Advances in the Study of Schistosomiasis: The Postgenomic Era

Transcription

1 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

2 Advances in the Study of Schistosomiasis: The Postgenomic Era

3 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

4 Copyright 213 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 Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

5 Editorial Board Takeshi Agatsuma, Japan Jeffrey Bethony, USA Dave Chadee, USA Kwang Poo Chang, USA Wej Choochote, Thailand Alvin A. Gajadhar, Canada C. Genchi, Italy Boyko B. Georgiev, Bulgaria Ana Maria Jansen, Brazil MariaV.Johansen,Denmark Nirbhay Kumar, USA D. S. Lindsay, USA Bernard Marchand, France Renato A. Mortara, Brazil Domenico Otranto, Italy Barbara Papadopoulou, Canada A. F. Petavy, France Benjamin M. Rosenthal, USA Joseph Schrevel, France José F. Silveira, Brazil M. J. Stear, UK Xin-zhuan Su, USA Kazuyuki Tanabe, Japan

6 Contents Advances in the Study of Schistosomiasis: The Postgenomic Era, Cristina Toscano Fonseca, Sergio Costa Oliveira, Andrea Teixeira-Carvalho, and Rashika El Ridi Volume 213, Article ID 84913, 2 pages Cytokine Pattern of T Lymphocytes in Acute Schistosomiasis mansoni Patients following Treated Praziquantel Therapy, Denise Silveira-Lemos, Matheus Fernandes Costa-Silva, Amanda Cardoso de Oliveira Silveira, Mauricio Azevedo Batista, Lúcia Alves Oliveira-Fraga, Alda Maria Soares Silveira, Maria Carolina Barbosa Alvarez, Olindo Assis Martins-Filho, Giovanni Gazzinelli, Rodrigo Corrêa-Oliveira, andandréa Teixeira-Carvalho Volume 213, Article ID 99134, 13 pages Cytokine and Chemokine Profile in Individuals with Different Degrees of Periportal Fibrosis due to Schistosoma mansoni Infection, Robson Da Paixão DeSouza,Luciana Santos Cardoso, Giuseppe Tittoni Varela Lopes, Maria Cecília F. Almeida, Ricardo Riccio Oliveira, Leda Maria Alcântara, Edgar M. Carvalho, and Maria Ilma Araujo Volume 212, Article ID , 1 pages NewFrontiers inschistosoma Genomics and Transcriptomics, Laila A. Nahum, Marina M. Mourão, and Guilherme Oliveira Volume 212, Article ID , 11 pages Changes in T-Cell and Monocyte Phenotypes In Vitro by Schistosoma mansoni Antigens in Cutaneous Leishmaniasis Patients, Aline Michelle Barbosa Bafica, Luciana Santos Cardoso, Sérgio Costa Oliveira, Alex Loukas, Alfredo Góes, Ricardo Riccio Oliveira, Edgar M. Carvalho, and Maria Ilma Araujo Volume 212, Article ID 5238, 1 pages Transcriptional Profile and Structural Conservation of SUMO-Specific Proteases in Schistosoma mansoni, Roberta Verciano Pereira, Fernanda Janku Cabral, Matheus de Souza Gomes, Liana Konovaloff Jannotti-Passos, William Castro-Borges, and Renata Guerra-Sá Volume 212, Article ID 48824, 7 pages Schistosoma Tegument Proteins in Vaccine and Diagnosis Development: An Update, Cristina Toscano Fonseca,Gardênia Braz Figueiredo Carvalho, Clarice Carvalho Alves, and Tatiane Teixeira de Melo Volume 212, Article ID , 8 pages Pathogenicity of Trichobilharzia spp. for Vertebrates,LichtenbergováLucieandHorák Petr Volume 212, Article ID , 9 pages

7 Hindawi Publishing Corporation Journal of Parasitology Research Volume 213, Article ID 84913, 2 pages Editorial Advances in the Study of Schistosomiasis: The Postgenomic Era Cristina Toscano Fonseca, 1 Sergio Costa Oliveira, 2 Andrea Teixeira-Carvalho, 1 and Rashika El Ridi 3 1 Centro de Pesquisas RenéRachou,Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil 2 Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil 3 Zoology Department, Faculty of Science, Cairo University, Giza 12613, Egypt Correspondence should be addressed to Cristina Toscano Fonseca; ctoscano@cpqrr.fiocruz.br Received 3 March 213; Accepted 3 March 213 Copyright 213 Cristina Toscano Fonseca et al. 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. Schistosomiasis remains a serious public health problem in 76 countries and territories of the world, with more than 2 million individuals infected, causing annual losses of 1.4 million in DALYs (disability adjusted to life years). Although effective drugs against schistosomiasis are used in disease control programs, the prevalence of the disease remains unaltered, and new diseases foci and acute outbreaks are observed worldwide. Also, drug resistance by the parasites is a concern, and new drugs and alternative control measures need to be developed. An effective vaccine against schistosomiasis is an alternative and desirable tool to help in the disease control; however, thecomplexschistosomelifecycleandthecomplexityof its interaction with the host immune system turn vaccine development into a difficult task. Neither drug development nor vaccine development is sufficient to ensure the success of schistosomiasis control programs. As a matter of fact, the combination of an effective vaccine together with chemotherapy is the strategy of choice. Many studies also demonstrate that without a more sensitive diagnosis test, able to detect individuals with low parasite burden, no control strategy will achieve the desirable result which nowadays is diseases elimination. The access to the genome from Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium recently published is expected to increase rapidly our understanding of the parasite biology and also accelerate the development of new drugs, vaccine, and diagnosis methods by helping in the identification of new targets. In this special issue, a review by C. T. Fonseca et al. givesanupdateonhowparasitegenomestogetherwith bioinformatics tools have helped in vaccine and diagnosis development in the last few years. Since the parasite surface represents an interesting target for the host immune system, this review was focused on parasite tegument as a source of antigens. Although the genome from the three most socioeconomically important schistosome species had already been published, many questions continue to challenge researchers. The review by L. A. Nahum and coworkers discusses the advances on schistosome genomics and transcriptomics studies and point out the gaps and future directions in this research field. An example on how parasite genome database helps in understanding the parasite biology is presented in the paper by R. V. Pereira and colleagues. In their article, using in silico analysis, they identified in the S. mansoni genome orthologs of the mammalian Sentrin/SUMO-specific proteases (SENPs) 1 and 7, which are involved in many cellular processes. The S. mansoni SENPs transcriptional profile as well as its structural conservation was analyzed. Just as important as understanding the biology of the parasite is deciphering how the host immune system deals with schistosome infection and its relation to the pathogenesis of the disease. Clinically schistosomiasis is characterized bytwodistinctphases:theacuteandthechronicphase. The acute phase is commonly observed in individuals living in nonendemic area, while chronic phase is observed in endemic-area residents.

8 2 Journal of Parasitology Research D. Silveira-Lemos et al. evaluated in their article immunological parameters in patients with acute schistosomiasis before and after praziquantel treatment. Interesting differences are demonstrated by the authors between noninfected, acute, and treated group, and a role for CD8+ T cells as a source of cytokines in acute patients is pointed by this study. The cytokine and chemokine profile observed in PBMCs from individuals infected with S. mansoni in response to specific stimulation and its correlation with different degrees of periportal fibrosis is described by R. P. de Souza and colleagues in their research article. Their results indicate potential biomarkers for the progression of liver pathology duetoschistosomiasis. Many schistosome antigens have been described to modulate inflammatory diseases such as asthma. A. M. B. Baficaetal.evaluatedintheirarticletheimpactofthe S. mansoni antigens: Sm29, TSP-2, and PIII in PBMCs culture from cutaneous leishmaniasis patients stimulated with L. braziliensis antigen. Their results demonstrated that schistosomeantigensinduceamodulatoryphenotypewith decreased monocyte activation and increased expression of T-lymphocyte modulatory molecules. Trichobilharzia spp. infection results in bird schistosomiases, which causes severe pathogenic impact in birds and frequently results in cercarial dermatitis in humans. In their article, L. Lucie and H. Petr review the pathogenesis of bird schistosomiasis and the immune response triggered by Trichobilharzia spp. infection with emphasis on the new species T. regent. We hope you enjoy reading this special issue! Cristina Toscano Fonseca Sergio Costa Oliveira Andrea Teixeira-Carvalho Rashika El Ridi

9 Hindawi Publishing Corporation Journal of Parasitology Research Volume 213, Article ID 99134, 13 pages Research Article Cytokine Pattern of T Lymphocytes in Acute Schistosomiasis mansoni 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 Alda Maria Soares Silveira, 5 Maria Carolina Barbosa Alvarez, 6 Olindo Assis Martins-Filho, 1 Giovanni Gazzinelli, 2 Rodrigo Corrêa-Oliveira, 2 and Andréa Teixeira-Carvalho 1 1 LaboratóriodeBiomarcadoresdeDiagnósticoeMonitoração,CentrodePesquisasRenéRachou,FIOCRUZ,BarroPreto, Belo Horizonte, MG, Brazil 2 Laboratório de Imunologia Celular e Molecular, Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, MG, Brazil 3 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, UniversidadeFederaldeOuroPreto,OuroPreto,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; denise.lemos@gmail.com Received 12 July 212; Revised 24 September 212; Accepted 26 September 212 Academic Editor: Rashika El Ridi Copyright 213 Denise Silveira-Lemos et al. is 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. Acute schistosomiasis is associated with a primary exposure and is more commonly seen in nonimmune individuals traveling through endemic regions. In this study, we have focused on the cytokine pro le of T lymphocytes evaluated in circulating leukocytes of acute Schistosomiasis mansoni-infected patients (ACT group) before and a er praziquantel treatment (ACT-TR group). Our data demonstrated increased values of total leukocytes, eosinophils, and monocytes in both groups. Interestingly, we have observed that patients treated with praziquantel showed increased values of lymphocytes as compared with noninfected group (NI) or ACT groups. Furthermore, a decrease of neutrophils in ACT-TR was observed when compared to ACT group. Analyses of short-term in 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 cells demonstrated higher percentage of IFN-γγ and IL-4 cells in both ACT and ACT-TR groups apart from increased percentage of IL- 1 cells only in the ACT group. is study is the rst one to point out the relevance of CD T lymphocytes in the immune response induced during the acute phase of schistosomiasis. 1. Introduction Schistosomiasis is a tropical parasitic chronic disease, caused bywormsofthegenusschistosoma. e infection is endemic in tropics and subtropics around the world and is present in Africa, South America, Arabia, and Asia. ere are approximately 2 million people infected worldwide, and the number of deaths caused by schistosomiasis is estimated at around 41. annually [1]. Schistosoma mansoni worms dwell in peri-intestinal venules and cause intestinal, hepatointestinal, and hepatosplenic schistosomiasis [2]. Brazil is the country most affected by schistosomiasis in the Americas, with 1.5 million people infected and more than 36 million at risk of acquiring infection [3, 4]. In the

