphism[57].

Later, studies profiled the S. mansoni gene expressionin different life stages broadening our understanding offactors controlling schistosome development [8, 53, 58].While comparing different parasite life stages, informationacquired from microarray datasets highlighted putativeantischistosome candidate molecules to be used as vaccinetargets including Dyp-type peroxidases, fucosyltransferases,G-protein coupled receptors, leishmanolysins, tetraspanins,and the netrin/netrin receptor complex [59].

Some of these candidates (fucosyltransferases, leishman-olysins, and tetraspanins) are members of protein families,which are expanded in the S. mansoni predicted proteomein comparison with other metazoans as inferred by phyloge-nomic analysis (http://phylomedb.org/). Leishmanolysins,which are members of the metallopeptidase M8 family,were analysed in detail corroborating the potential of theseenzymes as future therapeutic targets [45].

Another study identified upregulated genes during thefirst five days of schistosomula development in vitro thatare involved in blood feeding, tegument and cytoskeletaldevelopment, cell adhesion, and stress responses [8]. Thehighly upregulated genes included a tegument tetraspaninSm-tsp-3, a protein kinase, a novel serine protease andserine protease inhibitor, and intestinal proteases belongingto distinct mechanistic classes. Other groups have analysedimmunity towards Schistosoma using protein microarraysas a vaccine discovery tool. They selected a subset ofproteins from genomic, transcriptomic, and proteomic datato perform experimental validation aiming at identifyingnovel antischistosome vaccine candidates [60, 61].

Recently, an elegant approach to investigate specifictissues such as gastrodermis and both genders reproduc-tive tissues was performed by applying laser microdissec-tion microscopy combined to microarray. These studiesbrought the perspective of expediting tissue-specific profiling[62, 63].

Additionally, expression profiles of S. japonicum wereexplored in studies comparing two Chinese isolates (Anhuiand Zhejiang), as well as the Anhui Chinese and Philippinesisolates [55, 64]. Later, an interspecies study compared theS. japonicum and S. mansoni transcriptomes. Surprisingly,despite their large phenotypic differences, there was areduced number of differentially expressed genes based onthe expression profile, which might reflect a limitation ofthe technique (preferential hybridization related to speciespolymorphisms) [8]. Bypassing these pitfalls, the next gener-ation sequencing platforms allow exploiting expression data(RNA-seq) of any organism without previous knowledge ofthe genome or transcriptome and, further, will reveal newfeatures of the parasite transcriptional landscape.

The most recent achievement in this area was therefinement of the genomics and transcriptomics data using acombination of Sanger capillary sequencing, next generationDNA sequencing, genetic markers, and RNA-seq data fromseveral S. mansoni life stages [19]. A previous study, explored


the transcriptome of S. mansoni adult males obtaining 11novel microexon genes (MEGs), as well as mapping 989 and1196 contigs to intergenic and intronic genomic regions,respectively. This data could represent new protein-codinggenes or noncoding RNAs (ncRNAs) [65].

Together, these approaches have significantly advancedour understanding of the dynamics parasite transcriptomes[9, 65]. However, there is a need to polish and translatesequence data and gene expression profiles into functionalinformation by computational predictions and experimentalcharacterization. The vast amount of available data openeda myriad of possibilities towards the elucidation of genefunctions. It is time for data integration.

4.3. Posttranscriptional Mechanisms. Transcriptomics is alsoa key approach to study posttranscriptional regulatorymechanisms such as alternative and transsplicing [57, 66–68]. It is known that schistosomes use such mechanisms tokeep their intricate life cycle.

Alternative splicing of transcripts of secreted proteins,like MEGs and polymorphic mucin isoforms, has beenlargely studied in schistosomes [51, 66]. This posttranscrip-tional processing has been proposed as a distinct geneticsystem to generate protein variation that would allowparasite adaptation to the immune response of differenthosts. Additionally, alternative splicing has been shown togenerate protein variation involved in transcriptional controland splicing [57]. In the case of SmCA150, a spliceosomeinteracting transcriptional cofactor, it could impact manyother transcripts leading to several effects in the parasite,especially in sexual dimorphism.

Spliced leader (SL) transsplicing has been shown to bean important posttranscriptional regulation mechanism forS. mansoni [69]. At least 11% of all transcripts has beendescribed as undergoing transsplicing in this organism [19].SL-RNAs are small ncRNAs, products of small intronlessgenes, transcribed by DNA polymerase II and tandemlyrepeated in the genome [67].

Differently from nematodes, S. mansoni carries a singleSL sequence, which is composed by 36 nucleotides with an3′-terminal AUG codon. Although the role of transsplicingis poorly understood, there is an evidence suggesting that SLtranssplicing would function providing an initiator methio-nine for translation initiation of some recipient mRNAs inPlatyhelminthes [68]. This mechanism is also involved inthe resolution of polycistronic operons in monocistronictranscripts in S. mansoni [19, 70].

4.4. Transcriptome Data and Gene Function. Despite thegreat potential revealed by the increasing number of genomeand transcriptome studies in schistosomes, there are stillseveral Schistosoma species that remain neglected. In contrastto the vast amount of data available in public databases,limited data has been decoded in functional studies and/orin the development of successful tools to fight schistosomes.This slow evolving scenery might be due to several reasons.Some of them include the convolution of schistosomeslife cycle, the complex interaction with their hosts, time

and labor-demand functional studies, the difficulties tonurture the complete life cycle in vitro, and the absence ofimmortalized cell lines, which has delayed the developmentof transgenesis tools and models [71].

Lately, the posttranscriptional suppression of genesthrough RNA interference (RNAi) techniques has been apromise to fetch progress on new interventions for schis-tosomiasis and other helminthiases. In 2003, two pioneerinvestigations marked the use of this technique in S.mansoni research. Simultaneously gene knockdown of glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH) and aglucose transporter (SGTP1) in the larval stage (sporocysts)[72] and cathepsin B in adult worms [73] of S. mansoni werereported.

Currently, this powerful technique is widely used inschistosomes and has been employed in two studies of func-tional screening. The first study encompassed the exposureof sporocysts by soaking parasites to dsRNA targeting 33genes, from calcium binding proteins, transcription factors,to receptors, and antioxidant enzymes [74]. In this work,despite the efficacy of this tool to deliver suppression of 11different genes, it showed that, in S. mansoni, there is a gene-to-gene variability and exposure to dsRNA could triggergene specific upregulation. Later, in a different study usingelectroporation to expose schistosomula to dsRNA of 11target genes (expressed in distinct tissues), the efficiency ofRNAi in a target specific fashion was observed [75]. Otherissues with the use of RNAi have been widely discussed, notonly in schistosomes, but also in other helminth parasites,showing that care should be taken when conducting andevaluating such experiments [76].

Despite the difficulties in exploring gene function byRNAi in schistosomes, many interesting advances have beenachieved. Many proteases (cathepsins and aminopeptidases)[77–79], kinases [80], multidrug resistance transporters [81],and type V collagen [82], among other proteins have beenstudied in diverse life stages of S. japonicum and S. mansoni.

Recently, this tool has been described targetingtetraspanin 2 in diverse developmental stages of S.haematobium indicating the RNAi feasibility in this species[83]. Tetraspanins belong to a family of integral membraneproteins present in the outer surface membranes of theparasite tegument. Studies have shown that tetraspanins playimportant roles in the tegument development, maturation,or stability [84].

Importantly, the knockdown of peroxiredoxin [85]and thioredoxin glutathione reductase [86], antioxidantenzymes, were amongst the first reports of lethality aftergene silencing in the parasite, demonstrating the potentialof RNAi on drug target discovery. Moreover, enzymes suchas glutathione-S-transferases (GST26 and GST28), peroxire-doxins (Prx1 and Prx2), and superoxide dismutase have beenshown to play important roles in the protection of S. mansonisporocysts in the host-parasite interplay. Knockdown ofthose targets increased parasite susceptibility to oxidativestress and to intermediate host immune cells as describedelsewhere [87].

Recently, significant progress has been made in the areaof schistosome transgenesis. Researchers are developing new


tools for the introduction and expression of homologousor heterologous gene constructs via lentiviruses, replication-defective retroviruses, transposons, retrotransposons, andDNA plasmid vectors [9]. Sophisticated systems applied toassess gene function in the parasite are being achieved [88–91]. To date, eggs and miracidia are indicated as the preferredlife stages for successful transformation approaches in orderto enter the germline [92].

In summary, results from such independent studieshave shown that schistosome transgenesis works in all thedevelopmental stages tested and that some success has beenachieved in integrating genes into the germline [88–92].Thus, genetic knockdown experiments in schistosomes arepossible.

5. Genomic and Transcriptomic Databases

Altogether, the increasing information on schistosomegenomes and transcriptomes is now raising the possibilitiesto better explore molecular techniques in the field ofparasitology, genomics, and computational biology allowingthe development of approaches to unravel mechanisms ofparasite infection, host-parasite interaction, and so forth[21, 23, 24].

In order to accelerate both parasite research and devel-opment of effective tools for schistosomiasis diagnostics andtreatment, user-friendly databases have been created andmade publicly available to the scientific community [9, 93–95]. Here, we highlight some of these major resources.

SchistoDB integrates genome and proteome sequencedata along with functional annotation of genes and geneproducts of S. haematobium, S. japonicum, and S. mansoni[93]. This resource also covers results from large-scaleanalysis including ESTs, metabolic pathways, and candidatedrug targets.

HelmCoP (Helminth Control and Prevention) integratesfunctional, structural, and comparative genomics data ofhelminths from different taxonomic groups and distincthosts (plant, animal, and human) [94]. HelmCoP offers acomprehensive suite of structural and functional annotationsto support comparative analyses and host-parasite interac-tion studies.

GeneDB is an annotation database that brings togetherdata from a broad range of pathogens and closely relatedorganisms [95]. GeneDB offers database-driven annotationtools and pipelines and includes manually curated informa-tion maintained by computational and biological scientists.

Collectively, these computational resources support bio-logical and biomedical research and open new frontiersin evolutionary and functional genomics, transcriptomics,and proteomics of Schistosoma species and other parasites.Besides biodiversity assessment at the molecular level, theseresources provide a framework for the identification andprioritization of vaccine and drug targets against humandiseases. As the scientific community progresses in dataintegration and interpretation, we will meet the goals ofcurrent and future initiatives to improve human health [9,12, 23, 24].

6. Conclusions and Future Directions

The -omics studies came along with the aim to fill the gapsof long-established approaches, enabling the use of a broadrange of experimental and computational methods andcontributing to dissect the biological basis of host-parasiteinteraction, diagnostics improvement, and the discovery ofnew drug and vaccine targets.

Nuclear and mitochondrial genomes of some Schisto-soma species were completely sequenced opening new fron-tiers for comparative analyses, which will improve our under-standing of parasite biology and evolution. Other genomes tobe analysed include Schistosoma species from each of the sixclades currently recognized, and other representatives of theSchistosomatidae. Improving accuracy/quality of genomeassembly and annotation will be crucial for such analyses.

Despite the efforts in the characterization of genomesand transcriptomes of distinct Schistosoma species, manyquestions regarding the biology and evolution of this impor-tant taxon remain unanswered. These difficulties might bedue to the size of schistosome genomes (∼10-times largercompared to protozoan parasites), the complexity of its lifecycle, the presence of rare features in posttranscriptionalregulation (e.g., transsplicing), and little integration of thecurrent information available, among other reasons.

There is an urgent need for data integration to help inter-preting the massive amount of data generated over the yearsin this and other areas or research. The need for biocurationis essential to guide this process. We envision that, in thefuture, more transcriptomic and proteomic data will be usedto improve the understanding of the origin and evolution ofSchistosomatidae. The analysis of structural data generatedby experimental or computational approaches will also shedlight in the biodiversity of schistosomes at the molecularlevel.

In conclusion, the availability of schistosome genomeand transcriptome data has provided an unprecedentedresource for many research areas. Limitations do exist andshould be addressed to allow advances in understandingschistosome biology and evolution and ultimately supportvaccine and drug development against schistosomiasis.

List of Abbreviations

atp6: ATP synthase F0 subunit6atp8: ATP synthase F0 subunit 8bp: Base pairscob: Apocytochrome bcox1: Cytochrome c oxidase subunit 1cox1–3: Cytochrome c oxidase subunit 1 to 3DALY: Disability-adjusted life yearsdbEST: Database of expressed sequence tagsdsRNA: Double strand DNAESTs: Expressed sequence tagsMEGs: Microexon genesnad1–6: NADH dehydrogenase subunit 1 to 6nad4L: NADH dehydrogenase subunit 4LncRNAs: Noncoding RNAsORESTEs: Open reading frame expressed sequence tags


RNAi: RNA interferenceRNA-seq: RNA sequencingSAGE: Serial analysis of gene expressionSL: Spliced leaderTDR: Special Programme for Research and Training

in Tropical DiseasesUNDP: United Nations Development ProgrammeUNICEF: United Nations Children’s FundWHO: World Health Organization

Acknowledgments

The research leading to these results has received fund-ing from the European Community’s Seventh Frame-work Programme (FP7/2007–2013 FP7/2007–2011) underGrant agreement number 241865. This work was alsopartially supported by the National Institutes of Health-NIH/Fogarty International Center (TW007012 to GO), theNational Council for Research and Development, CNPq(CNPq Research Fellowship 306879/2009-3 to GO, INCT-DT 573839/2008-5 to GO, CNPq-Universal 476036/2010-0to LAN, and 480576/2010-6 to MMM), and the ResearchFoundation of the State of Minas Gerais (FAPEMIG) (CBB-1181/08 and PPM-00439-10 to GO and CBB-APQ-01715/11to MMM). The authors are very grateful to Larissa LopesSilva and two anonymous reviewers for their constructivefeedback and valuable suggestions, which greatly improvedthis paper.