10 2 Journal of Parasitology Research state of Minas Gerais, Schistosomiasis mansoni is prevalent in 519 municipalities, with an estimated number of one million people infected [5]. e propagation and maintenance of schistosomiasis in a given region are conditional of many factors such as appropriate climate, socioeconomic conditions, rapid spread of intermediate hosts, sanitary conditions, and water supply [6]. Tourist activity in endemic areas may be an important contributing factor to the propagation of outbreaks/cases of schistosomiasis [7 1]. In endemic areas for schistosomiasis, people develop the chronic phase of infection (which begins around six months a er exposure) and have different clinical manifestations. People who had never had previous contact with the parasite develop acute schistosomiasis when contracting the infection [11, 12]. e acute phase starts a er cercarial infection, and local urticaria may appear in a few hours. Between one and four weeks a er infection, the migrating and maturing schistosomula can cause a systemic hypersensitivity reaction with fever, general weakness, headache, nausea, vomiting, diarrhea, and dry cough [2, 9]. In this phase, individuals present a mixed cytokine pro le with predominance of 1 type of CD4 + T-cell differentiation [13, 14]. In the context of cytokine milieu triggered by S. mansoni infection, it has been suggested that interleukin (IL)-4 upregulates broblast chemokine, matrix protein expression, and collagen, which implies that IL-4 is a crucial cytokine for granuloma formation [15] and reduces the cellular proliferative response to soluble egg antigens (SEA) [13]. IL-5 in schistosomiasis induces liver brosis [16]. IL-1 has been shown to be a major cytokine during infection with downregulatory activity of both 1 and 2 T cell subpopulations [17]. Interferon gamma (IFN-γγ) is related to the activation of macrophages and plays a key role in the protective mechanism against periportal brosis, whereas the proin ammatory tumor necrosis factor-alpha (TNF-αα) may aggravate the disease [18]. As regards the cytokine pro le a er speci c chemotherapy, it has been suggested that peripheral blood mononuclear cells (PBMC) from patients with acute infection responded to SEA and soluble worm antigen preparation (SWAP) by producing signi cantly higher amounts of IFN-γγ and IL- 1. However, IL-5 was detected only in SEA-stimulated cultures, and little or no IL-4 was detected in SEA or SWAPstimulated cells [19]. De Jesus et al. [9] suggested that most patients a er speci c chemotherapy for schistosomiasis spontaneously released high levels of TNF-αα, IL-1, and IL- 6. In addition, detectable levels of IFN-γγ were present in the supernatants of unstimulated PBMC from these patients. Stimulation of PBMC from patients with acute disease only induced 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-1) of T lymphocytes and their subsets as well as the ultrasonographic features of patients with acute schistosomiasis infection before and a er praziquantel treatment. 2. Population, Material, and Methods 2.1. Study Population. e patients evaluated in this study were infected accidentally by S. mansoni in the country village of São Geraldo da Piedade, an endemic area for schistosomiasis, located in the east of the state of Minas Gerais (MG), Brazil, during the traveling in a holiday. is people reported swimming in a stream that probably harbors cercariae released by infected snails. In this area 6% of Biomphalaria glabrata are infected and the chronic disease is frequent in the population. ese individuals went to the hospital because they felt very bad, with acute symptoms. e patients did not live in areas of active transmission of S. mansoni. All patients showed clinical symptoms associated with early S. mansoni infection such as fever, diarrhoea, headache, and nausea. Only patients who had positive results for quantitative parasitological stool examinations for S. mansoni by the Kato-Katz method [2] and signed an informed consent were included in this study. e group of acutely S. mansoni-infected patients (ACT) without praziquantel treatment consisted of twenty-one individuals, ve women and sixteen men, with ages ranging from 13 to 44 years, with parasite load ranging from 12 to 1812 eggs per gram of feces (epg). A er blood sample collection, all S. mansoni-infected individuals received speci c therapeutic treatment with praziquantel in the standard Brazilian dose (5 6 mg/kg praziquantel), regardless of whether or not they were going to participate in this study [21]. One month a er praziquantel treatment, blood was collected from a group of patients (ACT-TR) composed of three women and four men, aged between 12 and 4 years. An additional noninfected group (NI) composed of nineteen individuals, eleven woman and eight men, with age ranging from 19 to 45 years, who were healthy blood donors contacted at the Hemominas Blood Bank Foundation in Belo Horizonte, MG, Brazil, participated in the study. All noninfected volunteers were included a er three consecutive negative parasitological exams for S. mansoni infection apart from negative serology for Chagas disease, leishmaniasis, human immunode ciency virus infection, and hepatitis. is study was approved by the Ethics Committees at the Oswaldo Cruz Foundation (FIOCRUZ), the School of Medicine at the Federal University of Minas Gerais (UFMG), and the Brazilian National Committee on Ethics in Research (CONEP) Ultrasonographic Analysis. Ultrasonographic evaluation was performed in all individuals using a Nemio SSA/55A ultrasound machine (Toshiba) with a 3 MHz sector probe. Liver size, portal vein diameter, thickness of central walls and peripheral portal branches, spleen size, and splenic vein diameter were assessed as described in previous studies [22, 23]. Liver span was measured both in the midclavicular line and the midline. e liver was also examined for surface smoothness. Portal vein diameter was measured at its entrance into the liver and its bifurcation to the liver. Oblique and longitudinal scans of the le upper quadrant were used to evaluate the spleen. e gallbladder was examined for wall thickness and stones. Periportal thickness was evaluated according to established criteria [22 24]. All individuals were examined by the same physician.

11 Journal of Parasitology Research Evaluation of Hematological Parameters. Hemograms were performed with an automated blood cell counter (Coulter MD18, USA), using whole blood collected in 5 ml vacutainer tubes containing the anticoagulant ethylenediamine tetraacetic acid (EDTA) (Becton Dickinson Biosciences, San Diego, CA, USA). e parameters measured were total leukocytes counts and differential analysis of leukocyte subsets including the absolute counts of eosinophils, neutrophils, lymphocytes, and monocytes Preparation of Antigens. S. mansoni eggs were isolated from the livers of infected mice, exposed 8 weeks previously to cercariae, homogenized, and ground in cold phosphatebuffered saline (PBS). e clear supernatant uid resulting from high-speed centrifugation of the homogenate at 5. g for 1 h at room temperature was named soluble egg antigens (SEA) and stored at 7 Cuntiluse[25] Monoclonal Antibodies. e experiments used monoclonal antibodies (mabs) against both human cell surface markers, including CD3 (UCTH-1), CD4 (SK3), and CD8 (SK1) labeled with uorescein isothiocyanate (FITC), and intracytoplasmic cytokines including IL-4 (MP4-25D2), IL- 5 (TRFK5), IL-1 (JES3-9D7), IFN-γγ (B27), and TNF-αα (Mab11) labeled with phycoerythrin (PE). Isotypic controls were 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 from Becton Dickinson Biosciences Pharmingen (San Diego, CA, USA) Short-Term Whole Blood Culture In Vitro and Intracellular Cytokine Immunostaining. Short-term cultures in vitro and cytokine immunostaining were performed as described by Silveira-Lemos et al. [26]. Brie y, ve hundred microliters of peripheral blood samples collected into Vacutainer tubes containing heparin sodium salt (Becton Dickinson, Mountain View, CA, USA) were dispensed into individual 17 1 mm polypropylene tubes (Falcon Becton Dickinson, Mountain View, CA, USA). Short-term cultures were performed in the absence (control cultures) or in the presence of S. mansoni eggs derived antigens (SEA). For this purposes, 5 μμl of whole blood samples were incubated in the presence of 5 μμl of RPMI 164 plus Brefeldin A, BFA (Sigma Chemical Company, St Louis, MO, USA) at a nal concentration of 1 μμg/ml. e blood samples were incubated for 4 h at 37 Cina5% CO 2 humidi ed incubator. ese conditions were chosen considering that the detection of cytokines, particularly in the absence of exogenous stimuli, may re ect the events of cytokine production by blood leukocytes in vivo. Antigen-speci c stimulation was performed by previous in vitro incubation of 5 μμl whole blood aliquots in the presence of SEA at a nal concentration of 25 μμg/ml for 1 h at 37 Cina5% CO 2 humidi ed incubator. Following the addition of BFA (Sigma Chemical Company, St Louis, MO, USA) at a nal concentration of 1 μμg/ml, blood samples were incubated for an additional 4 h at 37 C in a 5% CO 2 humidi ed incubator. Prior to immunostaining for intracellular cytokine, all cultures, including control and SEA stimulated, were treated with 2 mm EDTA (Sigma Chemical Company, St. Louis, MO, USA) and kept at room temperature for 15 min, to stop cell activation process. Following the short-term in vitro stimulation, cultured whole blood samples were washed with 6 ml of FACS buffer containing PBS supplemented with.5% Bovine Serum Albumin (BSA) and.1% sodium azide (Sigma Chemical Company, St. Louis, MO, USA), by centrifugation at 6 g at room temperature for 7 min. A er resuspension in 1 ml of FACS buffer, 4 μμl aliquots were dispensed into one mm polystyrene tube (Becton Dickinson, Mountain View, CA, USA) and labeled in the dark for 3 min at room temperature with the manufacture s recommended amount of mabs anti-cd3, anti-cd4, and anti-cd8-fitc. Following the erythrocyte lyses step, the cells were incubated with 3 ml of FACS permeabilizing solution, containing FACS buffer supplemented with.5% of saponin (Sigma Chemical Company, St. Louis, MO, USA) in the dark for 1 min at room temperature. Following incubation, the samples were centrifuged at 6 g at room temperature for 7 min, the supernatant gently decanted and 3 ml of FACS buffer added to the resuspended pellet. A er centrifugation, the pellet was resuspended and 3 μμl aliquots of cell suspension distributed in 96 wells U bottom microplate (Falcon-Becton Dickinson, Mountain View, CA, USA) and stained with 2 μμl of PE-labeled anticytokine mab (anti-il-4, anti-il-5, anti-il-1, anti-ifn-γγ, and anti- TNF-αα,). e cells were incubated in the dark for 3 min at room temperature. A er incubation, the cells were washed with 15 μμl of FACS permeabilizing solution followed by 2 μμl of FACS buffer. A er washing, the stained cells were xed in 2 μμl of FACS x solution and the samples stored at 4 C in the dark and analyzed by ow cytometry Data Collection and Analysis. Immunostained samples were run in a FACScan three-color detection ow cytometer (Becton Dickinson, San Jose, CA, USA). Data were collected and analyzed with CellQuest so ware (a er collection of 5. events/sample). Cytokine-expressing T lymphocytes, including CD3 +, CD4 +, and CD8 + T cells, were identi ed by dual color immunophenotyping with FL1/FITC-labeled antihuman cell surface markers against CD3, CD4 and CD8 mabs together with FL2/PE-labeled anticytokine mabs. Lymphocyte gating was initially based on lymphocyte selection by forward scatter (FSC) versus side light scatter (SSC) properties on dot plot distributions, where they are con ned into a region of low size and complexity. Further analysis of lymphocytes subpopulations was based on their selective staining with FITC-labeled anti-cd3, anti-cd4, and anti- CD8 mabs. Cytokine-expressing cell subsets were quanti ed using FL1 cell surface markers versus FL2 anti cytokine-pe dot plots by setting quadrants to segregate FL2 positive and negative cells based on the negative control immunostaining. e results are expressed as the percentage of lymphocyte subsets expressing a given cytokine Statistical Analysis. e statistical analysis was performed with so ware GraphPad PRISM 5. for Windows (La Jolla, CA, USA). Considering the parametric nature of the

12 4 Journal of Parasitology Research TABLE 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 ± 16. ( ) 99.7 ± 2.9 ( ) Anteroposterior le lobe of liver ± 4.4 ( ) 53.7 ± 7.6 ( ) Longitudinal right lobe of liver ± 1.5 ( ) ± 11.5 ( ) Anteroposterior right lobe of liver ± 1.6 ( ) 84.3 ± 9.6 ( ) Longitudinal spleen ± 7. ( ) ± 15.6 ( ) Anteroposterior spleen ± 3.9 ( ) 46.6 ± 8.5 ( ) Portal vein < ± 1.2 ( ) 1.5 ± 1.3 ( ) Hilar portal vein wall < ±.3 ( ) 2. ±.1 ( ) Splenic vein < ± 1.3 ( ) 6.5 ±.9 ( ) Superior mesenteric vein < ±.8 ( ) 6.2 ±.8 ( ) Values are represented in mm as mean ± standard deviation and 95% con dence intervals. Represents a statistically signi cant difference (PP P PPPP)amonggroupsCTandACT. data, all results were analyzed by the ANOVA test followed by Tukey s multiple comparison test. Signi cance was de ned at PP P PPPP. 3. Results 3.1. Ultrasonographic Features of the Study Group. Table 1 shows the most important ultrasonographic features from ACT in comparison with control group. Our data demonstrated that ACT had signi cant increase in the measurement (in mm) of the longitudinal right lobe of liver (ACT: ± 11.5; CT: 134.3±1.5), anteroposterior le lobe of liver (ACT: 53.7 ± 7.6; CT: 42.7 ± 4.4), the anteroposterior right lobe of liver (ACT: 84.3±9.6; CT: 7.3±1.6), size of longitudinal and the anteroposterior spleen (ACT: ± 15.6; CT: 88.1 ± 7. and ACT: 46.6 ± 8.5; CT: 33.7 ± 3.9, resp.), dimension of Hilar portal vein wall (ACT: 2. ±.1; CT: 1.5 ±.3, resp.) as compared with the CT group. No signi cant differences were found according to the sex categories Hematological Parameters during Acute Schistosomiasis mansoni Infection before and a er Treatment with Praziquantel. e results showed an increase in the values of total leukocytes from ACT and ACT-TR as compared with NI. A differential analysis of leukocyte subsets provides a more detailed description of the speci c differences across cell types. e data showed increased counts of eosinophils and monocytes in the ACT and ACT-TR groups as compared with NI. Moreover, increased values were detected for lymphocytes from ACT-TR as compared with NI and ACT. Interestingly, a er speci c therapy, count of neutrophils in ACT-TR was decreased in comparison with NI (Figure 1) Cytokine Producing T Lymphocytes following In Vitro SEA Stimulation. Aiming to evaluate the impact of SEA stimulation in triggering proin ammatory and regulatory cytokines by T lymphocytes and the effect of the speci c therapy for this pro le, we have performed a detailed single cell ow cytometric analysis to uantify the fre uency of cytokine + cells for IFN-γγ, TNF-αα, IL-4, IL-5, and IL-1 a er in vitro short-term incubation of whole blood samples focusing on T lymphocytes and their subsets (CD4 + or CD8 + T cells). Regardless of the addition of SEA, the data revealed increased synthesis for IFN-γγ and IL-4 by T cells in ACT as compared with NI (Figures 2(a) and 2(c), resp.). A er SEA 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 cultures detected increased synthesis of TNF-αα in ACT (Figure 2(b)). No signi cant differences were found according to the sex categories Cytokine Producing T CD4 + Lymphocytes following In Vitro 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 compared with NI (Figure 3(c)). Furthermore, a er SEA stimulation, we observed increased synthesis of TNF-αα by CD4 + T lymphocytes in ACT as compared with the control culture (Figure 3(b)) Cytokine Producing T CD8 + Lymphocytes following In Vitro SEA Stimulation. e analysis of the cytokine pro le in the CD8 + T subset revealed a higher number of differences than in the CD4 + T cells. Regardless of SEA stimulation, increased synthesis of IFN-γγ and IL-4 by CD8 + T lymphocytes was observed in ACT as compared with NI (Figures 4(a) and 4(c), resp.). Moreover, in cultures stimulated with SEA, increased synthesis of IL-4 was detected in ACT-TR as well as increased synthesis of IL-1 in ACT as compared with NI (Figures 4(c) and 4(e), resp.). A er SEA stimulation, data analysis also showed increased synthesis of IFN-γγ or IL-5 by CD8 + T lymphocytes as compared with the control culture (Figures 4(a) and 4(d), resp.). When the impact of SEA addition in the cultures from ACT-TR patient was investigated, increased synthesis of IFN-γγ or IL-4 by CD8 + T lymphocytes were observed as compared with the control cultures (Figures 4(a) and 4(c), resp.).

13 Journal of Parasitology Research 5 Number of leukocytes subpopulations (mm 3 ) 2 1 Total leukocytes =.21 =.12 Number of leukocytes subpopulations (mm 3 ) Eosinophils <.1 =.2 NI ACT ACT-TR NI ACT ACT-TR (a) (b) Number of leukocytes subpopulations (mm 3 ) 7 35 Neutrophils =.14 Number of leukocytes subpopulations (mm 3 ) 6 3 Lymphocytes =.68 =.112 NI ACT ACT-TR NI ACT ACT-TR (c) (d) 15 Monocytes Number of leukocytes subpopulations (mm 3 ) 75 =.49 =.29 NI ACT ACT-TR (e) FIGURE 1: Analysis of hematological pro le from patients with acute Schistosomiasis mansoni before (ACT, faint grey square = 21) and a er praziquantel treatment (ACT-TR, grey square = 7) and noninfected individuals (NI, white square = 19). e results are shown in bar-plot format highlighting the mean counts of datasets/mm 3 ± standard deviation. tatistically signi cant differences (connecting lines) observed between groups NI, ACT, and ACT-TR were considered at PP P PPPP. 4. Discussion Acute schistosomiasis is a severe disease and the mechanism involved in its clinical manifestations is not completely understood. In the present study, we evaluated patients exposed to the same place of contaminated water, 4 6 days a er exposure to cercaria. Different studies concerning this acute disease refer to groups of tourists, shermen, or sailors originally from a nonendemic country who has visited a tropical zone [27 29]. However, as schistosomiasis is a focally distributed disease [29 31], the acute form is also diagnosed in inhabitants from endemic countries who do not live in endemic areas. e main clinical ndings presented in our study such as headache, fever, diarrhea, and weight loss were consistent with those found by others authors [7, 9, 1, 29, 32]. etween two and eight weeks a er a rst contact with natural water infested by Schistosoma cercariae, susceptible infected patients may present a syndrome comprising of a period of 2 to 3 days of fever, diarrhea, toxemia and weakness, weight loss, abdominal pain, cough, myalgia, arthralgia, edema, urticaria, nausea/vomiting, and hepatosplenomegaly [33]. us, the diagnosis poses a challenge to physicians owing to the nonspeci city symptoms as well as the lack of positivity for S. mansoni eggs in feces from acute patients in

14 6 Journal of Parasitology Research Cytokines + total + T lymphocytes (%) Cytokines + total + T lymphocytes (%) IFN- =.5 =.6 NI ACT ACT-TR NI ACT ACT-TR TNF- =.395 NI ACT ACT-TR NI ACT ACT-TR Control culture SEA stimulation Control culture SEA stimulation (a) (b) Cytokines + total + T lymphocytes (%) IL-4 =.119 =.1 <.1 NI ACT ACT-TR NI ACT ACT-TR Cytokines + total + T lymphocytes (%) IL-5 NI ACT ACT-TR NI ACT ACT-TR Control culture SEA stimulation Control culture SEA stimulation (c) (d) Cytokines + total + T lymphocytes (%) IL-1 NI ACT ACT-TR NI ACT ACT-TR Control culture (e) SEA stimulation FIGURE 2: Cytokine pattern of circulating T lymphocytes from patients with acute Schistosomiasis mansoni before (ACT, faint grey square = 21) and a er praziquantel treatment (ACT-TR, grey square = 7) and noninfected individuals (NI, white square = 19), following short-term in vitro cultivation in the absence (control culture) or presence of SEA stimulation. e results are shown in bar-plot format highlighting the mean 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.

15 Journal of Parasitology Research 7 Cytokines + CD4 + T lymphocytes (%) Cytokines + CD4 + T lymphocytes (%) IFN- NI ACT ACT-TR NI ACT ACT-TR TNF- =.252 NI ACT ACT-TR NI ACT ACT-TR Control culture SEA stimulation Control culture SEA stimulation (a) (b) Cytokines + CD4 + T lymphocytes (%) IL-4 =.169 =.2 NI ACT ACT-TR NI ACT ACT-TR Cytokines + CD4 + T lymphocytes (%) IL-5 NI ACT ACT-TR NI ACT ACT-TR Control culture SEA stimulation Control culture SEA stimulation (c) (d) Cytokines + CD4 + T lymphocytes (%) IL-1 NI ACT ACT-TR NI ACT ACT-TR Control culture (e) SEA stimulation FIGURE 3: Cytokine pattern of circulating T CD4 + -lymphocytes from patients with acute Schistosomiasis mansoni before (ACT, faint grey square = 21) and a er praziquantel treatment (ACT-TR, grey square = 7) and noninfected individuals (NI, white square = 19), following short-term in vitro cultivation in the absence (control culture) or presence of SEA stimulation. e results are shown in bar-plot format highlighting the mean 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.

16 8 Journal of Parasitology Research Cytokines + CD8 + T lymphocytes (%) Cytokines + CD8 + T lymphocytes (%) IFN- =.381 =.258 =.112 <.1 NI ACT ACT-TR NI ACT ACT-TR TNF- NI ACT ACT-TR NI ACT ACT-TR Control culture SEA stimulation Control culture SEA stimulation (a) (b) Cytokines + CD8 + T lymphocytes (%) IL-4 =.8 =.173 <.1 <.1 NI ACT ACT-TR NI ACT ACT-TR Cytokines + CD8 + T lymphocytes (%) IL-5 =.299 NI ACT ACT-TR NI ACT ACT-TR Control culture SEA stimulation Control culture SEA stimulation (c) (d) Cytokines + CD8 + T lymphocytes (%) IL-1 =.156 NI ACT ACT-TR NI ACT ACT-TR Control culture (e) SEA stimulation FIGURE 4: Cytokine pattern of circulating T CD8 + -lymphocytes from patients with acute Schistosomiasis mansoni before (ACT, faint grey square = 21) and a er praziquantel treatment (ACT-TR, grey square = 7) and noninfected individuals (NI, white square = 19), following short-term in vitro cultivation in the absence (control culture) or presence of SEA stimulation. e results are shown in bar-plot format highlighting the mean 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.

17 Journal of Parasitology Research 9 the earlier stage of the infection. In this context, abdominal ultrasound imaging can be a complementary tool to assist the diagnosis of S. mansoni infection and its clinical monitoring. e ultrasonographic ndings presented in this study were consistent with those described by Barata et al. [34] and Costa-Silva [32], which showed nonspeci c increase in the size of liver and spleen (Table 1). Costa-Silva [32] showed that the ACT group had increased measurement of longitudinal le /right lobe of liver, size of longitudinal spleen as well as dimension of Hilar portal vein wall, and incipient periportal echogenic thickening, namely, grade I brosis. us, the data presented here suggest that alterations identi ed by analyses of ultrasonographic features, although not speci c, are compatible with the acute phase of Schistosomiasis mansoni and provide a reliable complementary tool for the diagnosis and clinical followup of the disease. Analyses of hematological parameters showed that ACT and ACT-TR groups presented increased values for total leukocytes, eosinophils, or monocytes. On the other hand, increased values for lymphocytes in addition to decreased values for neutrophils were observed only in the ACT-TR group (Figure 1). ese results suggest the existence of a systemic activation of various hematopoietic lineages, antigeninduced secretion of parasite eggs [35 37]. Smithers et al. [38] demonstrated in experimental models that the cellular in ltrate observed in hepatic granulomas is predominantly formed by eosinophils and monocytes. Different studies showed that acute schistosomiasis patients have increased levels of circulating eosinophils [26, 32, 39]. Previous studies have also demonstrated that eosinophilia occurs between the 5th and 7th weeks a er exposure to the parasite and may be induced by 2 cytokines, such as IL-4, IL-1, IL-13, IL- 9, and especially IL-5 [4]. Increased count of circulating monocytes was also observed by different studies [26, 41]. Moreover, Borojevic et al. [41] showed a delayed differentiation of bone marrow neutrophil granulocytes and blood monocytosis of S. mansoni-infected animals. ese changes are compatible with modi cations in the differentiation of bone marrow myeloid precursors, favoring the production of a monomacrophage cell lineage. Indeed, some previous studies show that during acute Schistosomiasis mansoni infection a higher percentage of neutrophil apoptosis is found, compared to patients with chronic illness or the control subjects [42]. Also, a er the praziquantel treatment, we have a higher exposure of cryptic antigens which could be contributing to increase lymphocyte rather than neutrophil counts. Another important point to be addressed is that, a er treatment, we have lesser levels of in ammatory cytokines such as IL-1-beta and TNF-αα, important inducers of neutrophil mobilization to the peripheral blood during the acute phase of the disease. Although our results do not show increased values for lymphocytes in the ACT group, in contrast with data obtained by Costa-Silva [32], a special interest in this study was the analysis of cytokines producing T lymphocytes, considering the importance of the speci c immune response to solve the infection. Analyses of proin ammatory cytokines (IFN-γγ and TNF-αα) and regulatory cytokines (IL-4, IL-5, and IL-1) demonstrated higher percentage of TNF-αα + T CD4 + lymphocytes in the ACT group a er SEA stimulation in comparison with the control culture. Furthermore, in the control cultures, the results showed increased percentage of IL-4 + T CD4 + lymphocytes in both ACT and ACT-TR group in comparison with NI (Figure 3). e 1-2 model of CD4 + T-cell differentiation is a well-established paradigm for understanding the basis of protective versus pathogenic immune responses for a host of important human pathogens [43]. 1 cells secrete IFN- γγ and IL-2 and promote cell-mediated immunity, while 2 cells secrete IL-4, IL-5, and IL-1, among others. In this context, an important concept that evolved from the study of 1 and 2 cell responses is the fact that both responses cross regulate each other. us, IFN-γγ downregulates 2 cell development, while IL-4 and IL-1 antagonize 1 cell differentiation [44]. In humans with acute schistosomiasis, higher levels of IFN-γγ and lower levels of IL-5 are found in supernatants from PBMC compared to patients with chronic disease [19]. However, the results by de Jesus et al. [9] showed only high production of the proin ammatory cytokines IL- 1, IL-6, and TNF-αα in cultures of unstimulated PBMC from patients with acute schistosomiasis. us, our results suggest a mixed cytokine pro le produced by CD4 + T cells. is cytokine milieu might be preventing tissue damage. In addition, development of a severe in ammatory disease during acute schistosomiasis has been documented in knockout mice for IL-4 and IL-1 genes [45, 46]. Interestingly, our results demonstrated predominant increased percentage of IFN-γγ + and IL-4 + T CD8 + lymphocytes, following shortterm incubation of whole blood with SEA in both ACT and ACT-TR groups and increase of IL-1 + cells only in the ACT group (Figure 4). is study is the rst one to point out the relevance of CD8 + T lymphocytes in the immune response of acute phase of schistosomiasis. CD8 + T cells have been implicated in several immunopathological events during helminthic infection including schistosomiasis [47]. e presence of CD8 + T cells was observed in both early and chronic murine granulomas with an increased ratio of CD8 + cells during the chronic phase of the disease [48]. Studies developed by Pancré et al. [49] in murine model demonstrated that SEA was able to stimulate IFN-γγ or IL- 2 producing CD8 + T cells, suggesting a type 1 response induced by SEA. In humans, Oliveira-Prado et al. [5] showed a smaller percent of CD4 + and a higher percent of CD8 + cells in peripheral blood from patients with chronic schistosomiasis. Moreover, our group has previously described the putative role of CD8 + T subsets in controlling morbidity during human schistosomiasis, which was evidenced by the increased levels of activated HLA-DR + CD8 + T lymphocytes in patients presenting intestinal clinical form (INT) of the disease and low levels of CD28 + CD8 + cells in hepatosplenic patients [51 53]. In addition, Teixeira-Carvalho et al. [54] observed increased percentage of IL-4 +, IL-5 +, and IL-1 + CD8 + lymphocytes following short-term SEA stimulation of whole blood samples in vitro in the INT group. Although the recruitment of CD8 + T cells by exogenous antigens (such as those derived from S. mansoni) primarily seems to be unexpected, recent studies have demonstrated that CD8 + T cells are prone to respond to extracellular antigens in infectious diseases [55, 56] via bystander activation triggered