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Clinical Study

Changes in T-Cell and Monocyte PhenotypesIn Vitro by Schistosoma mansoni Antigens in CutaneousLeishmaniasis Patients

Aline Michelle Barbosa Bafica,1 Luciana Santos Cardoso,1, 2

Sergio Costa Oliveira,3, 4 Alex Loukas,5 Alfredo Goes,3 Ricardo Riccio Oliveira,1

Edgar M. Carvalho,1, 4, 6 and Maria Ilma Araujo1, 4, 6

1 Servico de Imunologia, Complexo Hospitalar Universitario Professor Edgard Santos, Universidade Federal da Bahia,5 Rua Joao das Botas s/n, Canela, 40110-160 Salvador, BA, Brazil

2 Departamento de Ciencias da Vida, Universidade do Estado da Bahia, 2555 Rua Silveira Martins, Cabula,41.150-000 Salvador, BA, Brazil

3 Departamento de Bioquımica e Imunologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais,6627 Avenida Antonio Carlos, Pampulha, 31270-901 Belo Horizonte, MG, Brazil

4 Instituto Nacional de Ciencia e Tecnologia em Doencas Tropicais (INCT-DT-CNPQ/MCT), Rua Joao das Botas s/n,Canela, 40110-160 Salvador, BA, Brazil

5 Queensland Tropical Health Alliance and School of Public Health and Tropical Medicine, James Cook University,Cairns, QLD 4878, Australia

6 Escola Bahiana de Medicina e Saude Publica, No. 275 Avenida Dom Joao VI, Brotas, 40290-000 Salvador, BA, Brazil

Correspondence should be addressed to Aline Michelle Barbosa Bafica, [email protected]

Received 13 July 2012; Revised 24 September 2012; Accepted 8 October 2012

Academic Editor: Andrea Teixeira-Carvalho

Copyright © 2012 Aline Michelle Barbosa Bafica et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

High levels of proinflammatory cytokines such as IFN-γ and TNF are associated with tissue lesions in cutaneous leishmaniasis(CL). We previously demonstrated that Schistosoma mansoni antigens downmodulate the in vitro cytokine response in CL. Inthe current study we evaluated whether S. mansoni antigens alter monocyte and T-lymphocyte phenotypes in leishmaniasis.Peripheral blood mononuclear cells of CL patients were cultured with L. braziliensis antigen in the presence or absence of theS. mansoni antigens rSm29, rSmTSP-2- and PIII. Cells were stained with fluorochrome conjugated antibodies and analyzed byflow cytometry. The addition of rSm29 to the cultures decreased the expression of HLA-DR in nonclassical (CD14+CD16++)monocytes, while the addition of PIII diminished the expression of this molecule in classical (CD14++CD16−) and intermediate(CD14++CD16+) monocytes. The addition of PIII and rSmTSP-2 resulted in downmodulation of CD80 expression in nonclassicaland CD86 expression in intermediate monocytes, respectively. These two antigens increased the expression of CTLA-4 in CD4+ Tcells and they also expanded the frequency of CD4+CD25highFoxp3+ T cells. Taken together, we show that S. mansoni antigens,mainly rSmTSP-2 and PIII, are able to decrease the activation status of monocytes and also to upregulate the expression ofmodulatory molecules in T lymphocytes.

1. Introduction

American tegumentary leishmaniasis is a disease caused byparasites of the genus Leishmania. This disease representsa significative public health problem worldwide with anincidence of 1.5 million new cases in recent years [1].

Tegumentary leishmaniasis presents with a wide spectrumof clinical manifestations ranging from localized skin towidespread mucocutaneous lesions depending on the par-asite species [2] and the host immune response [3, 4]. Tcell-mediated immunity is crucial to host protection againstLeishmania sp. infection; however skin and mucosal lesions


occurs due to a deregulated T helper 1 (Th1) cell responsewith high production of proinflammatory cytokines such asIFN-γ and TNF [3, 4].

Experimental studies have shown that Schistosoma man-soni infection or parasite products, by inducing regulatorycells and cytokines, are able to prevent some Th1-mediatedautoimmune diseases in mice such as type I diabetes,experimental autoimmune encephalomyelitis, and psoriasis[5–7]. Recently, we demonstrated that the recombinant S.mansoni antigens Sm29 SmTSP-2, and also PIII down-modulated the production of IFN-γ and TNF in a groupof cutaneous leishmaniasis (CL) patients [8]. In the presentstudy, these antigens were tested regarding their ability toalter the monocyte and lymphocyte profiles. Studies haveshown that Sm29 and SmTSP-2 antigens are secreted by themembrane and/or tegument of the S. mansoni adult worm.Proteins secreted or localized on the surface of Schistosomaspp., which are in intimate contact with host tissues, mightbe more effective in triggering immunoregulatory processes[9]. The Sm29 is a membrane-bound glycoprotein located onthe tegument of the adult worm and lung stage schistosomula[10]. SmTSP-2 is a recombinant protein (tetraspanin) fromS. mansoni tegument. In a mice model, immunization withSmTSP-2 resulted in a 57% reduction in adult worm burdensand a 64% reduction in liver egg burdens compared withcontrol animals [11]. PIII is a multivalent antigen obtainedfrom S. mansoni adult worms that modulates granuloma sizein mice infected with the parasite [12, 13]. These antigenshave been evaluated by our group regarding their potential toinduce IL-10 production and suppress Th2 response in vitroin cells of asthmatic individuals [14].

Together with the Th1 immune response, monocytesand macrophage are key cells in controlling Leishmania sp.infection. Monocytes have been classifying into differentsubpopulations in mice models and also in humans. Anew nomenclature was recently published defining humanmonocytes into three subtypes. The major population ofhuman monocytes (90%) presents high expression of CD14and lack of expression of CD16 (CD14++CD16−). They arereferred to as classical monocytes. Intermediate monocytesare those which express CD14 and low CD16 expression(CD14++CD16+), while the nonclassical monocytes expressCD16 and relatively low CD14 (CD14+CD16++) [15]. Ina study conducted by Wong et al. [16] the characteris-tics of classical, intermediate and, nonclassical monocyteswere evaluated through the gene expression profiling. Theyobserved that classical monocytes express genes involvedin angiogenesis, wound healing, and coagulation, beinginvolved in tissue repair functions in addition to highexpression of proinflammatory genes [16]; intermediatemonocytes highly express MHC class II indicating theyhave antigen presenting cell function and T cell stimulatoryproperties. Nonclassical monocytes express genes involved incytoskeleton rearrangement, which may be responsible for itshigh motility observed in vivo [17].

A high frequency of CD16+ monocyte subsets havebeen demonstrated in Mycobacterium tuberculosis infectionand in viral infections such as hepatitis B (HBV) [18],hepatitis C (HCV) [19], HIV [20], and Dengue [21]. In

CL patients, the frequency of CD14+CD16+ monocytes wassignificantly higher compared to healthy controls and theywere positively correlated with the lesion size [22]. Thissubtype of monocytes is considered as antigen presentingcell [16], which produces cytokines and might be the mostimportant subtype in activating T cells response.

The activation of T cells requires the antigen recognitionin the MHC context and also signaling given by co-stimulatory molecules which interact with correspondingligands on antigen presenting cells (APC). One of the mostimportant co-stimulatory molecules on T cells is CD28,which is constitutively expressed and binds to CD80 andCD86 on the APC. CD86 is constitutively expressed at lowlevels and it is rapidly upregulated after primary antigenrecognition, whereas CD80 exhibits delayed expressionkinetics [23]. Other ligand for CD80 and CD86 is CTLA-4.The expression of this molecule is rapidly upregulated afterT cell activation and provides a negative signal limiting theimmune response [24–26]. In vitro study has shown that theaddition of CTLA-4-Ig to block CD28-B7 interaction in CLpatients PBMC cultures stimulated with Leishmania antigenled to a downmodulation of IFN-γ and TNF secretion [27].Other similar study showed that blocking the co-stimulatorymolecules CD80 and CD86 in human macrophages fromLeishmania-naive donors infected with L. major resulted ina significant reduction in IFN-γ, IL-5, and IL-12 production[28].

In this study we evaluated whether S. mansoni antigensalter the expression of CD80 and CD86 on monocytes of CLpatients and also the expression of CTLA-4 in T lymphocytes.Additionally, we accessed the frequency of regulatory T cellsinduced by S. mansoni antigens when they were added to thecell cultures stimulated with Leishmania antigens.

The CD4+CD25+ regulatory T cells have been extensivelystudied due to their critical function in maintaining self-tolerance. These cells can express both low and high CD25levels in mice models; however, only the CD4+CD25high

population exhibits a strong regulatory function in humans.These CD4+CD25high T cells also express FOXP3, a moleculeassociated with regulatory functions [29]. This T cell subsetin humans comprises ∼1.5–3% of circulating CD4+ T cells.They inhibit proliferation and cytokine secretion inducedby TCR cross-linking of CD4+CD25− responder T cells ina contact-dependent manner [30] and completely abrogateIL-2-dependent proliferation of NK cells [31]. Moreover,regulatory T cells are able to downregulate the intensityand duration of both Th1 and Th2 immune responses ininfectious diseases limiting damage to self-tissue [32, 33].

Our hypothesis in the present study is that the pathologyof cutaneous leishmaniasis results from monocyte and Tcell hypersensitivity due to impaired regulatory mechanisms.In this context, the use of S. mansoni antigens whichinduce regulatory cells and molecules would prevent theinflammatory process. To test this hypothesis we evaluatedwhether the addition of S. mansoni antigens to cell culturesof leishmaniasis patients would modify the phenotype andactivation status of monocytes and lymphocytes. Specifically,we evaluated the expression of HLA-DR, CD80, and CD86on classical, intermediate, and nonclassical monocytes and


CD28, CTLA-4, CD25, and Foxp3 in T lymphocytes fromCL patients in response to the soluble Leishmania braziliensisantigen (SLA) in the presence or absence of the S. mansoniantigens rSm29, rSmTSP-2, and PIII.


2.1. Patients and the Endemic Area. The study includedthe first 30 cutaneous leishmaniasis patients living in theendemic area of tegumentary leishmaniasis, Corte de Pedra,Bahia, Brazil, who attended the local Health Post from March2010 to March 2012 and agreed in participate. Corte dePedra is located in the southeast region of the State of Bahia,Brazil which is well known for its high rate of L. braziliensistransmission.

The diagnostic of cutaneous leishmaniasis was madeconsidering a clinical picture characteristic of CL, parasiteisolation or a positive delayed-type hypersensitivity (DTH)response to Leishmania antigen, and a histological feature ofCL.

The inclusion criteria to this study were patients with5 to 60 years of age diagnosis of CL with the presence ofactive skin lesions. The exclusion criteria were pregnancy,chronic diseases such as diabetes and asthma, and also HIVand HTLV-1 infection.

Immunological analyses were performed prior to thespecific therapy to leishmaniasis for all patients. There werenot enough cells to perform the whole experiment every timesince they require a larger number of cells more than whatcould be obtained from some patients.

The frequency of helminth infection in the leishmaniasisendemic area of Corte de Pedra is 88.3% and S. mansoniinfection presents in 16.7% of cutaneous leishmaniasispatients.

The Ethical Committee of the Maternidade Climerio deOliveira, Federal University of Bahia approved the presentstudy, and an informed consent was obtained from allparticipants or their legal guardians.

2.2. Antigen Preparation. The soluble Leishmania antigenused in this study was obtained from a Leishmania lysate(crude antigen). It was prepared from a L. braziliensis strain(MHOM/BR/2001) as previously described [34].

The S. mansoni antigens used in this study includedSm29, a Schistosoma mansoni recombinant protein [10];SmTSP-2, a recombinant protein (tetraspanin) from S.mansoni tegument [11]; a fraction of S. mansoni soluble adultworm antigen (SWAP) obtained by anionic chromatography(FPLC), called PIII. The Sm29 was cloned in E. coli andtested for lipopolysaccharide (LPS) contamination using acommercially available LAL Chromogenic Kit (CAMBREX).The levels of LPS in Sm29 were below 0.25 ng/mL. Howeverin order to neutralize potential effects of LPS presented inlow levels in the S. mansoni recombinant antigen, PolymyxinB was added to cell cultures every 12 hours accordingto the established protocol [35]. The SmTSP-2 used inthis study was produced in Pichia pastoris fermentationcultures and it has been kindly provided by Dr. Alex Loukas

[11, 36]. The proteins rSm29 and PIII were provided by theInstitute of Biological Science, Department of Biochemistryand Immunology, UFMG, Brazil.

2.3. Cell Culture and Intracellular and Surface Staining.Intracellular and surface molecules were evaluated by flowcytometry (FACSort, BD Biosciences, San Jose, CA). Inorder to perform the intracellular staining, peripheral bloodmononuclear cells (PBMC, 3 × 105) obtained through theFicoll-Hypaque gradient were cultured in vitro with SLA(5 μg/mL) in the presence or absence of the recombinantS. mansoni antigens Sm29, SmTSP-2, and PIII (5 μg/mL)for 20 h, 37◦C, and 5% of CO2. During the last 4 h ofculture, Brefeldin A (10 μg/mL; Sigma, St. Louis, MO),which impairs protein secretion by the Golgi complex, wasadded to the cultures. Afterwards, the cells were washedin PBS and fixed in 4% formaldehyde for 20 min at roomtemperature. Specific staining was performed with cychrome(CY)-labeled antibody conjugated with anti-CTLA-4 mAbsin saponin buffer (PBS, supplemented with 0.5% BSA and0.5% saponin) and phycoerythrin (PE)-labeled antibodyconjugated with anti-Foxp3 in Foxp3 staining buffer set(eBioscience). For double or triple staining, mAbs to humanCD4, CD8, and/or CD25 conjugated with FITC or CY wereused.

The evaluation of co-stimulatory molecule expressionwas performed in PBMCs stimulated for 60 h with thesame antigens aforementioned. Cells were incubated withfluorescein isothiocyanate (FITC)-, PE-, or CY-labeled anti-body solutions for 20 min at 4◦C in a volume of 20 μL inPBS. After staining, preparations were washed with 0.1%sodium azide PBS, fixed with 200 μL of 4% formaldehydein PBS and kept at 4◦C. The antibodies used for thestaining were immunoglobulin isotype controls-FITC, PEand CY, anti-CD4-FITC, anti-CD8-FITC, anti-CD25-FITC,anti-CD14-FITC, CD16-CY, anti-CD4-CY and anti-CD8-CY, anti-CD80-PE, anti-CD86-PE, anti-HLA-DR-PE, anti-CD28-PE (all from BD Biosciences Pharmingen). A totalof 50,000 and 100,000 events were acquired for surface andintracellular experiments, respectively.

2.4. Analysis of FACS Data. The frequency of positive cellswas analyzed by flow cytometry using the program flowjoin two regions. The lymphocyte region was determinedusing granularity (SSC) × size (FSC) plot. Monocytes wereselected based on their granularity and expression of CD14and CD16. Limits for the quadrant markers were alwaysset based on negative populations and isotype controls. Arepresentative density graph of one experiment showinglymphocyte and monocyte regions is shown in Figures 1(a)and 1(b), respectively.

2.5. Statistical Analyses. Statistical analyses were performedusing the software GraphPad Prism (GraphPad Software, SanDiego, CA). The frequency of positive cells was expressedas percentages and the intensity of expression as meanintensity fluorescence (MFI). The differences between means


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Figure 1: Strategy for T cell and monocytes evaluation by flow cytometry. The cell populations were defined by nonspecific fluorescencefrom the forward (FSC) and side scatter (SSC) as parameters of cell size and granularity, respectively. Lymphocytes region was determinedusing SSC × FSC plot (G1) and monocytes region (G2) were selected based on their granularity and expression of CD14 (a). (b) representsthe strategy for monocytes subsets classification through the expression of CD14 and CD16. Representative graph of one experiment.

Table 1: Demographic characteristics of the cutaneous leishmania-sis patients included in the study.