18 1 Journal of Parasitology Research by persistent antigenic stimulation, cytokines milieu [52, 57, 58], or interaction with antigen-presenting cells (APC) that acquire exogenous antigens by phagocytosis and present them throughout MHC class I molecules [59 62]. Similarly, data of Montenegro et al. [19] showedthatpbmcfrom acute schistosomiasis patients responded to SEA and SWAP by producing signi cantly higher amounts of IFN-γγ and IL- 1. A study carried out by de Jesus et al. [9] demonstrated the occurrence of higher levels of IFN-γγ production in patients with acute schistosomiasis than those who have chronic infection. However, fewer patients with acute disease produced IL-1 in response to SEA. ese authors also showed a positive correlation between IL-1 levels in SEAstimulated PBMC and time a er water exposure. On the other hand, Falcão et al. [63] showed that hepatosplenic schistosomiasis patients produced high levels of IFN-γγ and low levels of IL-1 as compared to INT, associating this latter cytokine with the establishment/maintenance of the severe clinical forms. Interestingly, studies by Pedras-Vasconcelos and Pearce [64] demonstrated that mice infected with schistosomes present a regulatory pathway in which type 1 CD8 + cells, under the control of IL-4, dampen immunopathologic type 2 responses. Corrêa-Oliveira et al. [13] showed that the blockage of IL-4 and IL-5 using anti-il-4 and anti-il- 5 antibodies signi cantly reduced the PBMC proliferative response to SEA antigens in acute, chronic intestinal, and hepatosplenic patients. Another interesting data found was the high IL-1 producing cells in response to SEA in uninfected individuals. Like SEA is a complex mix of proteins, glycoproteins, and polysaccharides, this composition inducing could be inducing cross-reactivity sharing some epitopes with other antigens that previously sensitized the uninfected individuals. Studies show that praziquantel is an effective drug in controlling infection with S. mansoni, promoting a reduction in egg excretion and regression of brosis in mice and humans [65 7]. We observed that one month a er speci c chemotherapy with praziquantel, the patients in this study showed similar changes in the number of eosinophils and monocytes, but had a speci c decrease in the number of both neutrophils and lymphocytes as compared with NI and ACT or only NI, respectively (Figure 1). Some authors have demonstrated that chemotherapy-induced immune responses are heterogeneous, depending on the type of antigen used and time of analysis a er treatment [7, 71]. In this context, we observed a higher number of IFN-γγ + and IL-4 + T CD8 + lymphocytes in response to SEA in the ACT- TR group (Figure 4). De Souza et al. [72] demonstrated six months a er speci c treatment with oxamniquine that the IFN-γγ levels increased signi cantly in response to SEA. Similarly, it was reported that patients with chronic disease living in endemic areas showed increased levels of IFN-γγ a er treatment in response to parasite antigens [73]. Probably, the increase in T-cell reactivity a er chemotherapy can be explained by exposure of released antigens to the immune system 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 acute schistosomiasis a er treatment with oxamniquine. In conclusion, the data showed that a mixed cytokine pro le is present during the acute phase of schistosomiasis mansoni. One month a er treatment with praziquantel there is a increase in the production of IL-4 and IL-1 cytokines following SEA stimulation in circulating lymphocytes from infected patients. is nding is associated with a reduction in their morbidity. Acknowledgments eauthorswouldliketothankthetechnicalstaffofthe Laboratório de Imunologia Celular e Molecular, Fundação Oswaldo Cruz and Núcleo de Pesquisa em Imunologia, UNI- VALE, Brazil, for their invaluable assistance during this study. eauthorsalsowishtothanktheprogramfortechnological Development in Public Health PDTIS FIOCRUZ for use of its facilities. is study was supported by FIOCRUZ, Conselho Nacional de Desenvolvimento Cient co e Tecnológico (CNPq), the Fundação de Amparo Pesquisa de Minas Gerais (FAPEMIG), the National Institutes of Health NIH-ICIDR/Grant AI , and the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases. O. A. Martins-Filho, A. Teixeira-Carvalho and R. Corrêa-Oliveira are thankful to CNPq for the fellowships received (PQ). ATC is thankful to the US Food and Drug Administration for the fellowship granted. References [1] WHO, World Health Organization, Geneva, Switzerland, 212, _eng.pdf. [2] B. Gryseels, Schistosomiasis, International Journal of Infectious Diseases, vol. 26, pp , 212. [3] P. Steinmann, J. Keiser, R. Bos, M. Tanner, and J. Utzinger, Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk, Lancet Infectious Diseases, vol. 6, no. 7, pp , 26. [4] J. R. Lambertucci, Acute schistosomiasis mansoni: revisited and reconsidered, Memorias Do Instituto Oswaldo Cruz, vol. 15, no. 4, pp , 21. [5] N. Katz, Schistosomiasis control in Brazil, Memorias Do Instituto Oswaldo Cruz, vol. 93, pp , [6] R. S. Do Amaral, P. L. Tauil, D. D. Lima, and D. Engels, An analysis of the impact of the Schistosomiasis Control Programme in Brazil, Memorias Do Instituto Oswaldo Cruz, vol. 11, no. 1, pp , 26. [7] C. S. Barbosa, S. M. L. Montenegro, F. G. C. Abath, and A. L. Coutinho Domingues, Speci c situations related to acute schistosomiasis in Pernambuco, Brazil, Memorias Do Instituto Oswaldo Cruz, vol. 96, pp , 21. [8] C. S. Barbosa, A. L. Domingues, F. Abath et al., An outbreak of acute schistosomiasis at Porto de Galinhas beach, Pernambuco, Brazil, Cadernos De Saude Publica, vol. 17, no. 3, pp , 21. [9] A. R. De Jesus, A. Silva, L. B. Santana et al., Clinical and immunologic evaluation of 31 patients with acute schistosomiasis mansoni, Journal of Infectious Diseases, vol. 185, no. 1, pp , 22.

19 Journal of Parasitology Research 11 [1] M. J. Enk, R. L. Caldeira, O. S. Carvalho, and V. T. Schall, Rural tourism as risk factor for the transmission of schistosomiasis in Minas Gerais, Brazil, Memorias Do Instituto Oswaldo Cruz, vol. 99, no. 5, pp , 24. [11] R. Veronsi and R. Focaccia, Tratado De Infectologia, Atheneu, Rio de Janeiro, Brazil, 2 edition, 22. [12] L. Q. Nguyen, J. Estrella, E. A. Jett, E. L. Grunvald, L. Nicholson, and D. L. Levin, Acute schistosomiasis in nonimmune travelers: Chest CT ndings in 1 patients, American Journal of Roentgenology, vol. 186, no. 5, pp , 26. [13] R. Corrêa-Oliveira, L. C. C. Malaquias, P. L. Falcão et al., Cytokines as determinants of resistance and pathology in human Schistosoma mansoni infection, Brazilian Journal of Medical and Biological Research, vol. 31, no. 1, pp , [14] S. A. Rezende, J. R. Lambertucci, and A. M. Goes, Role of immune complexes from pacients with different clinical forms of schistosomiasis in the modulation of in vitro granuloma reaction, Memorias Do Instituto Oswaldo Cruz, vol. 92, no. 5, pp , [15] X. Liu, T. Kohyama, H. Wang et al., 2 cytokine regulation of type I collagen gel contraction mediated by human lung mesenchymal cells, American Journal of Physiology, vol. 282, no. 5, pp. L149 L156, 22. [16] R. M. Reiman, R. W. ompson, C. G. Feng et al., Interleukin- 5 (IL-5) augments the progression of liver brosis by regulating IL-13 activity, Infection and Immunity, vol. 74, no. 3, pp , 26. [17] T. R. Mosmann and K. W. Moore, e role of IL-1 in crossregulation of TH1 and TH2 responses, Immunology Today, vol. 12, no. 3, pp. A49 A53, [18] M. Booth, J. K. Mwatha, S. Joseph et al., Periportal brosis in human Schistosoma mansoni infection is associated with low IL-1, Low IFN-γγ, high TNF-αα, or low RANTES, depending on age and gender, Journal of Immunology, vol. 172, no. 2, pp , 24. [19] S. M. L. Montenegro, P. Miranda, S. Mahanty et al., Cytokine production in acute versus chronic human schistosomiasis mansoni: the cross-regulatory role of interferon-γγ and interleukin-1 in the responses of peripheral blood mononuclear cells and splenocytes to parasite antigens, Journal of Infectious Diseases, vol. 179, no. 6, pp , [2] N. Katz, A. Chaves, and J. Pellegrino, A simple device for quantitative stool thick-smear technique in schistosomiasis mansoni, Revista Do Instituto de Medicina Tropical de Sao Paulo, vol. 14, no. 6, pp , [21] J. G. Hardman, L. E. Limbird, and A. G. Gilman, Eds., Goodman & Gilman s. e Pharmacological Basis of erapeutics, vol. 245, McGraw-Hill, New York, NY, USA, 1th edition, 21. [22] M. F. Abdel-Wahab, G. Esmat, A. Farrag, Y. A. El-Boraey, and G. T. Strickland, Grading of hepatic schistosomiasis by the use of ultrasonography, American Journal of Tropical Medicine and Hygiene, vol. 46, no. 4, pp , [23] M. Homeida, A. F. Abdel-Gadir, A. W. Cheever et al., Diagnosis of pathologically con rmed Symmers periportal brosis by ultrasonography: a prospective blinded study, American JournalofTropicalMedicineandHygiene, vol. 38, no. 1, pp , [24] G. G. Cerri, V. A. Alves, and A. Magalhaes, Hepatosplenic schistosomiasis mansoni: ultrasound manifestations, Radiology, vol. 153, no. 3, pp , [25] G. Gazzinelli, N. Katz, R. S. Rocha, and D. G. Colley, Immune responses during human schistosomiasis mansoni. X. Production and standardization of an antigen-induced mitogenic activity by peripheral blood mononuclear cells from treated, but not active cases of schistosomiasis, Journal of Immunology, vol. 13, no. 6, pp , [26] D. Silveira-Lemos, A. Teixeira-Carvalho, A. O. Martins-Filho et al., Eosinophil activation status, cytokines and liver brosis in Schistosoma mansoni infected patients, Acta Tropica, vol. 18, no. 2-3, pp , 28. [27] M. N. Lunde and E. A. Ottesen, Enzyme-linked immunosorbent assay (ELISA) for detecting IgM and IgE antibodies in human schistosomiasis, American Journal of Tropical Medicine and Hygiene, vol. 29, no. 1, pp , 198. [28] B. Evengard, L. Hammarstrom, C. I. E. Smith, and E. Linder, Early antibody responses in human schistosomiasis, Clinical and Experimental Immunology, vol. 8, no. 1, pp , 199. [29] A. Rabello, Acute human schistosomiasis mansoni, Memórias Do Instituto Oswaldo Cruz, vol. 9, no. 2, pp , [3] S. B. Pessoa and J. P. Amorim, Notas sobre a esquistossomose mansônica em algumas localidades de Alagoas, Revista Brasileira De Medicina, vol. 14, pp , [31] K. Kloetzel, Schistosomiasis in Brazil: does social development suffice? Parasitology Today, vol. 5, no. 12, pp , [32] M. F. Costa-Silva, Acompanhamento clínico, epidemiológico e imunológico de pacientes portadores da fase aguda da esquistossomose mansoni, su metidos terap utica especí ca com Praziquantel [Dissertação (Mestrado em Doenças Infecciosas e Parasitárias)], Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Programa de Pós-graduação em Ciências da Saúde, Belo Horizonte, Brazil, 29, ocruz.br/texto-completo/d_5.pdf. [33] J. R. Coura and R. S. Amaral, Epidemiological and control aspects of schistosomiasis in Brazilian endemic areas, Memorias Do Instituto Oswaldo Cruz, vol. 99, no. 5, pp , 24. [34] C. H. Barata, R. A. Pinto-Silva, and J. R. Lambertucci, Abdominal ultrasound in acute schistosomiasis mansoni, British Journal of Radiology, vol. 72, pp , [35] G. J. Race, R. M. Michaels, J. H. Martin, J. E. Larsh Jr., and J. L. Matthews, Schistosoma mansoni eggs: an electron microscopic study of shell pores and microbarbs, Proceedings of the Society for Experimental Biology and Medicine, vol. 13, no. 3, pp , [36] A. Sher, R. L. Coffman, S. Hieny, P. Scott, and A. W. Cheever, Interleukin 5 is required for the blood and tissue eosinophilia but not granuloma formation induced by infection with Schistosoma mansoni, Proceedings of the National Academy of Sciences of the United States of America, vol. 87, no. 1, pp , 199. [37] H. L. Lenzi, R. G. Pacheco, M. Pelajo-Machado, M. S. Panasco, W. S. Romanha, and J. A. Lenzi, Immunological system and Schistosoma mansoni: co-evolutionary immunobiology. what is the eosinophil role in parasite-host relationship? Memorias Do Instituto Oswaldo Cruz, vol. 92, pp , [38] S. R. Smithers, D. J. McLaren, and F. J. Ramalho-Pinto, Immunity to schistosomes: the target, e American Journal of Tropical Medicine and Hygiene, vol. 26, no. 6, pp , [39] E. Caumes and M. Vidailhet, Acute neuroschistosomiasis: a cerebral vasculitis to treat with corticosteroids not praziquantel, Journal of Travel Medicine, vol. 17, no. 5, pp , 21. [4] D. C. Cara, D. Negrao-Correa, and M. M. Teixeira, Mechanisms underlying eosinophil trafficking and their relevance