Characteristics (n = 30) Values

Age, mean± SD, years 29.1± 11.8

Sex, female/male 13/17

N◦ of lesion

1, no. (%) 24 (80.0)

≥2, no. (%) 06 (20.0)

Size of lesion, median mm2 (IQR) 130 (66.5–342)

IQR: interquartile range; SD: standard deviation.

were assessed using Wilcoxon matched pairs test. Statisticalsignificance was established at the 95% confidence interval.

3. Results

A total of 30 patients with cutaneous leishmaniasis wereenrolled in this study, 17 were male and 13 were female,with a mean age of 29.1 ± 11.8 years (range 6–48 years).The majority of patients presented with a single lesion (80%)and the median lesions size was 130 mm2 (IQR, 66.5–342;Table 1).

3.1. The Effect of the Addition of S. mansoni Antigens on Mono-cyte Phenotype and Expression of Co-Stimulatory Molecules.We evaluated the frequency of different monocyte subsets(classical, intermediate, and nonclassical) and also theexpression of co-stimulatory molecules in these monocytesafter in vitro stimulation with SLA in the presence orabsence of S. mansoni antigens. The addition of the antigens

rSm29 and PIII to the cultures expanded the frequency ofnonclassical (CD14+CD16++) (mean ± SEM = 7.4± 1.2%and 8.1± 1.9%, resp.) compared to cultures stimulated withSLA alone (5.7 ± 0.9%; P < 0.05. Figure 2(A)). We alsoobserved that the frequency of classical monocytes washigher in cultures without S. mansoni antigens (Figure 2(A)).Moreover, in the presence of PIII there was a reduction inthe expression of HLA-DR in classical (CD14++CD16−) andintermediate (CD14++CD16+) monocytes (496±72 MFI and544± 78.6 MFI, resp.) compared to cultures stimulated withSLA alone (611 ± 91 MFI and 771 ± 128 MFI, respectively;P < 0.05. Figure 2(B)). There was also a reduction inHLA-DR expression on CD14+CD16++ monocytes in thepresence of rSm29 (547 ± 140.5 MFI) in comparison toSLA alone (718 ± 188.4 MFI; P < 0.05. Figure 2(B)).Additionally, the expression of HLA-DR was higher inclassical and intermediate monocytes in the presence of SLAor SLA plus S. mansoni antigens compared to nonstimulatedcultures.

The expression of CD80 was also reduced in nonclassicalmonocytes compared to cultures stimulated with SLA. Theaddition of PIII to the cultures also resulted in reducedexpression of the co-stimulatory molecule CD80 onnonclassical monocytes (61.2 ± 19.5 MFI) compared to cul-tures without S. mansoni antigens (82.3±23.3 MFI; P < 0.05.Figure 2(C)). Also a decrease in the expression of CD86 onintermediate monocytes from 562 ± 149.7 MFI to 447.8 ±112.5 MFI was observed when rSmTSP-2 was added to thecultures (P < 0.05; Figure 2(D)). There was no significantdifference in the levels of CD86 expression on monocytes inculture stimulated with SLA after the addition of rSm29 andPIII (Figure 2(D)).


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3.2. The Effect of the Addition of S. mansoni Antigens on TCell Phenotype and Expression of Co-Stimulatory Molecules.PBMC of CL patients were incubated with SLA in the pres-ence or absence of S. mansoni antigens and the phenotype ofT cells were evaluated. The addition of rSm29 antigen to thecultures increased the frequency of CD4+ T cells (mean ±SEM = 40.8 ± 2.8%) in comparison to cultures with SLAalone (34.8 ± 2.8%, P < 0.05; Figure 3(A)). There was nosignificant difference in the frequency of CD4+ T cells afterthe addition of rSmTSP-2 or PIII antigens to the cultures(37.8 ± 2.7% and 35.8 ± 2.6%, resp.) compared to SLAalone (34.8 ± 2.8%; Figure 3(A)). Likewise, there was nosignificant variation in the frequency of CD8+ T cells by thepresence of rSm29, rSmTSP-2, and PIII antigens (5.9±1.2%,4.6±0.8%, and 6.2±1.1%, resp.) to the cultures in relation tothe cultures stimulated with SLA without S. mansoni antigens(5.7± 1.2%; Figure 3(A)). The frequency of CD4+ T cells wasdiminished by the presence of SLA and SLA plus rSmTSP-2or PIII, while the frequency of CD8+ T cells was higher in

the presence of SLA and SLA plus S. mansoni antigenscompared to nonstimulated cultures (Figure 3(A)).

We also evaluated the expression of CD28+ on lympho-cytes in cultures stimulated with SLA with or without theaddition of S. mansoni antigens. The expression of CD28+ onCD8+ T cells was higher in cultures stimulated with rSm29(91 ± 14 MFI, resp.) compared with SLA alone (84 ± 14MFI; P < 0.05. Figure 3(B)). The addition of rSmTSP-2 andPIII antigens to the cultures did not alter significantly theexpression of CD28 on CD4+ and CD8+ T cells (Figure 3(B)).

Regarding the expression of CTLA-4, rSmTSP-2 and PIIIantigens were able to increase the expression of this moleculein CD4+ T cells (58±5 MFI and 53±4.6 MFI, resp.) in com-parison with SLA alone (49.6±4 MFI, respectively; P < 0.05.Figure 3(C)). The expression of CTLA-4 was also higherin CD4+ T cells and CD8+ T cells in cultures stimulatedwith SLA plus rSmTSP-2 or PIII compared to nonstimulatedcultures (Figure 3(C)). Moreover, the addition of rSmTSP-2and PIII antigens to the cultures expanded the frequency of


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(A) (B)

(C) (D)

Figure 3: Phenotype of T cells and co-stimulatory molecules expression on T lymphocytes of CL patients (n = 17) stimulated in vitrowith SLA in the presence or absence of S. mansoni antigens rSm29, rSmTSP-2 or with a fraction of S. mansoni soluble adult worm antigen(SWAP) PIII. Frequency of CD4+ and CD8+ T cells (A), expression of CD28 and CTLA-4 in CD4+ and CD8+ T cells (B and C, resp.) and thefrequency of CD4+CD25highFoxp3+ T cells (n = 13) (D). Cells were stained for surface expression of CD4, CD8, CD25 and CD28, while theexpression of CTLA-4 and Foxp3 were evaluated intracellularly using flow cytometry. aP < 0.05 cultures without stimulation (WS) versusSLA + S. mansoni antigens; bP < 0.05 SLA versus SLA + S. mansoni antigens (Wilcoxon matched pairs test).

CD4+CD25highFoxp3+ T cells (10 ± 2.4% and 10.3 ± 1.9%,resp.) compared to cultures with SLA in the absence ofS. mansoni antigens (8.1 ± 1.8%; P < 0.05. Figure 3(D)).The frequency of CD4+CD25highFoxp3+ T cells was alsohigher in cultures stimulated with SLA plus PIII comparedto nonstimulated cultures (Figure 3(D)).

4. Discussion

The Th1 immune response associated with macrophage acti-vation and killing of Leishmania sp. is paradoxically related tothe development of cutaneous and mucosal leishmaniasis. Inan attempt to control parasite growth, activated macrophages

release high levels of proinflammatory cytokines and nitro-gen and oxygen reactive intermediates, leading to a tissuelesion.

A balance between the proinflammatory response char-acterized by the production of IFN-γ and TNF, and theregulatory response with the production of IL-10 hasbeen observed in individuals exposed to the Leishmaniabraziliensis in endemic areas of leishmaniasis in Brazil.Those individuals do not develop the disease and theyare designated as “subclinical” subjects [37]. The balancedimmune response described in these individuals may be thekey to achieving a harmless host-parasite relationship.

There are other evidences that IL-10 is capable ofdownmodulating the inflammatory response associated with


human tegumentary leishmaniasis [4, 38–40]. However,mononuclear cells of mucosal leishmaniasis (ML) patientshave a decreased ability to produce this cytokine and torespond to IL-10 after in vitro restimulation with L. brazilien-sis antigen [3].

Recently it has been shown that during chronic Schis-tosoma mansoni infection, cells from the innate immuneresponse, such as monocytes and regulatory T cells producehigh levels of IL-10 [24, 41–43]. Our group has performedstudies in an attempt to identify S. mansoni antigens withregulatory properties that enable them to downregulatethe inflammatory process associated with certain immune-mediated diseases. For instance, we have shown that the S.mansoni antigens rSm29, rSmTSP-2, and PIII induce IL-10and IL-5 production by PBMC of cutaneous leishmaniasispatients and that they are able to control the in vitroinflammatory response in a group of patients, independentof the clinical features, such as number and size of lesions[8]. In this current study we extended the assessmentof the potential of the antigens rSm29, rSmTSP-2, andPIII in modifying the immune response during cutaneousleishmaniasis. Specifically, we evaluated the impact of theaddition of these antigens to the cell culture stimulated withLeishmania antigens on monocyte and lymphocyte profileand activation status.

CD4+ T lymphocytes and monocytes are key cells in theprotection against leishmaniasis; however they have beenassociated to the inflammatory process and tissue lesion inthe cutaneous form of disease [20, 44–46]. In leishmaniasisand also in some others diseases the role of different subsetsof monocytes in the pathology has been described [47].For more than two decades, monocytes have been classifiedinto classical (CD14+CD16−) and inflammatory, those whichexpress the molecule CD16 and produce high levels of TNF-α [48]. The frequency of CD14+CD16+ in CL patients wasfound to be significantly higher compared to healthy controlsand they were positively correlated with the lesion size [22].

Recently, a new nomenclature has been used to clas-sify human blood monocytes into three subsets: classi-cal (CD14++CD16−), intermediate (CD14++CD16+), andnonclassical (CD14+CD16++) [15]. The classical monocytesaccount for about 85% of the total monocytes and arecharacterized by the expression of IL-13Rα1, IL-10, andRANTES and by the expression of genes associated with anti-apoptosis and wound healing properties. The intermediatemonocytes represent 5% of monocytes, express high levelsof HLA-DR and are associated with antigen processing andpresentation to T cell and T cell activation. Nonclassicalsubset are characterized by a low expression of CD14 andhigh expression of CD16 (CD14+CD16++). They represent10% of human monocytes and studies have shown that theyexpress genes associated with cytoskeletal rearrangementwhich account for their highly mobility and for the patrollingbehavior in vivo [16, 47].

Since the roles of the different monocyte subsets incutaneous leishmaniasis remain unclear, we decided toevaluate not only their frequency in leishmaniasis patients,but also whether the addition of S. mansoni antigens wouldalter the profile of these cells in an in vitro study.

The addition of the antigens rSm29 and PIII to thePBMC cultures stimulated with SLA increased the frequencyof nonclassical monocytes. This was not expected as a cellwith high mobility and migratory capacity [16, 47], theymay contribute to Leishmania metastasis and consequentdevelopment of more severe forms of the disease, such as themucosal and disseminated forms.

We found, however, that the S. mansoni antigens usedin this study, particularly PIII, diminished the expressionof HLA-DR as well as the expression of the co-stimulatorymolecules B7.1 and B7.2 on different monocyte subsets.A downmodulation of monocyte activation is desirable inleishmaniasis and may result in a reduced inflammatoryprocess.

In this study, we also evaluated the expression of co-stimulatory molecules in T cells. We observed a significantincrease in the frequency of CD4+ T cells by the presence ofthe rSm29 antigen in the cultures. The addition of rSmTSP-2and PIII antigens to the cultures stimulated with SLAincreased the expression of CTLA-4 in CD4+ T cells and alsoexpanded the frequency of CD4+CD25highFoxp3+ T cells. Wepreviously showed that S. mansoni infection expands CD4+

T cell population of the regulatory profile in S. mansoni-infected asthmatic individuals [24]. In such allergic disease,S. mansoni antigens downmodulate the Th2 exacerbatedinflammatory response in vitro by inducing the productionof IL-10 and the expression of the regulatory molecules,CTLA-4 in T lymphocytes [9, 24, 43]. In a murine model ofovalbumin-induced asthma, inhibition of lung inflammatoryprocess, by S. mansoni eggs or by the S. mansoni antigens,Sm22.6, PIII, and rSm29 were associated with an increase inthe number of CD4+CD25+Foxp3+ T cells and high levels ofIL-10 production [49, 50].

T cell-mediated immunity is fundamental to host pro-tection against Leishmania sp. and the activation of T cellsdepends upon signals from the interaction between the co-stimulatory molecules CD28 to its ligands B7.1 and B7.2on antigen presenting cells (APCs) [23]. We evaluated theexpression of CD28 on lymphocytes in cultures stimulatedwith SLA with or without the addition of S. mansoniantigens and found that the expression of this moleculeon CD8+ T cells was higher in cultures stimulated withrSm29 compared with SLA alone. These results suggest thatthe CD8+ T cells became more activated in the presenceof rSm29. There was, however, an increased expression ofCTLA-4 in CD4+ T cells, a molecule which is expressedto inhibit the T cell activation when rSmTSP-2 and PIIIwere added to the cultures. In vitro study has shown thatthe addition of CTLA-4-Ig to block CD28-B7 interactionin CL patients, PBMC cultures stimulated with Leishmaniaantigen led to a downmodulation of IFN-γ, IL-10, and TNFsecretion [27]. Our findings suggest that the CD4+ T cellswere downmodulated by the presence of the rSmTSP-2 andPIII. These antigens were also able to increase the frequencyof the CD4+CD25highFoxp3+ regulatory T cells.

In a survey performed by O’Neal et al. [51], the preva-lence of S. mansoni infection in cutaneous leishmaniasispatients from the endemic area of Corte de Pedra, Bahia,Brazil was 16.7% [51]. The authors also showed that


coinfected individuals tended to have small lesion size,however in the univariated model, the presence of helminthcoinfection was associated with delayed lesion healing. Whenthey evaluated infection with S. mansoni and Strongyloidesstercoralis there was no difference in lesion healing comparedto patients infected with geohelminths such as Ascarislumbricoides, Ancylostoma duodenale, and Trichuris trichiura.

Studies conducted by the same group in the endemicarea of leishmaniasis in Brazil demonstrated that the use ofimmunomodulatory agents such as granulocyte/macrophagecolony stimulating factor (GM-CSF) and pentoxifylline, aninhibitor of TNF production, can lead to faster healing timeand higher cure rate in cutaneous leishmaniasis [52–55]. Ithas also been reported that intralesional injections of GM-CSF reduce healing time of CL ulcers by 50%. Moreover,Santos et al. [53] showed that GM-CSF applied locally inlow doses as an adjuvant to pentavalent antimonial therapy,significantly decreases the healing time of CL ulcers, reducingthe dose of antimony administered and/or the durationof antimonial therapy. These studies indicate that down-modulation of the inflammatory response in CL patients isassociated with lesion cure and give support to the idea thatinhibition of the strong inflammatory response early afterLeishmania infection could avert tissue damage.

In this context, the data presented herein suggest that S.mansoni antigens are capable of downregulating monocyteand T lymphocyte response to the Leishmania antigen invitro, possibly through mechanisms that involve negativesignaling by CTLA-4 and the action of regulatory T cells.This knowledge could contribute to the development offuture strategies to prevent and treat cutaneous and mucosalleishmaniasis.

Conflict of Interests

The authors have no conflict of interests concerning the workreported in this paper.

Acknowledgments

The authors would like to thank the people of theleishmaniasis-endemic area in Corte de Pedra-Bahia for theirparticipation in our study; Dr. Luiz Henrique Guimaraes,Dr. Paulo Machado, and Ednaldo Lago who assisted in datacollection and Hana Khidir and Whitney Oriana for thereview of the paper. This work was supported by the BrazilianNational Research Council (CNPq) and by the NIH Grantno. NIH AI 088650. M. I. Araujo, A. M. Goes, S. O. Costa,and E. M. Carvalho are investigators supported by CNPq.

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Research Article

Transcriptional Profile and Structural Conservation ofSUMO-Specific Proteases in Schistosoma mansoni

Roberta Verciano Pereira,1 Fernanda Janku Cabral,2 Matheus de Souza Gomes,3

Liana Konovaloff Jannotti-Passos,4 William Castro-Borges,1 and Renata Guerra-Sa1

1 Departamento de Ciencias Biologicas/Nucleo de Pesquisas em Ciencias Biologicas, Instituto de Ciencias Exatas e Biologicas,Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro, 35400-000 Ouro Preto, MG, Brazil

2 Departamento de Parasitologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Av. Lineu Prestes, 1374-Butantan,05508-900 Sao Paulo, SP, Brazil

3 Instituto de Genetica e Bioquımica, Universidade Federal de Uberlandia, Av. Getulio Vargas, Palacio dos Cristais-Centro,38700-126 Patos de Minas, MG, Brazil

4 Fundacao Oswaldo Cruz, Centro de Pesquisas Rene Rachou, Laboratorio de Malacologia, Av. Augusto de Lima, 1715-Barro Preto,30190-002 Belo Horizonte, MG, Brazil

Correspondence should be addressed to Renata Guerra-Sa, [email protected]

Received 24 April 2012; Revised 9 August 2012; Accepted 23 August 2012

Academic Editor: Cristina Toscano Fonseca

Copyright © 2012 Roberta Verciano Pereira et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Small ubiquitin-related modifier (SUMO) is involved in numerous cellular processes including protein localization, transcription,and cell cycle control. SUMOylation is a dynamic process, catalyzed by three SUMO-specific enzymes and reversed bySentrin/SUMO-specific proteases (SENPs). Here we report the characterization of these proteases in Schistosoma mansoni. Usingin silico analysis, we identified two SENPs sequences, orthologs of mammalian SENP1 and SENP7, confirming their identitiesand conservation through phylogenetic analysis. In addition, the transcript levels of Smsenp1/7 in cercariae, adult worms, andin vitro cultivated schistosomula were measured by qRT-PCR. Our data revealed upregulation of the Smsenp1/7 transcripts incercariae and early schistosomula, followed by a marked differential gene expression in the other analyzed stages. However, nosignificant difference in expression profile between the paralogs was observed for the analyzed stages. Furthermore, in orderto detect deSUMOylating capabilities in crude parasite extracts, SmSENP1 enzymatic activity was evaluated using SUMO-1-AMC substrate. The endopeptidase activity related to SUMO-1 precursor processing did not differ significantly between cercariaeand adult worms. Taken together, these results support the developmentally regulated expression of SUMO-specific proteases inS. mansoni.

1. Introduction

Reversible posttranslational modification by ubiquitin andubiquitin-like proteins (Ubls) plays a crucial role in dynamicregulation of protein function, fate, binding partners,activity, and localization. Small ubiquitin-related modifier(SUMO) is a member of Ubls family that is covalentlyattached to lysine residues of their protein targets incells, altering function and subcellular localization [1].SUMOylation has been implicated in the regulation ofnumerous biological processes including transcription [2]and cell cycle control [3]. SUMO conjugation mechanismis a highly dynamic and reversible process closely related

to ubiquitylation. The sequential actions of E1, E2, and E3enzymes catalyze the attachment of SUMO to target pro-teins, while deconjugation is promoted by SUMO-specificproteases. In addition, like other Ubls, SUMO polypeptideis synthesized as an inactive precursor, which requires a C-terminal cleavage to expose the glycine residue for substrateconjugation. This process is also catalyzed by SUMO-specificprotease through its hydrolase activity. The catalytic activityis maintained within a highly conserved 200 amino acidregion at the C-terminus of the proteases [4–6].

Recently, the importance of reversible SUMOylation fornormal cell physiology has been demonstrated. ExcessiveSUMO conjugation using knockouts for either SENP1 or


SENP2 induced an embryonic lethal phenotype [7, 8] andderegulation of either SUMO conjugation or deconjugationcan contribute to cancer progression as reviewed by [9]. Inhumans there are six SENP enzymes (SENP1, -2, -3, -5,-6, and -7) that deconjugate mono-SUMOylated proteinsor disassemble polymeric SUMO side chains, while inSaccharomyces cerevisiae there are only two deSUMOylatingenzymes, Ulp1 and Ulp2. Based on these activities andaffinities for SUMO isoforms, SENPs can be categorized intothree independent subfamilies: SENP1 and SENP2; SENP3and SENP5; SENP6 and SENP7 [9].

The initial molecular characterization of SUMOylationpathway in S. mansoni showed the presence of two SUMOparalogs called SMT3C and SMT3B [10], suggesting a varietyof targeted substrates for these modifications. Recently, ourgroup reported the conservation of SUMO conjugatingenzyme SmUBC9 by phylogeny, primary and modeled ter-tiary structures, as well as mRNA transcription and proteinlevels throughout the S. mansoni life cycle. Our data showedthat Smubc9 transcription levels are upregulated in earlyschistosomula, suggesting the utilization of this posttrans-lational modification mechanism S. mansoni development,particularly at the initial phase of invasion of the vertebratehost [11].

To extend the investigation of the SUMOylation pathwayin S. mansoni, we firstly retrieved through homology-basedsearches putative SmSENPs using the publically available S.mansoni databases. Furthermore, the levels of Smsenp1/7transcripts were evaluated by qRT-PCR using mRNA fromcercariae, adult worms and mechanically-transformed schis-tosomula (MTS) in vitro-cultivated during 3.5 h, 1, 2, 3, 5,and 7 days. We also measured the endopeptidase activitiesof SmSENPs in cercariae and adult worm crude extracts.The present study reveals differences in the gene expressionprofile of Smsenp1/7 during schistosomula development.In addition, similar SmSENP1 activity was observed incercariae and adult worms, suggesting the importance of thisproteolytic activity during the parasite’s life cycle.


2.1. Ethics Statement. All experiments involving animalswere authorized by the Ethical Committee for Animal Careof Federal University of Ouro Preto, and were in accordancewith the national and international regulation accepted forlaboratory animal use and care.

2.2. Parasites. S. mansoni. LE strain was maintained byroutine passage through Biomphalaria glabrata snails andBALB/c mice. The infected snails were induced to shedcercariae under light exposure for 2 h and the cercariae wererecovered by sedimentation on ice. Adult worm parasiteswere obtained by liver perfusion of mice after 50 days ofinfection. Mechanically transformed schistosomula (MTS)were prepared as described by Harrop and Wilson [12].Briefly, cercariae were recovered, and washed in RPMI 1640medium (Invitrogen), before vortexing at maximum speedfor 90 s and immediately cultured during 3.5 h at 37◦C, 5%

CO2. Then the recovered schistosomula were washed withRPMI 1640 until no tails were detected. For subsequentincubations, the parasites were maintained in M169 mediumsupplemented with 10% FBS, penicillin, and streptomycin at100 μg/mL and 5% Schneider’s medium [13] at 37◦C on 5%CO2 during 3.5 h, 1, 2, 3, 5, and 7 days.

2.3. Identification and Computational Analysis ofSmSENP1/7. SmSENP1/7 sequences were retrieved fromthe S. mansoni genome database version 5.0 availableat http://www.genedb.org/genedb/smansoni/ throughBLAST searches. Amino acid sequences from Drosophilamelanogaster, Caenorhabditis elegans and Homo sapiensSENP orthologs were used as queries. The BLASTpalgorithm, underpinned by the Pfam (v26.0), alloweddetection of conserved protein domains or motifs from S.mansoni sequences. Multiple alignments of SmSENP1/7were performed by ClustalX 2.0 and phylogenetic analysiswas conducted in MEGA 5 [14]. A phylogenetic tree ofthese sequences was inferred using the Neighbor-Joiningmethod [15]. The bootstrap consensus tree inferred from1000 replicates was used to represent the evolutionaryhistory of the taxa analyzed. Branches corresponding topartitions reproduced in less than 50% bootstrap replicatesare collapsed. The percentage of replicate trees in which theassociated taxa clustered together in the bootstrap test (1000replicates) is shown next to the branches. The tree was drawnto scale, with branch lengths in the same units as those ofthe evolutionary distances used to infer the phylogenetictree. All positions containing gaps and missing data wereeliminated from the dataset.

2.4. RNA Preparation and Expression Analysis of Smsenp1/7by qRT-PCR. Total RNA from adult worms, cercariae, andschistosomula was obtained using a combination of theTrizol reagent (GIBCO-BRL) and chloroform for extraction,and then on column purified using the “SV total RNAIsolation System” (Promega, Belo Horizonte, Brazil). Thepreparation was treated with RNase-free DNase I in 3different rounds by decreasing enzyme concentration (RQ1DNase; Promega). RNA was quantified using a spectropho-tometer and an aliquot containing 1 μg of total RNA reversetranscribed using an oligodT primer from the ThermoscriptRT-PCR System (Invitrogen Sao Paulo, Brazil) as describedby the manufacturer. The efficiency of DNAse I treatmentwas evaluated by PCR amplification of the cDNA reactionmix without the addition of the Thermoscript enzyme. S.mansoni specific SENP1/7 primers were designed using theprogram GeneRunner for SmSENP1 (GeneDB access num-ber Smp 033260.2) (forward 5′-AGGAAACGGAGGCGG-GATTC-3′, reverse 5′-ACACTGGAGACACGGGATGAGC-3′) and for SmSENP7 (GeneDB access number Smp 159120)(forward 5′-TCAGTTACACGGCCCTTTATC-3′, reverse 5′-CCTGAGAAGTGGATGCGATC-3′). Reverse-transcribedcDNA samples were used as templates for PCR amplificationusing SYBR Green Master Mix UDG-ROX (Invitrogen)and 7300 Real Time PCR System (Applied Biosystems,Rio de Janeiro, Brazil). Specific primers for S. mansoni


α-tubulin were used as an endogenous control (GenBankaccess number M80214) (forward 5′-CGTATTCGCAAG-TTGGCTGACCA-3′, reverse 5′-CCATCGAAGCGCAGT-GATGCA-3′) [16]. The efficiency of each pair of primers wasevaluated according to the protocol developed by the AppliedBiosystems application (cDNA dilutions were 1 : 10, 1 : 100and 1 : 1000). For all investigated transcripts three biologicalreplicates were performed and their gene expression normal-ized against the α-tubulin transcript according to the 2−ΔCt

method [17] using the Applied Biosystems 7300 software.

2.5. Endopeptidase Activity. To determine the enzymaticactivity of SENP proteases present in adult worms andcercariae crude extracts we used the fluorogenic substrateSUMO-1-AMC that is specific for SENP1 (Enzo Life Sci-ences, Sao Paulo, Brazil). In these assays 10 μg of totalprotein were used and 0,45 μM of the fluorogenic substrate in50 mM Tris-HCl pH 8, 10 mM MgCl2± 0,45 μM SUMO-1-aldehyde (Enzo Life Sciences) to control for specific enzymeinhibition. Each enzymatic assay was conducted in a finalvolume of 100 μL for both extracts, followed by 60 minutesincubation at 37◦C. The reaction was stopped by addition of2 mL of 99.5% ethanol. The fluorometric readings were takenat wavelengths of 380 nm (excitation) and 440 nm (emission)in a spectrofluorimeter (Turner QuantechTM Fluorometer),and the results expressed in fluorescence units per μg of totalprotein.

2.6. Statistical Analysis. Statistical analysis was performedusing GraphPad Prism version 5.0 software package (Irvine,CA, USA). Normality of the data was established usingone way analysis of variance (ANOVA). Tukey post testswere used to investigate significant differential expressionof SUMO proteases throughout the investigated stages. Inall cases, the differences were considered significant when Pvalues were <0.05.

3. Results

3.1. SUMO Specific-Proteases: The Conservation ofSmSENP1/7. In silico analysis of SmSENPs revealed theconserved putative domains of SUMO proteases in S.mansoni. The parasite database has two sequences encodingputative SENPs: Smp 033260.2 (putative SENP1) andSmp 159120 (putative SENP7). SENP1 has an alternativespliced form annotated as Smp 033260.1. We evaluatedthe conservation of these proteins throughout evolutionusing a phylogenetic approach. Our results showed thateach SENP was grouped in 2 distinct clades, reinforcingtheir structural conservation among the thirteen analyzedorthologs (Figure 1).

To confirm that these predicted proteases are cysteine-family members, analyses using the Pfam protein domaindatabase were conducted. For both SmSENPs entries aconserved Peptidase C48 domain was revealed. Our in silicoanalysis demonstrated SmSENP1 more closely related toHsSENP1 (GenBank access number NP 055369.1), whereasSmSENP7 is related to HsSENP7 (GenBank access number

NP 065705.3). Alignment of their catalytic domains with thehuman SENPs showed the presence of the three essentialcatalytic residues (Cys-His-Asp), highlighted in Figure 2.

3.2. Smsenp1/7 Are Differentially Expressed in S. mansoni.The gene expression profile of Smsenp1 and Smsenp7was determined using qRT-PCR. We designed specificprimers to amplify Smsenp1 and Smsenp7 transcripts inthe cercariae-schistosomula transition and adult worm(Figure 3). We observed that Smsenp1 and Smsenp7 tran-scripts are expressed in basal levels from MTS-2d to 7 daysand adult worms. The levels of the SENPs transcripts weresignificantly higher in MTS-3.5 h being approximately 10-fold higher when compared to levels in adult worms andduring schistosomula development (MTS-2, 3, 5, and 7 days)and 3-fold higher when compared to cercariae and MTS-1d.We also observed that Smsenp1 and Smsenp7 transcriptionlevels were not significantly different (P < 0.05) within agiven stage, except for MTS-3.5 h where Smsenp7 levels are3 fold-higher than Smsenp1.

3.3. SmSENP1 Enzymatic Activity in Cercariae and AdultWorms. In vitro endopeptidase assays were performed usingcrude extract from cercariae and adult worms (Figure 4). TheSmSENP1 enzymatic activity was slightly higher in cercariaecompared to adult worms, although the levels were notsignificantly different (P < 0.05). Enzyme activities werealso recorded in the presence of a commercially availableHsSENP1 inhibitor but significant differences were notobserved in the analyzed stages.