20 12 Journal of Parasitology Research in vivo, Histology and Histopathology, vol. 15, no. 3, pp , 2. [41] R. Borojevic, C. G. Pinto, M. C. el-cheikh, and H. S. Dutra, Experimental murine schistosomiasis mansoni: hyperplasia of the mono-macrophage cell lineage and stimulation of myeloid proliferation by peripheral macrophages, Brazilian Journal of Medical and Biological Research, vol. 22, no. 5, pp , [42] I. M. el-sharkawy, A. A. Ibrahim, and M. A. el-bassuoni, Neutrophil apoptosis in acute and chronic Schistosoma mansoni infection, Journal of the Egyptian Society of Parasitology, vol. 32, no. 2, pp , 22. [43] T. R. Mosmann, L. Li, H. Hengartner, D. Kagi, W. Fu, and S. Sad, Differentiation and functions of T cell subsets, CIBA Foundation Symposia, vol. 24, pp , [44] T. R. Mosmann and S. Sad, e expanding universe of T-cell subsets: 1, 2 and more, Immunology Today,vol.17,no.3, pp , [45] L. R. Brunet, F. D. Finkelman, A. W. Cheever, M. A. Kopf, and E. J. Pearce, IL-4 protects against TNF-αα-mediated cachexia and death during acute schistosomiasis, Journal of Immunology, vol. 159, no. 2, pp , [46] P. G. Fallon, E. J. Richardson, P. Smith, and D. W. Dunne, Elevated type 1, diminished type 2 cytokines and impaired antibody response are associated with hepatotoxicity and mortalities during Schistosoma mansoni infection of CD4-depleted mice, European Journal of Immunology, vol. 3, no. 2, pp , 2. [47] P. Raso, Esquistossomose mansônica, in Patologia, L. Bogliolo, Ed., pp , Guanabara Koogan, Rio de Janeiro, Brazil, 4th edition, [48] P. J. Perrin and S. M. Phillips, e molecular basis of granuloma formation in schistosomiasis. I. A T cell-derived suppressor effector factor, Journal of Immunology, vol. 141, no. 5, pp , [49] V. Pancré, M. Delacre, J. Herno, and C. Auriault, Schistosomal egg antigen-responsive CD8 T-cell population in Schistosoma mansoni-infected BALB/c mice, Immunology, vol. 98, no. 4, pp , [5] R. Oliveira-Prado, I. R. Caldas, A. Teixeira-Carvalho et al., CD4 + and CD8 + distribution pro le in individuals infected by Schistosoma mansoni, Scandinavian Journal of Immunology, vol. 69, no. 6, pp , 29. [51] O. A. Martins-Filho, J. R. Mello, and R. Correa-Oliveira, e spleen is an important site of T cell activation during human hepatosplenic schistosomiasis, Memorias Do Instituto Oswaldo Cruz, vol. 93, supplement 1, pp , [52] O. A. Martins-Filho, J. R. Cunha-Melo, J. R. Lambertucci et al., Clinical forms of human Schistosoma mansoni infection are associated with differential activation of T-cell subsets and costimulatory molecules, Digestive Diseases and Sciences, vol. 44, no. 3, pp , [53] A. Teixeira-Carvalho, O. A. Martins-Filho, Z. A. Andrade, J. R. Cunha-Mello, R. A. Wilson, and R. Corrêa-Oliveira, e study of T-cell activation in peripheral blood and spleen of hepatosplenic patients suggests an exchange of cells between these two compartments in advanced human schistosomiasis mansoni infection, Scandinavian Journal of Immunology, vol. 56, no. 3, pp , 22. [54] A. Teixeira-Carvalho, O. A. Martins-Filho, V. Peruhype- Magalhães et al., Cytokines, chemokine receptors, CD4 + CD25 HIGH+ T-cells and clinical forms of human schistosomiasis, Acta Tropica, vol. 18, no. 2-3, pp , 28. [55] J. M. M. Den Haan and M. J. Bevan, Antigen presentation to CD8 + T cells: cross-priming in infectious diseases, Current Opinion in Immunology, vol. 13, no. 4, pp , 21. [56] B. J. Manfras, S. Reuter, T. Wendland, and P. Kern, Increased activation and oligoclonality of peripheral CD8 + T cells in the chronic human helminth infection alveolar echinococcosis, Infection and Immunity, vol. 7, no. 3, pp , 22. [57] A. Kalinkovich, Z. Weisman, Z. Greenberg et al., Decreased CD4 and increased CD8 counts with T cell activation is associated with chronic helminth infection, Clinical and Experimental Immunology, vol. 114, no. 3, pp , [58] G. Borkow, Q. Leng, Z. Weisman et al., Chronic immune activation associated with intestinal helminth infections results in impaired signal transduction and anergy, Journal of Clinical Investigation, vol. 16, no. 8, pp , 2. [59] E. P. Grant and K. L. Rock, MHC class I-restricted presentation of exogenous antigen by thymic antigen-presenting cells in vitro and in vivo, Journal of Immunology, vol. 148, no. 1, pp , [6] K. L. Rock, S. Gamble, L. Rothstein, C. Gramm, and B. Benacerraf, Dissociation of ββ 2 -microglobulin leads to the accumulation of a substantial pool of inactive class I MHC heavy chains on the cell surface, Cell, vol. 65, no. 4, pp , [61] K. L. Rock, C. Fleischacker, and S. Gamble, Peptide-priming of cytolytic T cell immunity in vivo using ββ 2 - microglobulin as an adjuvant, Journal of Immunology, vol. 15, no. 4, pp , [62] M. Larsson, J. F. Fonteneau, and N. Bhardwaj, Dendritic cells resurrect antigens fromdead cells, Trends in Immunology, vol. 22, no. 3, pp , 21. [63] P. L. Falcão, L. C. Malaquias, O. A. Martins-Filho et al., Human schistosomiasis mansoni: IL-1 modulates the in vitro granuloma formation, Parasite Immunology, vol. 2, no. 1, pp , [64] J. A. Pedras-Vasconcelos and E. J. Pearce, Type 1 CD8 + T Cell responses during infection with the helminth Schistosoma mansoni, Journal of Immunology, vol. 157, no. 7, pp , [65] E. Doehring-Schwerdtfeger, I. M. Abdel-Rahim, R. Kardorff et al., Ultrasonographical investigation of periportal brosis in children with Schistosoma mansoni infection: reversibility of morbidity twenty-three months a er treatment with praziquantel, American Journal of Tropical Medicine and Hygiene, vol. 46, no. 4, pp , [66] G. F. Cota, R. A. Pinto-Silva, C. M. Antunes, and J. R. Lambertucci, Ultrasound and clinical investigation of hepatosplenic schistosomiasis: evaluation of splenomegaly and liver brosis four years a er mass chemotherapy with oxamniquine, American Journal of Tropical Medicine and Hygiene,vol.74,no.1,pp , 26. [67] Z. A. Andrade, A. P. Baptista, and T. S. Santana, Remodeling of hepatic vascular changes a er speci c chemotherapy of schistosomal periportal brosis, Memorias Do Instituto Oswaldo Cruz, vol. 11, no. 1, pp , 26. [68] J. H. Kihara, N. Muhoho, D. Njomo et al., Drug efficacy of praziquantel and albendazole in school children in mwea division, central province, kenya, Acta Tropica, vol. 12, no. 3, pp , 27.

21 Journal of Parasitology Research 13 [69] P. Martins-Leite, G. Gazzinelli, L. F. Alves-Oliveira et al., Effect of chemotherapy with praziquantel on the production of cytokines and morbidity associated with schistosomiasis mansoni, Antimicrobial Agents and Chemotherapy, vol. 52, no. 8, pp , 28. [7] C. M. Fitzsimmons, S. Joseph, F. M. Jones et al., Chemotherapy for schistosomiasis in ugandan shermen treatment can cause a rapid increase in interleukin-5 levels in plasma but decreased levels of eosinophilia and worm-speci c immunoglobulin E, Infection and Immunity, vol. 72, no. 7, pp , 24. [71] S. Joseph, F. M. Jones, K. Walter et al., Increases in human T helper 2 cytokine responses to Schistosoma mansoni worm and worm-tegument antigens are induced by treatment with praziquantel, Journal of Infectious Diseases, vol.19,no.4,pp , 24. [72] J. R. De Souza, C. N. Morais, M. L. Aroucha et al., Treatment of human acute schistosomiasis with oxamniquine induces an increase in interferon-γγ response to Schistosoma mansoni antigens, Memorias Do Instituto Oswaldo Cruz, vol. 12, no. 2, pp , 27. [73] R. Corrêa-Oliveira, I. Rodrigues Caldas, O. A. Martins-Filho et al., Analysis of the effects of treatment of human Schistosoma mansoni infection on the immune response of patients from endemic areas, Acta Tropica, vol. 77, no. 1, pp , 2. [74] M. I. Araújo, A. R. De Jesus, O. Bacellar, E. Sabin, E. Pearce, and E. M. Carvalho, Evidence of a T helper type 2 activation in human schistosomiasis, European Journal of Immunology, vol. 26, no. 6, pp , [75] J. L. Grogan, P. G. Kremsner, A. M. Deelder, and M. Yazdanbakhsh, Elevated proliferation and interleukin-4 release from CD4 + cells a er chemotherapy in human schistosoma haematobium infection, European Journal of Immunology, vol. 26, no. 6, pp , [76] C. F. Brito, I. R. Caldas, P. Coura Filho, R. Corrêa-Oliveira, and S. C. Oliveira, CD4 + T cells of schistosomiasis naturally resistant individuals living in an endemic area produce interferon-γγ and tumour necrosis factor-αα in response to the recombinant 14KDA Schistosoma mansoni fatty acid-binding protein, Scandinavian Journal of Immunology, vol. 51, no. 6, pp , 2.