4. Discussion

Previous reports from our group showed the presence oftwo SUMO paralogs (SMT3C and SMT3B) in S. mansoni,exhibiting differential expression between larvae and adultworms. The electrophoretic pattern of SUMO-conjugatedmolecules also differed comparing the analyzed stages [10].Furthermore, our group demonstrated a differential expres-sion profile for UBC9 throughout the parasite life cycle [11].The structural SUMO conservation in S. mansoni reinforcesSUMO as a candidate for a transcription regulator and adegradation signal by facilitating ubiquitylation of certaintarget proteins during parasite development.

In order to understand the role of SUMOylation in S.mansoni, we evaluated the conservation of SUMO proteasesand their gene expression profile in cercariae, schistosomula,and adult worm. Firstly, we retrieved, through homology-based searches, two sequences containing conserved domainsof SUMO proteases. A phylogenetic approach then revealedtwo putative proteases closely associated to human SENP1and SENP7. Alignment of parasite sequences with theirhuman counterparts also demonstrated conservation of thecatalytic triad for both proteases [18]. The active site residueswithin the core domain of Ulp1 and Ulp2 have been provento be necessary for catalytic activity and in vivo function inyeast [19, 20]. Furthermore, Ulp1 lacks obvious sequencesimilarity to any known deubiquitinating enzyme so it is


99

94

94

86

85

83

78

55

3831

00.20.40.60.811.21.41.6

Smp 033260.2

Smp 159120

NP 001254524.1 (Homo sapiens)

NP 659100.1 (Mus musculus)

XP 001343517.1 (Danio rerio)

XP 002596928.1 (Branchiostoma floridae)

XP 797423.3 (Strongylocentrotus purpuratus)

XP 003251079.1 (Apis mellifera)

NP 573362.1 (Drosophila melanogaster)

AAX27951.2 (Schistosoma japonicum)

XP 002648991.1 (Caenorhabditis briggsae)

NP 498095.3 (Caenorhabditis elegans)

NP 191978.3 (Arabidopsis thaliana)

NP 015305.1 (Saccharomyces cerevisiae S288c)

NP 195088.2 (Arabidopsis thaliana)

NP 012233.1 (Saccharomyces cerevisiae S288c)

XP 003727099.1 (Strongylocentrotus purpuratus)

XP 002630625.1 (Caenorhabditis briggsae)

NP 494914.1 (Caenorhabditis elegans)

XP 002590318.1 (Branchiostoma floridae)

XP 001122456.2 (Apis mellifera)

NP 572827.1 (Drosophila melanogaster)

CAX72652.1 (Schistosoma japonicum)

XP 003201158.1 (Danio rerio)

NP 001070671.1 (Homo sapiens)

NP 001003971.1 (Mus musculus)

SENP7 orULP2-like

SENP1 orULP1-like

Figure 1: Consensus phylogenetic tree based on amino acid sequences of SmSENPs. The tree construction and analysis of bootstrap wereperformed using ClustalX 2.0 and MEGA 4.0. For the consensus tree and reliability of the branches formed test was used phylogeneticbootstrap using 1000 replicates for each sequence, and 50% the minimum for considering the branch reliably. Organisms: Hs (Homo sapiens),Mm (Mus musculus), Rn (Rattus norvegicus), Xl (Xenopus laevis), Sm (Schistosoma mansoni), Sj (Schistosoma japonicum), Dm (Drosophilamelanogaster), Cb (Caenorhabditis briggsae) and Ce (Caenorhabditis elegans).


: : .: : .:: : :: . . : :VPVNESSHWYLAVIC---------------RPCILILDSLKA----------VPKQDNSSDCGVYLLQYIPINECAHWFLGLVC---------------MPCVLLFDSLPC----------VPVQSNLVDCGIYLLHY

: : : : : : .:

VPIHLG-VHWCLAVVD-----ITYYDSMG----------IPQQMNGSDCGMFACKY

IPIHDRGMHWCLSCID-----ITYYDSMG----------VPQQYNGSDCGVFLCTF

∗ ∗∗ ∗∗ ∗ ∗ ∗∗ ∗ ∗∗∗ ∗∗ ∗ ∗ ∗∗∗ ∗∗∗ ∗

∗∗∗ ∗∗∗∗ ∗ ∗∗∗∗∗∗∗∗ ∗∗∗ ∗∗∗∗ ∗∗ ∗ ∗

Hs SENP1Sm SENP1

Hs SENP7Sm SENP7

Figure 2: S. mansoni has two predicted SUMO-Specific Proteases. ClustalX 2.0 alignment of SENP1 and SENP7 catalytic residues fromhuman and S. mansoni. The domains position of proteins in S. mansoni were based on Pfam database as well as the e-value score. The greyboxes represent the conserved domains based on Pfam database. Aligned catalytic residues are denoted by the black box.∗, :, and . indicate,respectively, identical residues, highly conserved amino acid substitution, and conserved amino acid substitution.

SENP1SENP7

90

75

60

45

30

15

543210

Rel

ativ

e ex

pres

sion

of

SmSE

NPs

∗

∗∗∗

∗∗

∗∗∗

∗∗∗∗

∗∗∗

∗∗∗ ∗∗∗

∗∗

∗∗∗

∗∗∗∗∗

∗∗∗∗∗

∗∗∗∗∗

Adu

lt w

orm

Cer

cari

ae

MT

S-3,

5 h

MT

S-1

d

MT

S-2

d

MT

S-3

d

MT

S-5

d

MT

S-7

d

∗∗∗∗∗

Figure 3: SmSENPs are differentially expressed throughout the S. mansoni life cycle. The mRNA expression levels were measured basedon three replicates, for each of the following stages: adult worms, cercariae, MTS-3.5 h, 1, 2, 3, 5, and 7 days using quantitative RT-PCR. Expression levels were calibrated according to the comparative 2−ΔCt method, using the constitutively expressed Smα-tubulin as anendogenous control (one-way variance analysis followed by Tukey pairwise comparison P < 0.05). ∗Different from adult worm, ∗∗differentfrom cercariae, and ∗∗∗different from MTS-3.5 h.

30

20

10

0

Flu

ores

cen

ce (µ

g/m

in)

Cercariae Adult worms

SUMO1-AMCSUMO1 aldehyde

∗

Figure 4: SmSENP1 enzymatic activity in S. mansoni. The fluores-cence levels were measured based on three replicates, in cercariaeand adult worm stages. Statistical analysis was performed using ttests followed by unpaired test P < 0.05. ∗Different from cercariae.

unlikely that it is able to process ubiquitin-linked substrates[21].

Considering that the SUMOylation pathway dictates thefunction of a variety of substrates [22], and the existenceof two SUMO paralogs in S. mansoni [10], it is plausiblethat the worm utilizes distinct SUMO proteases for SUMOmaturation and deSUMOylation of target substrates. Dis-crimination between SUMO-1 and SUMO-2/3 paralogs mayalso account for distinct functions of SENP proteases inS. mansoni. In mammalian cells, proteomic studies haveidentified proteins that are selectively modified by eitherSUMO-1 or SUMO-2/3 [23]. It is well established thatSENP1 processes SUMO-1 in preference to SUMO-2, andhas a very low catalytic constant for SUMO-3 [24]. Incontrast, the recent characterization of the catalytic domainof SENP7 in humans demonstrated a greater deconjugatingactivity for SUMO-2/3 chains [25]. As discussed by Shen [26]SENP7 acts as a SUMO-2/3 specific protease that is likelyto regulate the metabolism of polySUMO-2/3 rather thanSUMO-1 conjugation in vivo.


In the present investigation, the transcriptional profileof SUMO proteases was also evaluated by qPCR, usingtotal RNA from distinct stages of the S. mansoni lifecycle. The gene expression data revealed similar expressionprofile for Smsenp1 and Smsenp7 at any given stage, butwith a remarkable differential expression for both proteasescomparing the larvae and adult stages. The differential geneexpression profile observed for Smsenp1/7 throughout the S.mansoni life cycle attests for the distinct patterns of SUMOconjugates observed during parasite development [10]. Poly-SUMOylation has been reported to increase during heat-shock treatment and stress conditions, with SENP7 beingresponsible for dismantling SUMO polymers [25, 27]. Giventhat the levels of Smsenp1/7 transcripts were significantlyhigher in cercariae and MTS-3.5 h, we suggest a stress-induced expression of the SUMOylation machinery duringthe parasite’s body adaptation to the new metabolic andmorphological alterations it undergoes [28]. Our findings arealso in agreement with Pereira [11] which demonstrated asimilar pattern of gene expression profile for Smubc9 in theaforementioned parasite stages.

We have not observed a positive correlation betweendifferential transcriptional levels of Smsenp1 and enzymaticactivity evaluated using synthetic SUMO-1-AMC substrate.The levels of enzymatic activity for SmSENP1 were slightlyhigher in extracts of cercariae compared to adult worms. Inthe presence of a specific inhibitor, no significant change inenzymatic activity was detected. This may account for thelimited amino acid sequence identity (28%) shared betweenSmSENP1 and its human ortholog, from which the synthesisof SUMO-1 aldehyde is based. Additional studies aimed atelucidating substrate selection of SmSENP1/7 in S. mansoniand a detailed kinetic analysis of their enzymatic activities,particularly during the cercarie to schistosomula transitionmay provide further insights into the biological activitiesof these enzymes and the role of SUMO conjugation in S.mansoni.

Acknowledgments

The authors thank the transcriptome initiatives: Sao PauloTranscriptome Consortium; Minas Gerais GenomeNetwork;Wellcome Trust Genome Iniatiative (UK). This work wassupported by the following Brazilian research agencies:FAPEMIG (Fundacao de Amparo a Pesquisa do Estado deMinas Gerais, CBB 0558/09), Nucleo de Bioinformatica daUniversidade Federal de Ouro Preto (NuBio UFOP) andCNPq (Conselho Nacional de Desenvolvimento Cientıfico eTecnologico).

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Review Article

Schistosoma Tegument Proteins in Vaccine andDiagnosis Development: An Update

Cristina Toscano Fonseca,1, 2 Gardenia Braz Figueiredo Carvalho,1

Clarice Carvalho Alves,1 and Tatiane Teixeira de Melo1

1 Laboratorio de Esquistossomose, Centro de Pesquisas Rene Rachou, Fundacao Oswaldo Cruz, Avenida Augusto de Lima 1715,Belo Horizonte,MG 30190-002, Brazil

2 Instituto Nacional de Ciencias e Tecnologia em Doencas Tropicais (INCT-DT), Avenida Augusto de Lima 1715,Belo Horizonte, MG 30190-002, Brazil

Correspondence should be addressed to Cristina Toscano Fonseca, [email protected]

Received 27 July 2012; Accepted 24 September 2012

Academic Editor: Andrea Teixeira-Carvalho

Copyright © 2012 Cristina Toscano Fonseca et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

The development of a vaccine against schistosomiasis and also the availability of a more sensitive diagnosis test are importanttools to help chemotherapy in controlling disease transmission. Bioinformatics tools, together with the access to parasite genome,published recently, should help generate new knowledge on parasite biology and search for new vaccines or therapeutic targetsand antigens to be used in the disease diagnosis. Parasite surface proteins, especially those expressed in schistosomula tegument,represent interesting targets to be used in vaccine formulations and in the diagnosis of early infections, since the tegumentrepresents the interface between host and parasite and its molecules are responsible for essential functions to parasite survival. Inthis paper we will present the advances in the development of vaccines and diagnosis tests achieved with the use of the informationfrom schistosome genome focused on parasite tegument as a source for antigens.

1. Introduction

Schistosomiasis is still a significant public health problemin tropical countries despite the existence of effective drugsagainst the parasite [1]. Chemotherapy as a strategy for dis-ease control has proved ineffective in controlling transmis-sion [1] therefore, the development of a vaccine against thedisease and also a more sensitive diagnosis test is necessary toassist chemotherapy in control programs [1, 2].

In this context, the recent availability of schistosomegenomes information represents an important toll to beused in the discovery of new targets for vaccine and diag-nosis. Schistosoma mansoni genome, published in 2009 [3]described 11.809 genes while Schistosoma japonicum genome[4] has been described to be composed of 13.469 genes.Their assemblies were generated by conventional capillarysequencing resulting in 19.022 scaffolds (S. mansoni) and25.048 scaffolds (S. japonicum). More recently an improvedversion of the S. mansoni genome was published [5], utilizing

a combination of traditional Sanger capillary sequencing anddeep-coverage Illumina sequencing that refined gene pre-diction resulting in a reduction in the number of predictedgenes from 11.809 to 10.852. Illumina-based technology wasalso used in Schistosoma haematobium genome sequencing,which described 13.073 genes [6].

Simultaneously to genome publication, an importanttool to access and analyze parasite genome has been devel-oped, the SchistoDB (http://www.schistodb.net/) database[7]. The SchistoDB enables access to information on theparasite genome even to those researchers not specialized incomputer language. The current 3.0 database version pro-vides access to the latest draft of S. mansoni genome sequenceand annotation and also to S. japonicum and S. haematobiumgenome annotation.

The bioinformatics tools, together with the availability toaccess parasite genome, should have helped the knowledge ofparasite biology and the search for new vaccines, therapeutictargets, and antigens to be used in the disease diagnosis. In


this paper we will present the advances in the developmentof vaccines and diagnostics tests achieved with the use of theinformation from schistosome genome, focus will be givento the parasite tegument as a source for antigens.

2. Host-Parasite Relationship: Role forthe Parasite Tegument

Highly adapted to parasitic life, schistosomes can live foryears or decades even in a hostile environment as the circu-latory system from vertebrate host where the parasite has anintimate contact with circulating elements of the immunesystem [8].

In this successful host-parasite relationship, the hostimmune system plays an important role in both parasitedevelopment and elimination. CD4+ cells, hormones, andcytokines as TNF-α, TGF-β, and IL-7 produced by the host,seem to assist the parasite development [9–15]. While CD4+cells, B cells, IFN-γ, and TNF-α has been described to beinvolved in parasite elimination in the irradiated cercariaevaccine model [16–18].

Moreover, the highly adapted relationship between schis-tosomes and the mammalian definitive host also involvesthe effective mechanisms for evading the immune responsethat they provoke. In this context, the parasite tegumentplays an important role [19, 20]. After penetration, the para-site surface undergoes a profound change that allows parasiteadaptation into the host internal microenvironment wherethe parasite switches from its immune-sensitive to animmune-refractory state [21]. In cercariae, the surface ischaracterized by a single bilayer membrane covered by adense glicocalyx. During penetration, the glicocalyx is lostand the membrane transforms into a double bilayer mem-brane [22]. Evading mechanisms as antigenic mimicry,membrane turnover, production of immunomodulatorymolecules and modulation of surface antigens expressionalso takes place in the parasite surface and contributes toschistosome survival [23, 24].

Trying to eliminate the parasite, host immune systemtargets the antigens in parasite surface. Studies in mice haveshown that the developmental stage most susceptible to thehost immune system attack is the schistosomula stage. Veryearly after infection, schistosomula are susceptible to cellularand humoral immunity, however, in the course of parasitedevelopment the susceptibility is rapidly lost [25, 26]. Theresistance to host immune response acquired by parasitescan be in part explained by surface changes independentlyof host antigens adsorption [27–29]. In addition, El Ridiand colleagues [30], demonstrated that lung-stage schisto-somulum protect themselves from the host immune systemby confining antigenic molecules in lipid-rich sites of surfacemembrane. In contrast, McLaren, in 1989 [31], demon-strated that both skin and lung schistosomula phases are tar-gets of the immune system in the radiation-attenuated vac-cine model which trigger an inflammatory reaction aroundthe larvae inhibiting their migration.

Since schistosomula is the major target of the hostimmune system attack and its tegument represents theinterface between parasite and host, also performing vital

functions that ensure parasite survival [32], the study of itsstructure and how it interacts with the host immune systemcan provide important information about disease control,especially to those related to the search for new drugs andvaccine development. We have recently demonstrated thatthe schistosomula tegument from S. mansoni (Smteg) is rec-ognized by TLR4 in dendritic cells (DC) leading to DC acti-vation and production of proinflammatory cytokines as IL-12 and TNF-α [33]. In contrast to this inflammatory profile,Smteg also induce IL-10 production by DC in a TLR (Toll likereceptors) 2, 3, 4, and 9 independent manner (unpublisheddata) once again demonstrating that schistosomula tegumentcan both activate or modulate host immune system.

3. The Tegument as Antigen Source forVaccine Development

Most of the studies that aimed to identify membrane proteinsin parasite tegument were performed in adult worms [34–36]. Although schistosomula is the major target for hostimmunity, its tegument proteins have still not been charac-terized, mainly due to the difficulty in obtaining sufficientquantities of material for such protein studies [37]. Indeedprotective antigens are found in S. mansoni schistosomulategument (Smteg) since mice immunization with Smtegformulated with Freunds’ adjuvant [38] or Alum + CPG-ODN (unpublished data) is able to reduce significantly wormburden and egg elimination with the feces. The characteri-zation of these protective antigens is being performed usingimmune-proteomics analysis and genome databases to iden-tify candidates to be used in a vaccine formulation againstschistosomiasis. Other “omics” technologies are also beingused to identify schistosoma proteins, mainly those expressedin schistosomula. In this context, two studies, using cDNAmicroarrays technologies assessed the most relevant tran-scriptional changes in the schistosomula development phase.These studies demonstrated that tetraspanin, Sm22.6, Sm29,Sm200 and phosphadiesterase are membrane proteins arehighly expressed during schistosomula phase [39, 40]. Fur-thermore, the studies that used gene silencing through RNAitechnique could clarify the importance of some proteins,such as cathepsins [41, 42] and tetraspanins [43] for parasitedevelopment and survival. The same membrane protein wasidentified in adult worm tegument preparations using Massspectrometry (MS-)-based proteomics [33, 34] together withgenome, transcriptome and genetic maps information [3,44–46]. Recently a proteomic analysis demonstrated thatSm29 and Sm200 are linked to parasite surface membranethrough a GPI-anchor [47] while the most abundant proteinin adult worm tegument, among the investigated molecules,are aquaporin, dysferlin, TSP-2, and ATP diphosphohy-drolase [48]. Among this expressive catalogue of proteinexpressed in the schistosome tegument, some of them havebeen evaluated as vaccine antigen in immunization protocolsin mice. The Table 1 summarizes the results observed in thesepreclinical trials using tegument proteins.

Sm29 was identified by Cardoso and coworkers using insilico analysis to identify in S. mansoni transcriptome putativeexpressed proteins localized in the parasite tegument [49].


Table 1: Schistosome tegument protein evaluated as vaccine candidates in preclinical studies.

Protein Vaccine type Protection level Egg reductionBioinformatic tool used in

antigen selectionReferences

Sm 21.7 Recombinant protein 41%–70% ND ND [63]

Sm 21.7 DNA vaccine 41.5%62% (liver)

67% (intestine)ND [64]

Cu/Zn superoxidedismutase

DNA vaccine 44%–60% ND ND [65]

Sm TSP2 Recombinant protein 57%64% (liver)65% (feces)

BLAST [57, 83]

Sm29 Recombinant protein 51% 60% (intestine)

InterProScan, SignalIP 3.0,Signal IP Neural, NetNGlyc

1.0, BLAST, WolfpSORT,SOSUI, Compute pI/Mw tool,

[49, 50]

ECL (200 kDa protein) DNA vaccine 38.1% ND ND [61]

Sm 22.6 Recombinant protein 34.5% ND BLAST [53]

Sm TSP 1 Recombinant protein 34%52% (liver)

69% (intestine)BLAST [57, 83]

ND: not determined.

Sm29 recombinant form induces a Th1 profile in mice asso-ciated with a reduction of 51% in worm burden when usedin vaccine formulation [50]. The tegumental protein, Sm22.6and its homologue in S. japonicum (Sj22.6), are involved inresistance to reinfection in endemic areas [51, 52]. Immu-nization of mice with recombinant 22.6 formulated with Fre-und adjuvant resulted in 34.5% reduction on worm burden[53] while Sm22.6 formulated with alum failed to induceprotection against schistosomiasis but induced a regulatoryresponse able to modulate allergic asthma in mice [54, 55].

Tetraspanins (TSP) 1 and 2 were identified in a cDNAlibrary from S. mansoni based on their membrane-targetingsignal [56]. Immunization of mice with TSP1 recombinantprotein resulted in a reduction of 57% in worm burdenand reduction in the number of eggs in liver (64%) andintestine (65%), TSP2 recombinant protein was less effectivein reducing worm burden (34%) but had similar effects inreducing the number of eggs trapped in the liver (52%) andintestine (69%) [57]. The TSP-2 homologue in S. japonicumhas also been evaluated in murine immunization however noprotection was observed [58].

ECL or Sm200 is a GPI-anchored protein in the S. man-soni tegument that has also been associated with praziquantelefficacy, since antibodies against this protein can restoredrug efficacy in B cells depleted mice [59, 60]. Murine DNAvaccination with the gene encoding Sm200 elicited 38.1%protection while immunization of mice with enzymaticallycleaved GPI-anchored proteins from the S. mansoni tegu-ment, in which Sm200 represent the most abundant proteinresult in 43% reduction in adult worm burden [61, 62].Sm21.7 was tested as antigen in a recombinant vaccine [63]and DNA vaccine [64]. Immunization of mice with recom-binant Sm21.7 resulted in a decrease of 41%–70% in wormburden while DNA vaccination resulted in of 41.5% wormburden reduction [63, 64].

The schistosome antioxidant enzymes (Cu/Zn super-oxide dismutase-SOD, glutathione-S-peroxidase-GPX) are

developmentally regulated. The lowest level of gene expres-sion and enzyme-specific activity was found in the larvalstages while the highest level of gene expression was observedin adult worms [65–68]. This suggests that antioxidantenzymes are important in immune evasion by adult schis-tosome parasites [67]. Also RNAi assays demonstrated thatknocking down the antioxidants enzymes GPX and GSTresult in dramatic decreases in sporocysts survival indicatingthat these enzymes are capable of enhancing parasite survivalin an oxidative environment [69]. Mice immunized withthe antioxidant enzyme Cu-Zn superoxide dismutase in aDNA vaccine strategy resulted in 44–60% reduction in wormburden [65].

4. Antigens to Be Used in SchistosomiasisDiagnostic Test

Currently, all available techniques for the diagnosis of schist-osomiasis are characterized by having some limitations,especially when it becomes necessary to detect infection ina large number of patients with low parasite load [70]. Oneof the initial difficulties in the development of a test for thediagnosis of schistosomiasis is the choice of an appropriateantigen. There are several factors that influence this choice:easily of production, high stability in sample storage, immu-nogenicity, specificity, and ability to be incorporated to lowcosts test platforms [71].

In this context, the availability of the complete genomesequences in combination with other technologies such asbioinformatics and proteomics, provides important tolls toseek for an ideal candidate to compose an efficient immun-odiagnostic test. With this in mind, our group have recentlydesigned an in silico strategy based in the principles of reversevaccinology, and using a rational criteria to mine candidatesin parasite genome to be used in the immunodiagnosis ofschistosomiasis [72]. Six antigens were selected based on theevidence of gene expression at different phases of the parasite


Table 2: Schistosoma mansoni protein selected by genome mining to be used in serological diagnosis for schistosomiasis.

ProteinSchistoDB

numberAnnotation

Number ofamino acid

Base pairsPredicted

molecular weightPredicted

isoelectric pointPredicted location

Sm200 Smp 017730200-kDa GPI-anchored

surface glycoprotein1656 4971 186,5 kDa 4.97

Tegument surfacemembranes

Sm12.8 Smp 034420.1 Expressed protein 117 354 12,8 kDa 6.88 Extracellular

Sm43.5 Smp 042910 Expressed protein 382 1149 43,5 kDa 8.43 Extracellular

Sm127.9 Smp 171300 Hypothetical protein 1143 3432 127,9 kDa 6.63 Extracellular

Sm18.9 Smp 184440Cytochrome oxidase

subunit, putative171 516 18,9 kDa 9.30 Extracellular

Sm16.5 Smp 184550Cytochrome oxidase

subunit, putative146 441 16,5 kDa 9.14 Extracellular

Adapted of Carvalho et al., 2011 [72].

life cycle in the definitive host, accessibility to host immunesystem (exposed proteins), low similarity with human andother helminthic proteins, and presence of predicted B cellsepitopes (Table 2) [72]. Although our in silico analysis led toidentification of six candidates, this strategy has not been yetexperimentally validated.

Other groups have also used bioinformatics analysis toselect target sequence from S. japonicum genome to be usedfor the detection of parasite DNA in blood samples. A 230-bp sequence from the highly repetitive retrotransposon SjR2was identified and it was demonstrated that PCR test todetect SjR2 is highly sensitive and specific for detectionS. japonicum infection in the sera of infected rabbits andpatients [73]. More recently the same group performed acomparative study to determine the best target to be usedin a molecular diagnosis test for schistosomiasis japonicumin 29 retrotransposons identified by bioinformatics analysis.A 303-bp sequence had the highest sensitivity and specificityfor the detection of S. japonicum DNA in serum samples [74].

Proteomics analysis has also been used in the identifica-tion of candidates to the immunodiagnosis of schistosomia-sis. Western Blot with sera from S. japonicum infected rabbitin a two-dimensional gel loaded with adult worm prepara-tion identified 10 spots that were demonstrated by LC/MS-MS to correspond to four different proteins: SjLAP (Leucineaminopeptidases), SjFBPA (fructose-1,6-bisphosphate aldo-lase), SjGST (Glutathione-S-transferase) and SJ22.6 [75].Recombinant SjLAP and SjFBPA were tested in ELISA assayand presented high efficacy for the diagnosis of S. japonicuminfection, with 96.7% specificity for both proteins and 98.1%or 87.8% sensitivity to detect acute and chronically infectedindividuals, respectively, when SjLAP was used as antigen ora sensitivity of 100% (acute) and 84.7% (chronic infection)when SjFBPA was used as antigen [75].

5. Other Membrane Proteins Candidatesto Be Used in Vaccine Formulation andDiagnosis Tests

Aquaporins are small integral membrane proteins involvedin the selective transportation of water and other solutesthrough plasma membranes of mammals, plants and lower

organisms [76]. This protein was described to be abundant inschistosome tegument and due to its physiological functionand abundance represent an interesting target to vaccinesand diagnosis tests [48]. Characterization of the S. japonicumaquaporin-3 using bioinformatics tools demonstrated thatthis 32.9 kDa transmembrane protein has predicted B cellsepitopes with the most likely epitopes present in the N-terminal portion of the protein, located outside the mem-brane [77]. Other abundant protein in schistosoma tegumentis dysferlin, based on analogy with homologues from otherorganisms, this protein seems to be involved in membranerepair and/or vesicle fusion in tegument surface [34].

ATP-diphosphohydrolases are enzymes involved in ADPand ATP hydrolysis that has been related to host immune sys-tem evasion, since this enzyme could hydrolyze the ATP pro-duced in response to parasite induced stress in the endothe-lio thus modulating the DAMP (danger associated mole-cular pattern)-mediated inflammatory signaling [78, 79].In schistosomes two different proteins have been describedSmATPDase 1 and SmATPDase2 with approximately 63 and55 kDa [80, 81]. SmATPDase 1 is located in the borderof the tegument while SmATPDase2 is located in internalstructure of the tegument syncytium and can be secreted[81]. The immunogenicity of the synthetic peptide (r175–190) from SmATPDase2 has been demonstrated in Balb-cmice, however the protection induced by this epitope has notbeen evaluated [82].

Although most tegument protein listed in this paper hasbeen identified in adult worm tegument, an in silico anal-ysis performed in SchistoDB (http://www.schistodb.net/)demonstrates that some of them are also expressed in theschistosomula stage as demonstrated in Figure 1 reinforcingtheir potential to be used in a vaccine formulation or in theearly diagnosis of schistosome infection.

6. Conclusion

So far the genome, transcriptome, and proteome informa-tion provided many targets to be tested in schistosomiasisvaccine and diagnosis and also new knowledge about schisto-some biology. However approximately 40% of the schisto-some genome is composed of hypothetical proteins withunknown function that represents interesting targets to be


0

5

10

15

20

25

30

35

40

Sm200 Sm29 TSP-2 TSP-1 Dysferlin Sm22.6 Sm21.7

CercariaeSchistosomula

Adult wormEgg

Nu

mbe

rs o

f E

ST

Figure 1: Predicted expression of schistosome tegument proteinsin the different parasite life stage in the definitive host. schistosometegument protein identified by proteomics analysis of the adultworm tegument was analyzed in SchistoDB database (http://www.schistodb.net/). Bars represent the numbers of EST in each parasitelife stage whose annotation correspond to Sm200, Sm29, TSP-2,TSP-1, Dysferlin, Sm22.6, or Sm21.7.

tested and characterized. An increase in the knowledge aboutparasite biology, pathogenesis, and host-parasite relationshipcan be expected for the next years.

Acknowledgments

This work was supported by CNPq, INCT-DT/CNPq, Ripag/CPqRR-Fiocruz, and Papes/Fiocruz. G. B. F. Carvalho andC. C. Alves both received fellowship from Fapemig. C. T.Fonseca received a fellowship from PQ/CNPq.