22 Hindawi Publishing Corporation Journal of Parasitology Research Volume 212, Article ID , 1 pages doi:1.1155/212/ Research Article Cytokine and Chemokine Profile in Individuals with Different Degrees of Periportal Fibrosis due to Schistosoma mansoni Infection Robson Da Paixão De Souza, 1 Luciana Santos Cardoso, 1, 2, 3 Giuseppe Tittoni Varela Lopes, 1 Maria Cecília F. Almeida, 1 Ricardo Riccio Oliveira, 1, 3, 4 Leda Maria Alcântara, 1 Edgar M. Carvalho, 1, 3, 4, 5 and Maria Ilma Araujo1, 3, 4, 5 1 Serviço de Imunologia, Complexo Hospitalar Universitário Professor Edgard Santos, Universidade Federal da Bahia, Rua João das Botas s/n, Canela, Salvador, BA, Brazil 2 Departamento de Ciências da Vida, Universidade do Estado da Bahia, 2555 Rua Silveira Martins, Cabula, Salvador, BA, Brazil 3 Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais (INCT-DT-CNPQ/MCT), Rua João das Botas s/n, Canela, Salvador, BA, Brazil 4 Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, UFBA, Avenida Adhemar de Barros s/n, Ondina, Salvador, BA, Brazil 5 Escola Bahiana de Medicina e Saúde Pública, Rua Frei Henrique No. 8, Nazaré, Salvador, BA, Brazil Correspondence should be addressed to Robson Da Paixão De Souza, robson.imuno@gmail.com Received 13 July 212; Accepted 7 November 212 Academic Editor: Sergio Costa Oliveira Copyright 212 Robson Da Paixão De Souza et al. 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. Periportal fibrosis in schistosomiasis has been associated to the host immune response to parasite antigens. We evaluated the immune response in S. mansoni infected individuals with different degrees of periportal fibrosis. Cytokine and chemokines were 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 (31.9 pg/ml) compared to individuals without fibrosis (36.8 pg/ml; P =.418). There was also a higher production of TNF-α in cultures stimulated with SWAP in patients with insipient fibrosis (1446 pg/ml) compared to those without fibrosis (756.1 pg/ml; P =.319). The serum levels of IL-13 and MIP-1α were higher in subjects without fibrosis than in those with moderate to severe fibrosis. However a positive association between serum levels of IL-13, TNF-α, MIP-1α, and RANTES and S. mansoni parasite burden was found. From these data we conclude that IL-5 and TNF-α may participate in liver pathology in schistosomiasis. The positive association between IL-13, TNF-α, MIP-1α, and RANTES with parasite burden, however, might predict the development of liver pathology. 1. Introduction Schistosomiasis is a chronic parasitic infection that affects 2 million people in Africa, South America, and Asia and it is estimated that roughly 7 million people in the world live at risk of infection [1, 2]. In Brazil, the only species described is Schistosoma mansoni, and it is estimated that seven million people are infected by the parasite and about 25 million are at risk of infection [3]. The liver pathology results from the host immune response to parasite antigens from the eggs that become trapped in the portal venous system, and it is associated to the morbidity and mortality described in schistosomiasis [4]. The granuloma formation around S. mansoni eggs is complex and represents an interaction between products secreted by the miracidia, which are released from the egg

23 2 Journal of Parasitology Research and the host immune response [5]. Consequently, the granulomas formed act as barriers that prevent the dispersion of egg antigens of S. mansoni, and the consequent damage to the liver parenchyma. However, when caused by deposition of large numbers of eggs, the inflammatory process may progress to severe fibrosis, which leads to interruption of normal blood flow in the venous system to the sinusoids resulting in portal hypertension, hepatosplenomegaly, and formation of gastric and esophageal varices that can lead to bleeding and even death. This severe form of the disease occurs in five to ten percent of infected subjects living in endemic areas [6 8]. Some studies have evaluated the immune response associated with granuloma formation and development of periportal fibrosis due to schistosomiasis, both in experimental models and in vitro models of granuloma or tissue biopsies [9 11]. Recently described, the cytokine interleukin-17 (IL-17) is considered as an inflammatory mediator and may be associated to the pathology of several chronic illnesses [12]. IL-17 stimulates the production of IL-6, nitric oxide, and prostaglandin E2 (PGE2) and acts in synergy with other cytokines, such as IL-1, TNF-α, and IFN-γ. Furthermore, this cytokine promotes the proliferation and recruitment of monocytes and neutrophils to inflammatory sites. Th17 cells may have been involved in the pathogenesis of schistosomiasis in experimental models [4, 13 15]. There are few studies evaluating the serum levels of cytokines and chemokines in schistosomiasis patients with different degrees of periportal fibrosis and the participation of IL-17 in the formation of granuloma and progression to fibrosis in human schistosomiasis remains unclear. Thus, in this study apart from assessment of Th1, Th2, and T regulatory 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 was carried out in an endemic area from schistosomiasis named Água Preta, in the state of Bahia, Brazil. Água Preta is located 28 km south of Salvador, the capital of the State Bahia. It is composed of a residential area in the center of the village and some surrounding farms. A total of approximately 8 people live in the community. They live in poor sanitary conditions 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 the residents to high risk of Schistosoma infection. A cross-sectional parasitological surveys using Kato-Katz [16] and sedimentation techniques were conducted in three different stool samples collected on different days. The current degree of exposure to S. mansoni infection was assessed by a previously developed questionnaire, which provides four categories of reported level of exposure to infested water: no exposure, low exposure (<1 h/week), medium exposure (1 3 h/week), or high exposure (1 3 h/day) [17 19]. The inclusion criteria for this study were individuals from endemic areas who have at least one positive parasitological exam for S. mansoni. From the 537 individuals who agreed to participate in this study 334 were infected with S. mansoni (62.5%). The frequency of other helminthic infections was 43.4% 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, 22 agreed to perform abdominal ultrasound, in order to determine the degree of periportal fibrosis. They also agreed to donate blood for the study of the immunological response. For this particular aim, we did not include individuals under five or above 6 years old. We also did not include alcoholic individuals and those with positive serology for HIV, HTLV- 1, or hepatitis virus types B and C, all of which are conditions that could interfere with the immunological response Ultrasound Examination. Abdominal ultrasound (USG) were performed using the Quantum 2 Siemens and Elegra Siemens ultrasound with a convex transductor of Mhz. Liver span was measured in the midclavicular line and midline. The liver was also examined for smoothness of surface, echogenicity and posterior attenuation of the sound bean, and portal vein diameter outside the liver midway between its entrance into the portal hepatic and its first bifurcation in the liver. Periportal fibrosis was observed as multiple diffuse echogenic areas. Grading of periportal fibrosis was determined by the mean total thickness of four portal tracts after the first division from the right and left branches of portal vein (PT1) as follow: degree, mean thickness <3mm; degreei, meanthickness 3to5mm; degree II, mean thickness >5 to 7mm; and degree IIImean thickness >7mm[2 22]. Of the 22 individuals evaluated 62 (28.2%) had some degree of periportal fibrosis as shown in Table 1. The scores of periportal fibrosis were grouped according to the severity, being degree without periportal fibrosis. Incipient periportal fibrosis was considered to individuals with degree I and moderate to severe periportal fibrosis to those with degrees II and III [23]. To perform the immune response we included 3 individuals with degree and 25 individuals with degree I, while all individuals with severe forms of the disease characterized by the grade II (n = 13) or III (n = 4) were included Schistosoma mansoni Antigens. The S. mansoni antigens used in this study were the soluble extract of the adult worm of S. mansoni (SWAP) and the soluble egg antigen (SEA), prepared as previously described [24] Cell Culture and Cytokine Measurements. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll- Hypaque gradient sedimentation and adjusted to a concentration of /ml in RPMI 164 medium containing 1% normal human serum (AB positive and heat inactivated), 1 U/mL of penicillin, 1 mg/ml of streptomycin, 2 mmol/l of L-glutamine, and 3 mmol/l of HEPES (all from Life Technologies GIBCO, BRL, Gaithersburg, MS). Cells were cultured without any stimulation or stimulated

24 Journal of Parasitology Research 3 Table 1: Demographic characteristics of the individuals enrolled in the study (n = 22). Characteristic Without fibrosis (n = 158) Incipient fibrosis (n = 45) Moderate to severe fibrosis (n = 17) P Age (median/range) 19 (5 6) 3 (9 58) 4 (21 59) <.1 a,b Gender n (%) Male 83 (52.5) 23 (51.1) 8 (47.1) Female 75 (47.5) 22 (48.9) 9 (52.9) ns Burden parasite for Schistosoma mansoni (EPG/range) 72 ( ) 42 (24 138) 6 (24 2).451 a Coinfection (Schistosoma mansoni and other helminth infections) (%) Yes No ns Kruskal-Wallis; Chi-squared Test; a without fibrosis versus incipient fibrosis; b without fibrosis versus moderate to severe fibrosis. EPG: eggs per gram of feces; ns: not significant (P >.5). with 1 mg/ml of SWAP, SEA, or Phytohemagglutinin (PHA). Plates were incubated at 37 Cinanatmosphere containing 5% CO 2. Supernatants were collected after 72 hours of incubation and maintained at 2 Cformeasurement of cytokines by Enzyme-linked immunosorbent assay (ELISA). Levels of IL-5, IL-13, IL-17, IL-1, IFN-γ, and TNFα were determined (R&D Systems, Inc, Minneapolis, MN), and results were expressed as pg/ml on the basis of standard curves Serum Cytokine and Chemokine Measurement. Levels of the cytokines IL-5, IL-13, IFN-γ, TNF-α, IL-17, IL-1, TGF-β (R&D systems Inc., Minneapolis) and the chemokines MIP- 1α/CCL3 and RANTES/CCL5 were measured in serum using sandwich ELISA (ebioscience). The results are expressed as pg/ml, based in standard curves Statistical Analysis. Statistical analyses were performed using the Statistical Package for the Social Sciences software (version 9. for Windows, SPSS). Statistical differences between groups were assessed using the Kruskal-Wallis analysis of variance test. Fisher s exact test was used to compare proportions. To assess the association between the serum levels of cytokines and chemokines and the parasite burden of S. mansoni the Linear Regression tests using Graphpad PRISM 3.3 software (La Jolla, CA, USA) were used. All statistical tests were two-tailed and the statistical significance was established at the 95 percent confidence interval and significance was defined to P<.5. The Ethical Committee of Climério de Oliveira Maternity, Federal University of Bahia, approved the present study. Informed consent was obtained from all study participants or their legal guardians (License number 24/28). 3. Results 3.1. Features of the Studied Subject. The demographic characteristics, parasite burden, and the ultrasonography evaluation for periportal fibrosis measurement are shown in Table 1. It was observed that the mean age of individuals with incipient and moderate to severe periportal fibrosis was higher than in individuals without fibrosis (P <.5). There was no significant difference in gender distribution among groups. There was also no significant difference in the levels of exposure to the contaminated water, with medium to high contact to the water being found in 6.5%, 55.3%, and 8.% of individuals without periportal fibrosis, incipient fibrosis, and moderate to severe fibrosis, respectively (P >.5). The S. mansoni parasite burden of individuals without periportal fibrosis was higher than in patients with incipient fibrosis (P <.5). There was no statistically significant difference of being coinfected with other helminths in all groups of patients (Table 1) Cytokine Responses Induced by S. mansoni Antigens. Levels of IL-5, IL-13, IL-17, IFN-γ, TNF-α, and IL-1 were measured in supernatant of PBMCs cultures unstimulated and stimulated with SWAP and SEA as shown in Table 2. A significantly higher levels of IL-5 in cultures stimulated with SEA was observed in the group of patients with moderate to severe fibrosis (median levels = 31.9 pg/ml) compared to those without fibrosis (median levels = 36.8 pg/ml; P =.418). Levels of TNF-α were significantly higher in supernatants from SWAP-stimulated PBMC of subjects with incipient periportal fibrosis (median levels = 1446pg/mL) than in individuals without fibroses (median levels = pg/ml; P =.319). Moreover, the levels of TNF-α in nonstimulated cultures were higher in individuals with moderate to severe fibrosis (median levels = pg/ml) compared to subjects without periportal fibrosis (median levels = 61.9 pg/ml; P =.182). The levels of IL-13, IL-17, IFN-γ, and IL-1 did not differ significantly between individuals of the different groups (Table 2) Serum Levels of Cytokines. The levels of serum cytokines in patients with different degrees of periportal fibrosis are shown in Figure 1. With the exception of IFN-γ and IL-17, whose levels were below the detection limit of 15.6 pg/ml