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Review Article

Pathogenicity of Trichobilharzia spp. for Vertebrates

Lichtenbergova Lucie and Horak Petr

Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 44 Prague 2, Czech Republic

Correspondence should be addressed to Lichtenbergova Lucie, [email protected]

Received 13 July 2012; Accepted 13 September 2012

Academic Editor: Rashika El Ridi

Copyright © 2012 L. Lucie and H. Petr. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Bird schistosomes, besides being responsible for bird schistosomiasis, are known as causative agents of cercarial dermatitis.Cercarial dermatitis develops after repeated contact with cercariae, mainly of the genus Trichobilharzia, and was described as a typeI, immediate hypersensitivity response, followed by a late phase reaction. The immune response is Th2 polarized. Primary infectionleads to an inflammatory reaction that is insufficient to eliminate the schistosomes and schistosomula may continue its migrationthrough the body of avian as well as mammalian hosts. However, reinfections of experimental mice revealed an immune reactionleading to destruction of the majority of schistosomula in the skin. Infection with the nasal schistosome Trichobilharzia regentiprobably represents a higher health risk than infections with visceral schistosomes. After the skin penetration by the cercariae,parasites migrate via the peripheral nerves, spinal cord to the brain, and terminate their life cycle in the nasal mucosa of waterfowlwhere they lay eggs. T. regenti can also get over skin barrier and migrate to CNS of experimental mice. During heavy infections,neuroinfections of both birds and mammals lead to the development of a cellular immune response and axonal damage in thevicinity of the schistosomulum. Such infections are manifest by neuromotor disorders.

1. Introduction

Despite their worldwide distribution, avian schistosomeswere neglected by parasitologists who assumed that theyhave no or minor pathogenic impact on birds or mammals,including humans. Nowadays, many studies focus on theseparasites since it has been recognized that they can be severepathogens of birds. Moreover, their larval stages (cercariae)frequently infect humans and cause cercarial dermatitis. Themost reported agents of swimmer’s itch are cercariae of thegenus Trichobilharzia [1].

Human infections by bird schistosomes are associatedmostly with the development of cercarial dermatitis (swim-mer’s itch), an allergic skin response, which develops afterrepeated contact with cercariae penetrating into the skin. Fora long time, it was assumed that the reaction eliminated themajority of the schistosomes that had penetrated into theskin. However, the studies on mice infected experimentallywith bird schistosomes showed that soon after the penetra-tion, the cercariae transform to schistosomula. Under certaincircumstances, these schistosomula are able to resist hostimmune response, escape from the skin, and migrate further

to target organs [2, 3]. In mammals, bird schistosomes cansurvive for several days or weeks, but they never mature[4, 5]. The exact reason why bird schistosomes die in mam-malian hosts has not been known until the present.

Studies on bird schistosomes disclosed a new species—Trichobilharzia regenti [4]— with unusual behavior in com-patible as well, noncompatible hosts. In comparison to themajority of bird schistosome species living in the bloodsystem of visceral organs, mature T. regenti occur in thenasals of their definitive host where they lay eggs. Migrationof the worms from the skin to the nasals is via the spinal cordand brain [4]. Experimental infections of mice showed that T.regenti schistosomula can evade attack by immune cells in theskin of mammalian hosts allowing them to migrate furtherthrough the central nervous system (CNS) where immatureworms die after several days [5, 6]. Migration of the parasitesthrough CNS of both bird and mammalian hosts causessevere tissue injuries [6, 7] that can result in leg paralysis,balance, and orientation disorders and even host death [4, 7].

Nowadays, mainly two species of bird schistosomes, T.szidati and T. regenti, are studied under laboratory conditionswith regard to their development, physiology (including


enzymes participating in host tissue degradation and diges-tion), immunomodulation of the host immune response,and pathogenicity towards natural and accidental hosts.In field studies, the emphasis is on the study of speciesspectrum, inter- and intraspecific variability, and prevalenceof bird schistosomes (see, e.g., Brant and Loker [8], Jouetet al. [9], and Korsunenko et al. [10]; see also review byHorak and Kolarova [1]). With regard to T. szidati andT. regenti, their occurrence has been reported from severalcountries. In particular, T. regenti cercariae have been foundin freshwater ponds for example, in Russia [11] and cercariaeof T. szidati in Russia, Belorussia [11], Germany [12] andFrance [13]. Several findings of T. szidati infections in birdswere reported, for example, from France [9], Poland, andCzech Republic [14]. Infections of birds with T. regenti weredetected for example, in Iceland [15] and in France, wherethe prevalence on three studied localities reached 40% [9].Based on findings of Rudolfova et al. [14, 16], prevalence ofT. regenti infection of waterfowl was 14% in Czech Republic(one studied locality) [16] and 22% in Gdansk area in Poland[14]. Although T. szidati cercariae are mostly distributedthroughout Europe, there is a report of their occurrence insnails collected from Michigan and Montana in the UnitedStates [8].

The main aim of our review is to summarize the presentknowledge of the pathogenesis of bird schistosomiasis andthe immune reactions to bird schistosomes presence in avianand mammalian hosts, with a special emphasis on T. regenti.The neurotropic species T. regenti, due to its unusual mode ofmigration and potential pathogenic impact on avian as wellas mammalian hosts, deserves more attention. Therefore,a major part of this review is dedicated to this species ofschistosome.

2. Skin Infection

After leaving the snail intermediate hosts, bird schistosomecercariae have a tendency to cling to the water surface andwait for their definitive host. They react to shadow stimuliand start to swim with a negative phototactic orientationfrom the water surface toward the definitive host [17]. Exceptfor physical stimuli such as shadow, water turbulence, andwarmth, cercariae respond to host chemical cues like duck-foot skin lipids—cholesterol and ceramides [17, 18].

Once attached to the host skin, cercariae creep on the skinand search for a suitable penetration site [19]. In contrastto human schistosomes, such as Schistosoma mansoni, whichpenetrate smooth skin, bird schistosome cercariae preferskin wrinkles and hair follicles for penetration [20]. Studieson cercarial behavior of S. mansoni and T. szidati revealeddifferences in the speed of migration through the host skin.For example, cercariae of T. szidati invade human skin moreefficiently than S. mansoni such that they are able to locateentry sites and penetrate through the skin more rapidly thanS. mansoni [20].

Skin penetration by cercariae is stimulated by fatty acids[19]. According to the study of Haas and Haeberlein [20],T. szidati cercariae respond to linolenic acid with highersensitivity if compared to S. mansoni. This feature seems to

represent an adaptation to invade duck skin that has a lowercontent of free fatty acids compared to human skin [20].Therefore, human skin with higher amount of surface lipidsis likely more attractive to bird schistosome cercariae thanduck skin [19].

Penetration through the skin is facilitated by a numberof proteolytic enzymes, which are released from cercarialcircum- and postacetabular glands immediately after attach-ing to the host skin. In the case of bird schistosomes,glandular secretion is stimulated mainly by fatty acids,ceramides, and cholesterol [20]. Cercarial glands fill aboutone-third of the cercarial body [21] and contain manyof the potentially antigenic proteins. The most importantpenetration enzyme of S. mansoni is probably a serineprotease, elastase [22]. Nevertheless, Mikes et al. [23] andKasny et al. [24] did not find any elastase activity in thesecretions of T. szidati and T. regenti cercariae, and it wasnot found in the congener S. japonicum [25]. However,cathepsin B-like activity was detected in the aforementionedspecies. This enzyme from cercarial penetration glands isconsidered to be the main component in the cercarialpenetration process [23–25]. The same types of enzymescould be the reason for similar penetration speed of S.japonicum and Trichobilharzia cercariae [20]. In addition, sixisoforms of cathepsin B1 (TrCB1.1–TrCB1.6) and cathepsinB2 (TrCB2) were identified in an extract of migrating T.regenti schistosomula [26, 27]. Two isoforms, TrCB1.1 andTrCB1.4, degrade myelin basic protein, but do not efficientlycleave hemoglobin [26]. The recombinant form of TrCB2is able to cleave protein components of the skin (keratin,collagen, and elastin) as well as nervous tissue (myelin basicprotein), but has negligible activity towards hemoglobin[27]. The enzyme could, therefore, serve as a tool formigration through the host skin and subsequently throughthe nervous tissue.

Host fatty acids seem to stimulate not only the penetra-tion of cercaria through the host skin, but also transforma-tion of their tegument as a part of parasite immune evasion[19]. Penetration of the cercariae into the host skin isaccompanied by cercaria/schistosomulum transformationwith reconstruction of tegumental surface. Transformationstarts with loss of tail, a process supported by a sphinctermuscle in cercarial hindbody [19], then the cercariae shedthe glycocalyx and start to form a surface double membrane.Creation of a new surface is accompanied by the disappear-ance of lectin and antibody targets on the surface of theschistosomula [28].

In the skin of the bird hosts, schistosomula move throughthe skin towards deeper layers and, therefore, require infor-mation for orientation. Studies on visceral schistosomesinvading humans, S. mansoni, and birds, T. ocellata, showedthat schistosomula use negative photo orientation to moveaway from light source [29]. The other stimulus involvedin navigation of the visceral schistosomula is represented bythe concentration gradient of chemicals, such as D-glucoseand L-arginine [30]. Unfortunately, data about orientationof the nasal species T. regenti are not complete, but there is anindication that the stimuli differ from those used by visceralspecies (unpublished).


3. Cercarial Dermatitis

In humans, the skin infection is a result of the developmentof an inflammatory reaction known as swimmer’s itch orcercarial dermatitis. Cercarial dermatitis can occur aftercontact with water containing cercariae from snails infectedby bird schistosomes. Chances of getting cercarial dermatitisincrease with repeated exposures to the parasite. Higherincidence of the infection is connected with bathing inshallow water, which is the preferred habitat for water snailsand, therefore, a place where cercariae accumulate [31].

Penetration of cercariae into the skin may result in animmediate prickling sensation that lasts for about 1 hour[32]. Severity and intensity of cercarial dermatitis dependon various factors including the number and duration ofexposures to the cercariae, and host immune status, that is,history of cercarial dermatitis, and individual susceptibilityto the infection [32]. After a primary infection, the skinreaction is unapparent or mild with small and transientmacules or maculopapules, which develop after 5–14 days[32]. The most pronounced disease occurs after repeatedexposures that result in a strong inflammatory reactionagainst the parasites [33]. The skin disease manifests bymaculo-papulovesicular eruptions accompanied by intenseitching and, occasionally, by erythema, fever, local lymphnode swelling, oedema. Massive infections may also causenausea and diarrhea (for a review see Horak et al. [34]). Skinlesions develop only on those parts of body where there wascercarial penetration [35].

Diagnosis of cercarial dermatitis is based on anamnesisand clinical findings [36]. Some work has been done usingserological tests for confirmation of the diagnosis [37].Nevertheless, immunological tests are not routinely availableand laboratory confirmation of causative agents of thedermatitis remains difficult.

4. Skin Immune Response

Clinical pattern of cercarial dermatitis is linked with histo-pathological reactions to the infection. In the case of thehuman schistosome, Schistosoma mansoni, infections innaive mice led to a mild skin response with contributionof neutrophils and mononuclear cells. In contrast, a moresevere cellular reaction developed in the mouse skin afterrepeated infections with S. mansoni [38]. Similarly as forhuman schistosomes, infections of mice with the birdschistosomes T. szidati (T. ocellata) and T. regenti initiate thedevelopment of a skin immune response [2, 39]. Primarymouse infection with T. regenti initiates an acute inflamma-tion with oedema, vasodilatation, and tissue infiltration byneutrophils, macrophages, mast cells, and MHC II antigenpresenting cells (APCs), and a weak infiltration by CD4+

lymphocytes; repeated infections cause substantially elevatedinfiltration of all cells mentioned above [2, 6]. Trichobilharziaregenti primoinfection leads to the development of aninflammatory reaction in the murine skin within 1–6 h afterexposure [2]. Detection of cytokine production by in vitrocultured biopsies of pinnae (later skin biopsies) obtainedfrom primoinfected mice revealed that the inflammation is

accompanied by a transient release of acute phase cytokines(IL-1β and IL-6) and increasing amounts of IL-12 [2].Increased production of IL-12 in the skin correlated withhigher Th1-associated IFN-γ production by cells from skin-draining lymph nodes [2]. Similarly, the study of Hogg et al.[40] using S. mansoni illustrates rapid host immune responseto parasite penetration with production of acute phasecytokines, such as IL-1β and IL-6 which were detected in thesupernatants of skin biopsies from wild-type mice. Both IL-1β and IL-6 promote Th17-cell differentiation [41], thereforeimplying that primary mouse infection with human as wellas avian schistosomes induces a Th17 polarized response.

Skin immune response to challenge infections leadsto capture and elimination of the majority of schistoso-mula in the skin [2]. In the early phase after re-infec-tion with T. regenti cercariae, infiltration of mouse skinwith inflammatory cells (high density of granulocytes andneutrophils, abundant MHC II APCs, macrophages andCD4+ lymphocytes) was also accompanied by oedema caus-ed by local vascular permeability that was initiated byhistamine produced by activated mast cells and basophils[2]. Degranulation of mast cells and basophils with releaseof histamine and IL-4 is realized after binding of IgE-antigencomplex via high affinity receptors FcεRI on the cell surface[42, 43]. Histamine has been described previously as a potenteffector of Th1 and Th2 responses as well as immunoglobulinsynthesis [44, 45]. Repeated infections with T. regenti evokedominant production of Th2-type cytokines, and the firstand most abundant cytokine detected in supernatants of skinbiopsies from mice after repeated infections is IL-6 [2], whichcan initiate Th2-type polarization via induction of IL-4 [46].Within 1 hour after the penetration by T. regenti cercariae amassive upregulation of IL-4 and IL-10 can be observed inthe skin biopsies, and the level of these cytokines declinesafter 48 h. This upregulation during T. regenti infection isaccompanied by release of histamine and proliferation ofmast cells [2]. Production of histamine and IL-4 detectedin skin biopsies immediately after the last infection of re-infected mice was realized via IgE-dependent mast celldegranulation [2]. IL-4 plays also a crucial role in thedevelopment of Th2-type immune responses to S. mansoniantigens and regulation of immunoglobulin isotype switch toIgE [47]. CD4+ cells, numbers of which significantly increasein the skin after challenge infections, are potential sources ofIL-4 associated with Th2 response [2].

Like mast cells, basophils possess high-affinity IgE sur-face receptors (FcεRI) that, after antigen-specific cross-link, induce production and release of mediators such ashistamine and IL-4 [48]. In vitro stimulation of basophilsobtained from healthy (nonsensitized) humans by homo-genate of cercariae and excretory/secretory (E/S) productsof T. regenti cercariae revealed that these antigens inducebasophil degranulation and release of IL-4 [49]. Antigensstimulated basophil release of IL-4 in a dose-dependentmanner, and antigens from E/S products were more potentinducers of IL-4 release than cercarial homogenate [49].Elevated levels of skin mast cells [2] and high titers of serumIgE in mice infected repeatedly with T. regenti [49] showedthat the cells of mast cell/basophil lineage play an important


role in development of Th2 responses during Trichobilharziainfections.

5. Antibody Response and Antigens ofBird Schistosomes

Domination of Th-2 polarization of the immune responseafter repeated T. regenti infections was confirmed by meas-urement of antigen-specific antibody levels. Similarly as inthe study of Kourilova et al. [2], the increase of Th2-asso-ciated antigen-specific IgG1 and total serum IgE antibodieswith concurrent decline of Th1-associated IgG2b antibodywas demonstrated in sera of mice repeatedly infected with T.regenti [49].

During T. regenti primary infection, IgM responseagainst glycan structures of the cercariae and their excre-tory/secretory (E/S) products was observed [49]. Thisindicates that early antibody response is directed againstcomponents of highly antigenic cercarial glycocalyx as well asagainst glycoproteins contained in E/S products of circum-and postacetabular glands of cercariae [49]. The glycocalyxof T. regenti cercariae was described as the most antigenicstructure and its remnants were still present on 1-dayold schistosomula transformed in vitro [50], therefore IgMantibodies could recognize components of the glycocalyxduring primary infection.

After penetration into the host skin, cercariae transformto schistosomula by shedding their tails, releasing E/S pro-ducts, and rebuilding their surface [51]. The dominantpart of the cercarial surface is represented by glycocalyx,which is likely the main component responsible for com-plement activation [52]. Cercarial surface of human andbird schistosomes is recognized by antibodies from humansand mice infected with T. regenti, T. szidati, and S. mansoni[53]. In the skin, parasites need to avoid destruction bythe host immune system, thus the transformation of cercariato schistosomulum is accompanied by the decrease of surfacesaccharides and antigen epitopes which could be recognizedby lectins and antibodies, respectively [28]. Although asubstantial part of glycocalyx is removed by the cercariaeduring penetration [54], some of glycocalyx componentsremain associated with surface of the schistosomula for sometime after transformation [54, 55]. Therefore, not only thecercarial surface but also the surface of the early in vitrotransformed (5 h) schistosomula was strongly recognized byIgG and total Ig antibodies from mice repeatedly infectedby T. szidati [28]. E/S products of human as well asbird schistosome cercariae, mainly the products released bytransforming larvae, are rich in components of glycocalyxand secretions of circum- and post-acetabular glands [23,54].

Immunohistochemical staining of ultrathin sections ofparticular developmental stages revealed variable distribu-tion of antigens recognized by IgG antibodies from sera ofmice re-infected with T. regenti [50]. Except for antibodybinding to the surface of cercariae and 1-day-old schisto-somula, a positive reaction with spherical bodies located insubtegumental cells of cercariae and early schistosomula was

recorded [50]. In schistosomula, spherical bodies representedthe most reactive structure. Further development of theparasite was accompanied with loss of immunoreactivity. Inadult worms, the antibodies recognized the surface and sub-tegumental cells, but with lower intensity if compared to thelarval stages [50].

Spherical bodies primary located within subtegumentalcells were transferred via cytoplasmic bridges to the surfaceof schistosomula. As in human schistosomes [56], thesebodies probably release their content on surface of thetegument where the antigenic molecules could be recognizedby the host immune system [50]. However, composition ofthe spherical body content is not known till present.

Nevertheless, based on Western blot analysis of cercarialhomogenate probed with sera from re-infected mice, severalantigens (14.7, 17, 28, 34, and 50 kDa) were identified byIgG and IgE antibodies. Antigens of 34 and 50 kDa werealso recognized in cercarial gland secretions [49]. A preciseidentification of the 34 kDa molecule which is regarded as amajor immunogen is in progress.

6. CNS Infection

The bird schistosome T. regenti exhibits an unusual mode ofbehavior. After successful escape from the host skin, schis-tosomula migrate further to the CNS of both specific avianand accidental mammalian hosts [5, 7]. CNS represents anobligatory part on the migration pathway of T. regenti to thenasal mucosa [4]. In specific bird hosts, schistosomula growand mature during the migration, and their development iscompleted in the nasal area of the host [4]. In accidentalmammalian hosts, the parasite is not able to complete itsdevelopment and dies as an immature schistosomula in thespinal cord or brain [4].

Soon after penetration of cercariae into the skin ofspecific as well as accidental definitive hosts, schistosomulalocate peripheral nerves, enter nerve fascicles (Figure 1) orepineurium, and reach the spinal cord via spinal roots [5, 57].Schistosomula appear in the spinal cord from day 2 postinfection (p.i.); intact parasites can be detected in the duckspinal cord even 23 days p.i. [7], and 21–24 days p.i. in thespinal cord of mice [5]. Then, schistosomula migrate to thebrain where the parasites occur from day 12 p.i. to day 18 p.i.in the case of infected ducks, and from 3 days p.i. to 24 daysp.i. in the case of infected mice [5]. At the beginning of thebrain infection, schistosomula are located mainly in medullaoblongata and further in subarachnoidal area of cerebellum[5, 57]. Other localization in the brain is mostly restricted tothe subarachnoidal space and the area of the fourth ventricle[57, 58].

Migration through the host CNS requires parasitesadaptations to this environment. Light-brown-pigmentedgranules in the intestine of T. regenti schistosomula [59],which exhibit immunoreactivity with antibodies againstcomponents of CNS [57], proved that schistosomula utilizehost nervous tissue for nutrition during their migration viaCNS. For this purpose the parasite should have proteasescapable of cleaving components of the nervous tissue. Untilthe present only cathepsins B1 and B2 with the capability


(a) (b)

Figure 1: T. regenti schistosomula (arrows) migrating inside the peripheral nerve fascicles of an experimentally infected mouse: (a)haematoxylin and eosin staining, scale bar 100 μm; (b) myelin sheets visualized by binding of anti-MBP (myelin basic protein) antibodyand secondary anti-rabbit IgG-Cy3 (red), neurofilaments stained by anti-SMI-32 (neurofilament H nonphosphorylated) and secondaryanti-mouse IgG-Alexa Fluor (green), cell nuclei stained by DAPI (4′,6-diamidino-2-phenylindole) (blue).

to degrade myelin basic protein [26, 27] were identified ascandidate molecules.

Except localization in CNS that is typical for this species,schistosomula were accidentally observed in the lungs ofspecific avian [4] as well as non-compatible mammalianhosts [5, 57]. Moreover, schistosomula were also found inskin capillaries [53]. However, in vitro tests of blood vesselattractiveness did not show any positive results [5]. There-fore, it seems that schistosomulum presence in the lungsrepresents an ectopic localization of the parasite in heavilyinfected experimental animals [57].

7. Immune Response and Pathology in the CNS

Presence of the parasites in CNS initiates a cellular response.In the spinal cord of ducks 23 days p.i., the parasites werelocated mostly in meninges of thoracic and synsacral spinalcord and in the white and gray matter of synsacral spinal cord[7]. The surroundings of the worms were infiltrated witheosinophils, heterophils, and less frequently with plasmacells, histiocytes, lymphocytes, and macrophages [7]. Despitedense infiltration with the immune cells, the parasites werenot destroyed by host immune response [7]. The para-sites located in brain, predominantly in meninges, weresurrounded by macrophages and endothelial cells [7]. Atpresent, details about the immune response in the infectedducks are still missing.

More studies have been done on mice as a model ofnon-compatible host. Early infections (3 days p.i.) of themouse spinal cord did not initiate any inflammatory reactionor damage to the nervous tissue; only focal oedema wasobserved [6, 57]. Signs of the infection were observed 6 or 7days p.i., when the vicinity of schistosomula was infiltratedwith granulocytes, predominantly neutrophils, activatedmicroglia, macrophages and, less frequently, CD3+ lympho-cytes [6, 57]. It seems that microglia and macrophages, areresponsible for destruction of schistosomula in the mouse

CNS [57]. Similarly, microglia are the most important cellsin prevention against Toxoplasma gondii tachyzoite pro-liferation in the brain [60]. The presence of T. regentischistosomula in the nervous tissue triggered activation andproliferation of astrocytes, which were detected in the tracksafter migrating schistosomula [57]. Formation of glial scarsby astrocytic processes around inflammatory infiltrates wasalso observed in the brain of human patients infectedwith Taenia solium metacestodes [61]. The presence of themetacestodes was accompanied by astrocytic activation andincreased expression of glial fibrillary acidic protein (GFAP)[61]. Similar activation of astrocytes was detected in thebrain of Toxocara-infected mice [62]. Progress of T. regentiinfection led to the formation of inflammatory lesions sur-rounding, destroyed schistosomula in the white matter of thespinal cord. Lesions were formed by microglia, macrophages,eosinophils, neutrophils and CD3+ lymphocytes, and dam-aged axons were detected in the tissue surrounding thelesions. On the other hand, presence of the parasitesoutside of parenchyma, in subarachnoidal space of the spinalcord and brain, and in the cavity of the 4th ventricle ofthe brain, did not initiate any heavy inflammation, norpathological changes in the surrounding tissue. This impliesthat schistosomula are able to prolong their survival whilethey migrate outside of parenchyma [57]. Nevertheless,progression of the infection inevitably led to eliminationof the parasite. Most schistosomula were destroyed on day21 p.i. [57]. Challenge infections initiated development ofa strong immune response leading to rapid destruction andelimination of schistosomula. No intact worms, only parasiteremnants surrounded by inflammatory foci, were observedin CNS 3 days after the fourth infection [6, 57].

Infections of immunodeficient mice (SCID strain)revealed that deficiency in T- and B-cell production led todevelopment of mild immune reaction, which is not suffi-cient for elimination of the parasite [6, 57]. Even challengeinfections did not induce proper immune reactions leading


to the destruction of all worms. However, the tissue inthe vicinity of intact schistosomula was infiltrated withinflammatory cells, and also axonal damage as a result ofschistosomulum migration was observed [57]. A highernumber of schistosomula in CNS and prolonged survival ofworms indicate the importance of lymphocytes in thedestruction of schistosomula [57]. Lymphocyte infiltrationin CNS was also demonstrated for other disorders, includingneurocysticercosis [63], cerebral malaria [64], and toxo-plasmic encephalitis [65]. It was observed that T cellspotentiated an immune defense against Mesocestoides cortiinfection of mice by indirect activation of other immune cells(i.e., macrophages) and resident CNS cells (i.e., microglia,astrocytes) [63].

The increased number of T. regenti schistosomula mig-rating through the nervous tissue of immunodeficient micecaused axonal damage and activation and proliferation ofastrocytes. No significant differences in the number of dam-aged axons around the inflammatory lesions and in the vicin-ity of schistosomula were observed in immunocompetentand immunodeficient mice. Therefore, axonal injury wasprobably caused mechanically by migrating schistosomulaand not by the host immune defense [57]. These injuries ofthe spinal cord resulted in partial hind leg paralysis [57] oreven death (unpublished) of infected SCID mice.

8. Terminal Phase of the Infection

The exact migratory route from the brain to the site of finallocation in the nasals is not described. Two hypotheses havebeen published. Based on the presence of worms in bulbusolfactorius [5, 7] a hypothesis about migration via cranialnerves was formulated. Nevertheless, investigation of cranialnerves, n. olfactorius and n. opticus, did not reveal any par-asites or lesions; therefore this hypothesis was rejected [58].Histological studies showed an extravascular localization ofthe parasite in subarachnoidal space; several worms were alsolocated intravascularly [7, 58]. The intestine of the wormsin meninges contained a dark-brown pigment, probablyhematin produced by hemoglobin digestion [58, 59]. Thesefindings support the theory that T. regenti probably migratesfrom the meninges to the nasal cavity via blood vessels [58],but this hypothesis needs to be validated.

The first appearance of parasites in the nasal mucosawas noted 13 days p.i. [4]. Intact live worms were locatedintravascularly or extravascularly [66] in the connective tis-sue between cartilage of turbinate and glandular epitheliumof the nasal mucosa until 24 days p.i. [58]. The tissue aroundthe adult worms was infiltrated with various inflammatorycells [7]. Fifteen days p.i., immature eggs were detectedextravascularly in connective tissue close to cartilage. Thehighest number of eggs appeared 22 days p.i. and they weredistributed all over the nasal mucosa [58]. The eggs with fullydeveloped miracidia were surrounded by a focal dense massof eosinophils, heterophils, histiocytes, and multinucleatedgiant cells [7]. Miracidia hatch directly in the nasal tissueand leave the nasal cavity (bill) when the duck submerges itsbill in water to feed or drink [34]. Swimming miracidia then

actively search for their snail host and a new life cycle of T.regenti begin.

9. Conclusion

Avian schistosomes, in comparison to the extensive studieson human schistosomes, are neglected. The most studiedspecies of avian schistosomes belong to the genus Tri-chobilharzia. Their life cycle is connected to an aquaticenvironment and they use waterfowl as definitive hosts.Adult worms of the genus Trichobilharzia inhabit eithervisceral or nasal areas of their bird hosts. Visceral speciesrepresent the majority of avian schistosomes, whereas nasalspecies form a small group. Trichobilharzia regenti is the onlynasal species whose life cycle is described. After penetrationinto the skin, T. regenti is able to invade peripheral nervesand continue via CNS to the nasal cavity of birds where adultfemales lay eggs. Such neuroinfections of birds can resultin transient or permanent neuromotor disorders, especiallyduring heavy infections.

Experimental studies on mice revealed that bird schis-tosomes possess an ability to penetrate into the body ofmammals. In case of nonsensitized or immunodeficientmice, the parasites are not eliminated in the skin by the hostimmune system and can migrate further through the hostbody; the parasites never mature in mammalian host and dieafter a few days/weeks after infection. In case of infectionswith T. regenti, schistosomula reach the spinal cord andbrain of the experimental mice. Migrating schistosomulacause axonal damage in the mouse nervous tissue and initiatedevelopment of inflammatory reaction. Neuroinfections,mainly in case of immunodeficient animals, are manifest byhind leg paralysis and even death of the heavily infected host.

Under natural conditions, cercariae of bird schistosomesare attracted not only by the duck-foot skin, but theyalso positively react to human skin stimuli. In the humanskin, cercariae cause an allergic disease known as cercarialdermatitis. It develops after repeated contact with cercariaeas a response of the host immune system to antigensof penetrating parasites, and leads to the destruction ofparasites in the skin.

Based on recent reports of bird schistosomes and out-breaks of cercarial dermatitis in new areas, cercarial dermati-tis is considered as a reemerging disease [1]. Experimentalinfections of small mammals show that there might be apotential health risk for humans due to exposure to T. regenticercariae (particularly for immunodeficient patients). If theparasite is not killed in the skin, then its neurotropic behaviormight represent a serious problem. Unfortunately, no dataon such human infections are available, and an appropriatediagnostic tool is still missing. Immunological or molecularmethods for differential diagnosis of bird schistosomes arehighly desirable for verification/exclusion of T. regenti as acausative agent of undetermined neurological disorders ofanimal and human patients.

Acknowledgments

The authors wish to thank Dr Sara V. Brant (The Universityof New Mexico) for the linguistic corrections. The research


of coauthors has been supported by the Czech Science Foun-dation (Grant no. 502/11/1621) and the Charles Universityin Prague (the Projects PRVOUK P41 and UNCE 204017).

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