25 4 Journal of Parasitology Research Table 2: Levels of cytokines produced by peripheral blood mononuclear cells (PBMC) of studied individuals. Cytocines (pg/ml) Without fibrosis Incipient fibrosis Moderate to severe median (min max) (n = 3) (n = 25) Fibrosis (n = 17) P IL-5 Without antigen 31.2 ( ) 31.2 ( ) 31.2 ( ) ns SWAP ( ) 524 ( ) ( ) ns SEA 36.8 ( ) ( ) 31.9 ( ).418 a IL-13 Without antigen ( ) 93.8 ( ) 93.8 ( ) ns SWAP ( ) 19.3 ( ) ( ) ns SEA ( ) 93.8 ( ) 93.8 ( ) ns IL-17 Without antigen 15.6 ( ) 15.6 ( ) 15.6 ( ) ns SWAP 21.4 ( ) 57.1 ( ) 21.4 ( ) ns SEA 18.9 ( ) 15.6 ( ) 27.2 ( ) ns IFN-γ Without antigen 31.2 ( ) 31.9 ( ) 31.2 ( ) ns SWAP 73.1 ( ) 125. ( ) ( ) ns SEA 83.1 ( ) 23.8 ( ) 13.1 ( ) ns TNF-α Without antigen 61.9 ( ) ( ) ( ).182 a SWAP ( ) ( ) 12. ( ).319 b SEA ( ) ( ) ( ) ns IL-1 Without antigen 15.6 ( ) 48.6 ( ) 17.5 ( ) ns SWAP ( ) ( ) ( ) ns SEA ( ) ( ) ( ) ns Kruskal-Wallis; a without fibrosis versus moderate to severe fibrosis; b without fibrosis versus incipient fibrosis; ns: not significant (P >.5). 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 were observed in the different groups of individuals (Figure 1). While the median levels of IL-13 were higher in individuals without periportal fibrosis compared to those with moderate to severe fibrosis (113.3 pg/ml and 97.7 pg/ml, respectively; P =.6), there was no significant difference in the median levels of IL-5, IL-1, TNF-α and TGF-β betweengroups (P >.5; Figure 1) Serum Level of the Chemokines. The median levels of the chemokines MIP-1α and RANTES in serum of individuals with different degrees of periportal fibrosis are shown in Figure 2. It was observed that the median level of MIP- 1α was higher in individuals without periportal fibrosis (7.9 pg/ml) compared to those with moderate to severe fibrosis (7.8 pg/ml; P =.232). There was no significant difference in serum levels of RANTES in individuals with different degrees of periportal fibrosis Association between Serum Levels of Cytokines and Chemokines and S. mansoni Parasite Burden. We tested the possible association between serum levels of cytokines and chemokines and S. mansoni parasite burden in the studied population. A positive association between serum levels of IL-13, TNF-α, MIP-1α, and RANTES and parasite burden was observed (Figure 3). There were no significant associations between serum levels of IL-5 (R 2 =.3413; P>.5), IFN-γ (R 2 =.5831; P>.5), IL-1 (R 2 =.4651; P>.5), and TGF-β (R 2 =.3391; P>.5) and the parasite burden of S. mansoni. 4. Discussion The development of periportal fibrosis can occur as a result of chronic schistosomiasis infection and accounts for the severe forms of the disease [6]. This fact characterizes schistosomiasis as a serious public health problem. The present study aimed to evaluate cytokine and chemokine profile in individuals with different degrees of periportal fibrosis living in the schistosomiasis endemic area of Água Preta, Brazil. Additionally, possible personal and environmental features which could interfere with the development of periportal fibrosis were also evaluated. We showed that despite the high prevalence of S. mansoni infection in Água Preta, Bahia, the frequency of severe forms of the disease determined by ultrasonography was low. This could be due to intrinsic characteristics of this population, since there is no report of previous treatment for schistosomiasis in this region. Other possible explanation

26 Journal of Parasitology Research 5 8 IL-5 4 IL-13 IL-5 (pg/ml) IL-13 (pg/ml) Without fibrosis Incipient Moderate to severe (a) TNF-α Without fibrosis Incipient Moderate to severe (b) 8 TGF-β TNF-α (pg/ml) TGF-β (pg/ml) Without fibrosis Incipient Moderate to severe Without fibrosis Incipient Moderate to severe (c) (d) 8 IL-1 IL-1 (pg/ml) Without fibrosis Incipient Moderate to severe (e) Figure 1: Serum cytokine levels in schistosomiasis individuals with different degrees of periportal fibrosis: (first box) individuals without fibrosis (n = 3), (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-1 were determined by sandwich ELISA technique. Horizontal lines represent the median values, boxes represent the 25th to the 75th percentiles, and vertical lines represent the 1th to 9th percentiles. Asterisks indicate statistically significant differences (P <.5; ANOVA). would be the Cairo s methodology to classify the periportal fibrosis which considers only degrees II and III as severe. We opted to use the Cairo s classification because we have performed previous studies using these parameters and because the physicians who have performed the USG in our studies are very well familiar with this classification. The majority of individuals who had the most severe degree of periportal fibrosis were over 4 years of age, and there was no influence of gender on periportal fibrosis development. This is in agreement with other authors who have demonstrated that most individuals who develop periportal fibrosisareover5yearsofage[22]. This could be explained

27 6 Journal of Parasitology Research 4 MIP-1α 11 RANTES 3 15 MIP-1α/CCL3 2 1 RANTES/CCL Without fibrosis Incipient Moderate to severe 85 Without fibrosis Incipient Moderate to severe (a) (b) Figure 2: Serum chemokine levels in schistosomiasis individuals with different degrees of periportal fibrosis: (first box) individuals without fibrosis (n = 3), (second box) incipient fibrosis (n = 25), and (third box) moderate to severe periportal fibrosis (n = 17).Levels of MIP-1α and RANTES were determined by sandwich ELISA technique. Horizontal lines represent the median values, boxes represent the 25th to the 75th percentiles, and vertical lines represent the 1th to 9th percentiles. Asterisks indicate statistically significant differences (P <.5; ANOVA). 2 5 IL-13 (pg/ml) IL-13 R 2 =.1624 P<.5 TNF-α (pg/ml) TNF-α R 2 =.6642 P<.5 (pg/ml) MIP-1α EPG of feces (a) EPG of feces (c) MIP-1α R 2 =.3183 P<.5 RANTES (pg/ml) EPG of feces (b) RANTES R 2 =.713 P< EPG of feces Figure 3: Correlation between serum levels of IL-13, TNF-α, MIP-1α, and RANTES (n = 65) and S. mansoni parasite burden. P <.5 indicates statisticallysignificant differences (Linear Regression). (d)

28 Journal of Parasitology Research 7 by the immune response induced by constant reexposure to the parasite over lifetime, or by the slow process of fibrosis formation. Therefore, younger individuals probably have not been exposed long enough to the cumulative effects of collagen deposition in the periportal tract [25]. Many variables may influence the magnitude of the immune response in human schistosomiasis which includes gender and age [26], intensity of infection [27 3], and genetic characteristics of the population [31]. However, the reason why severe fibrosis develops only in a fraction of the population, which is under the same environmental conditions as others who do not develop fibrosis, remains not well understood. In this study, we found no association between level of exposure to contaminated water and degree of periportal fibrosis. Moreover, individuals without periportal fibrosis had a higher parasite burden than individuals with incipient periportal fibrosis. A possible explanation for this observation is that chronic infection can lead to intestinal fibrosis that impairs the migration of eggs to the intestinal lumen and thereby decreases eggs count in parasitological exams [32]. It has been described that the specific type 2 cytokine pattern is a marker of S. mansoni chronic infection in mice and humans, and that this response is involved in the development of periportal fibrosis due to schistosomiasis [21, 33 35]. In this study we evaluated cytokine production bypbmcstimulated withthes. mansoni antigens SWAP and SEA as well as its serum levels and correlation with parasite load. We observed a greater variability in cytokines levels, which shows heterogeneity of response to the S. mansoni antigens. Levels of IL-5 in supernatants of SEA-stimulated PBMCs were higher in cultures of individuals with moderate to severe periportal fibrosis, when compared to the group without fibrosis. These data are similar to what was observed in a study conducted in schistosomiasis patient s residents in other endemic areas of Bahia [36, 37]. These data suggest that the Th2 immune response is developedand directed to antigens from the eggs. IL-5 has been associated with granuloma formation around S. mansoni eggs in the liver, since this cytokine participates in eosinophil growth and activation contributing to a significant source of profibrotic mediators such as IL-13 [38]. Likewise, IL-5 may participate directly in tissue remodeling and fibrosis in schistosomiasis [38]. In contrast to our data that showed no significant difference or correlation between serum levels of IL-5 and S. mansoni parasite burden in individuals with different degrees of periportal fibrosis, it has been described that serum levels of IL-5 is higher in patients with severe degrees of periportal fibrosis than in those without fibrosis [39]. Many studies in experimental models have shown that IL-13 plays an important role in the development of liver fibrosis. The blockage of IL-13 and IL-4 receptors prevented granuloma development and liver fibrosis in mice [33]. Human studies suggest a correlation between production of high levels of IL-13 and development of more advanced degree of liver fibrosis [36, 37]. However, in this study we did not observe differences in IL-13 levels in supernatants of SWAP or SEA stimulated PBMCs in all groups evaluated. We found, however, higher levels of serum IL-13 in the group of individuals without periportal fibrosis compared to patients with severe fibrosis. It has been suggested that levels of IL- 13 tend to be higher during the prefibrotic phase, acting as an inducer of the lesion [35, 39, 4]. The parasite burden is one of many other variables that can influence the magnitude of the immune response in human schistosomiasis [32, 41] and a high parasite burden has been associated with the development of fibrosis [5, 6]. Corroborating with these observation, we also found a positive association between parasite burden and serum levels of IL-13. There are no published data about the role of IL-17 in development of hepatic fibrosis due to schistosomiasis in humans. As reviewed bytallima et al. (29), this cytokine is involved in the development of severe disease by recruitment of neutrophils and macrophages in inflammatory sites, demonstrated only in experimental models [42]. We observed production of IL-17 in cultures stimulated by all the antigens tested without, however, differences among the groups. It is suggested that induction of severe liver disease associated with expression of IL-17 in experimental models, with polarized immune response to a Th1 profile, is dependent on the presence of IL-23, another Th17 cytokine [12, 13]. In this study, we evaluated the IL-17 production in individuals chronically infected by S. mansoni, who have a predominantly Th2 immune response, which may explain the low and not detectable levels of IL-17. When evaluating serum levels of this cytokine we also found levels of IL-17 below the detection limit in most individuals, which corroborates the results observed when measuring the supernatants of PBMCs cultures. Moreover, there was no correlation between serum levels of IL-17 and S. mansoni parasite load. Some authors have suggested that IFN-γ has certain antifibrotic activities, since this cytokine acts by inhibiting production of extracellular matrix proteins, enhances the activity of collagenase in liver tissue, and downmodulates the Th2 response [1, 43]. In this study, we found no significant differences in levels of IFN-γ between groups, and no significant association between parasite burden and serum levels of this cytokine in individuals with different levels of periportal fibrosis. Although controversy, TNF-α is another cytokine that may participate in the granuloma formation and evolution of fibrotic tissue process.hoffmann and colleagues (1998) have demonstrated in experimental models that TNF-α exerts a protective effect, whereas other authors attribute to the TNF-α proinflammatory and profibrogenic effects [7, 44]. In the present study it was observed that cells of individuals with incipient or moderate to severe fibrosis, even without antigenic stimuli, produced higher levels of TNF-α when compared to those without fibrosis. Additionally, there was a positive association between serum levels of TNF-α and S. mansoni parasite burden, which suggests that this cytokine may contribute to the liver pathology observed in schistosomiasis [45]. Supporting the role of TNF-α in the development of liver pathology due to schistosomiasis, a study conducted in a schistosomiasis endemic area in Brazil has demonstrated that individuals with moderate to severe

29 8 Journal of Parasitology Research periportal fibrosis have higher serum levels of TNF-α than those without fibrosis [39]. Some studies have pointed out TGF-β as an important factor in periportal fibrosis development during chronic schistosomiasis. This cytokine is considered a multifunctional cytokine that regulates biological processes such as inflammation, development, and differentiation of many cell types, tissue repair, and tumor genesis. It is also associated with pro-inflammatory responses and immunosuppressive activities [46, 47] and participates in the process of Th17 cells differentiation [42]. In this study, we found no significant differences in serum levels of TGF-β between groups with different degrees of periportal fibrosis, nor did we find an association between parasite burden and levels of TGF-β. Recent studies in experimental models have shown a negative association between serum levels of TGF-β and S. mansoni parasite burden in chronically infected animals, suggesting the involvement of this cytokine in controlling the parasite load [48]. Other molecule assessed in this study was the regulatory cytokine IL-1, which is associated with the switch from the Th1 immune response observed during the acute phase to Th2 responses and consequently preventing the development of severe disease. In this study we found no significant difference in the serum levels of this cytokine,nor in the levels measured in supernatant of stimulated cultures among individuals with different degrees of periportal fibrosis. This is in agreement with Silva-Teixeira and coworkers (24) who demonstrated no association between levels of IL-1 and parasite burden, suggesting that IL-1 may not be involved in periportal fibrosis development during schistosomiasis [39]. Levels of cytokines in patients with schistosomiasis have been evaluated in serum or supernatants of cell cultures. In this study we decided to evaluate cytokine in both, serum and supernatants. We believe that serum cytokine levels represent the in vivo profile status. However, as paracrine cytokines, such as IL-5, IL-13, IFN-γ, and IL-1, are not well detected in serum using convenient technique such as ELISA, we decided to evaluate these molecules in supernatants of PBMC restimulated in vitro with parasite antigens. We believe that these two measurements are complementary and lead to a better knowledge of the cytokine profile in human schistosomiasis. The role of chemokines in periportal fibrosis due to schistosomiasis is also not completely understood [49]. In this study, we found higher levels of serum MIP-1α in individuals without periportal fibrosis than in patients with incipient or moderate to severe fibrosis, which is in contrast to what has been found by Souza and colleagues [5, 51]. We showed, however, a positive association between serum levels of MIP-1α and S. mansoni parasite burden. This finding is similar to what was obtained for IL-13 and reinforces the thought that individuals without fibrosis enrolled in this study may have been in a prefibrotic stage. Interestingly, it has been also demonstrated that MIP-1α induces production of Th2-pattern cytokines, including IL-13 [52, 53]. Many authors have suggested that high levels of IFNγ and TNF-α are associated with increased expression of RANTES, whereas Th2 cytokines, such as IL-4 and IL-13, are associated with decreased expression [54 56]. In agreement with these studies, it has shown that RANTES deficient mice showed a significant increase granulomatous response when compared to the control group [57]. These studies reinforce the idea that RANTES regulates negatively the development of granuloma and fibrosis. On the other hand, there are no data in the literature on the possible role of RANTES in the development of periportal fibrosis in human schistosomiasis. The present study showed high levels of IL-5 and TNF-α in individuals with periportal fibrosis. Higher levels of IL-13 and MIP-1α in individuals without periportal fibrosis were also documented. Considering that these former molecules werepositively associatedwith S. mansoni parasite burden, as were TNF-α and RANTES, they may represent biomarker for the progression of liver pathology in schistosomiasis. Larger casuistic further studies are needed however to confirm the role of these cytokines and chemokines in the development of 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 selection of patients, and Dr. Irismá Souza and Dr. Delfin Gonzalez for performing the ultrasound assessment. they are very grateful to all volunteers from the community of Água Preta, Gandu, who agreed to partipate in this study, to the local health agent Irene Jesus for her support, and to Michael Andrew Sundberg for the corrections and suggestions made in the text. They also thank the Fundação de Amparo à Pesquisa do Estado da (Bahia FAPESB) for the financial support. E. Carvalho andm.i.araujoareinvestigatorssupportedbytheconselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This work was supported by the Brazilian National Research Council (CNPq) Universal (482113/21-3). References [1] L. S. Iarotski and A. Davis, The schistosomiasis problem in the world: results of a WHO questionnaire survey, Bulletin of the World Health Organization, vol. 59, no. 1, pp , [2] Ministério da Saúde, Guia de Vigilância Epidemiológica, Ministério da Saúde, Brasília, Brasil, 25. [3] M. S. Wilson, M. M. Mentink-Kane, J. T. Pesce, T. R. Ramalingam, R. Thompson, and T. A. Wynn, Immunopathology of schistosomiasis, Immunology and Cell Biology, vol. 85, no. 2, pp , 27. [4] Z. A. Andrade, Schistosomiasis and liver fibrosis: review Article, Parasite Immunology, vol. 31, no. 11, pp , 29. [5]J.C.Bina, Estudo de variáveis que podem influenciar na evolução da esquistossomose mansônica: efeito da terapêutica específica e da interrupção da transmissào, Revista de Patologia Tropical, vol. 26, pp , [6] J. C. Bina and A. Prata, Schistosomiasis in hyperendemic area of Taquarendi. I- Schistosoma mansoni infection and severe clinikal forms, Revista da Sociedade Brasileira de Medicina Tropical, vol. 36, no. 2, pp , 23.

30 Journal of Parasitology Research 9 [7] S. Henri, C. Chevillard, A. Mergani et al., Cytokine regulation of periportal fibrosis in humans infected with Schistosoma mansoni: IFN-γ is associated with protection against fibrosis and TNF-α with aggravation of disease, Journal of Immunology, vol. 169, no. 2, pp , 22. [8]P.L.Falcão, L. C. C. Malaquias, O. A. Martins-Filho et al., Human Schistosomiasis mansoni: IL-1 modulates the in vitro granuloma formation, Parasite Immunology, vol. 2, no. 1, pp , [9] S. Gustavson, C. S. Zouain, J. B. Alves, M. F. Leite, and A. M. Goes, Modulation of granulomatous hypersensitivity against Schistosoma mansoni eggs in mice vaccinated with culture-derived macrophages loaded with PIII, Parasitology International, vol. 51, no. 3, pp , 22. [1] T. A. Wynn, Cytokine regulation of granuloma formation in schistosomiasis, Current Opinion in Immunology, vol. 7, no. 4, pp , [11] L. Steinman, A brief history of TH17, the first major revision in the T H1/TH2 hypothesis of T cell-mediated tissue damage, Nature Medicine, vol. 13, no. 2, pp , 27. [12] L. I. Rutitzky, L. Bazzone, M. G. Shainheit, B. Joyce-Shaikh, D. J. Cua, and M. J. Stadecker, IL-23 is required for the development of severe egg-induced immunopathology in schistosomiasis and for lesional expression of IL-17, Journal of Immunology, vol. 18, no. 4, pp , 28. [13] L. I. Rutitzky, J. R. L. Da Rosa, and M. J. Stadecker, Severe CD4 T cell-mediated immunopathology in murine schistosomiasis is dependent on IL-12p4 and correlates with high levels of IL-17, Journal of Immunology, vol. 175, no. 6, pp , 25. [14] L. I. Rutitzky and M. J. Stadecker, CD4 T cells producing pro-inflammatory interleukin-17 mediate high pathology in schistosomiasis, Memorias do Instituto Oswaldo Cruz, vol. 11, no. 1, pp , 26. [15] N.Katz,P.M.Coelho,andJ.Pellegrino, EvaluationofKato s quantitative method through the recovery of Schistosoma mansoni eggsaddedtohumanfeces, Journal of Parasitology, vol. 56, no. 5, pp , 197. [16] M. I. A. S. Araujo, B. Hoppe, M. Medeiros et al., Impaired T helper 2 response to aeroallergen in helminth-infected patiente with asthma, Journal of Infectious Diseases, vol. 19, no. 1, pp , 24. [17] A. J. Dessein, M. Begley, C. Demeure et al., Human resistance to Schistosoma mansoni is associated with IgG reactivity to a 37-kDa larval surface antigen, Journal of Immunology, vol. 14, no. 8, pp , [18] A. V. Grant, M. I. Araujo, E. V. Ponte et al., High heritability but uncertain mode of inheritance for total serum IgE level and Schistosoma mansoni infection intensity in a schistosomiasis-endemic Brazilian population, Journal of Infectious Diseases, vol. 198, no. 8, pp , 28. [19] M.F.Abdel-Wahab,G.Esmat,A.Farrag,Y.A.El-Boraey,and G. T. Strickland, Grading of hepatic schistosomiasis by the use of ultrasonography, American Journal of Tropical Medicine and Hygiene, vol. 46, no. 4, pp , [2] A. R. De Jesus, A. Silva, L. B. Santana et al., Clinical and immunologic evaluation of 31 patients with acute schistosomiasis mansoni, Journal of Infectious Diseases, vol. 185, no. 1, pp , 22. [21] A. R. De Jesus, D. G. Miranda, R. G. Miranda et al., Morbidity associated with Schistosoma mansoni infection determined by ultrasound in an endemic area of Brazil, Caatinga do Moura, American Journal of Tropical Medicine and Hygiene, vol. 63, no. 1-2, pp. 1 4, 2. [22] L. F. A. Oliveira, E. C. Moreno, G. Gazzinelli et al., Cytokine production associated with periportal fibrosis during chronic schistosomiasis mansoni in humans, Infection and Immunity, vol. 74, no. 2, pp , 26. [23] E. J. Pearce, S. L. James, and J. Dalton, Immunochemical characterization and purification of Sm-97, a Schistosoma mansoni antigen monospecifically recognized by antibodies from mice protectively immunized with a nonliving vaccine, Journal of Immunology, vol. 137, no. 11, pp , [24] C. Hirsch and A. M. Goes, Characterization of fractionated Schistosoma mansoni soluble adult worm antigens that elicit human cell proliferation and granuloma formation in vitro, Parasitology, vol. 112, no. 6, pp , [25] M. Booth, J. K. Mwatha, S. Joseph et al., Periportal Fibrosis in Human Schistosoma mansoni InfectionIsAssociatedwith Low IL-1, Low IFN-γ, High TNF-α, or Low RANTES, Depending on Age and Gender, Journal of Immunology, vol. 172, no. 2, pp , 24. [26] Q. Mohamed-Ali, N. E. M. A. Elwali, A. A. Abdelhameed et al., Susceptibility to periportal (Symmers) fibrosis in human Schistosoma mansoni infections: evidence that intensity and duration of infection, gender, and inherited factors are critical in disease progression, Journal of Infectious Diseases, vol. 18, no. 4, pp , [27] M. F. Abdel-Wahab, G. Esmat, S. I. Narooz, A. Yosery, J. P. Struewing, and G. T. Strickland, Sonographic studies of schoolchildren in a village endemic for Schistosoma mansoni, Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 84, no. 1, pp , 199. [28] E. Doehring-Schwerdtfeger, I. M. Abdel-Rahim, Q. Mohamed-Ali et al., Ultrasonographical investigation of peripheral fibrosis in children with Schistosoma mansoni infection: evaluation of morbidity, American Journal of Tropical Medicine and Hygiene, vol. 42, no. 6, pp , 199. [29] A. L. C. Domingues, A. R. F. Lima, H. S. Dias, G. C. Leao, and A. Coutinho, An ultrasonographic study of liver fibrosis in patients infected with Schistosoma mansoni in north-east Brazil, Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 87, no. 5, pp , [3] M. Homeida, A. F. Abdel-Gadir, A. W. Cheever et al., Diagnosis of pathologically confirmed Symmers periportal fibrosis by ultrasonography: a prospective blinded study, American Journal of Tropical Medicine and Hygiene, vol. 38, no. 1, pp , [31]A.J.Dessein,D.Hillaire,N.E.M.A.Elwalietal., Severe hepatic fibrosis in Schistosoma mansoni infection is controlled by a major locus that is closely linked to the interferon-γ receptor gene, American Journal of Human Genetics, vol. 65, no. 3, pp , [32] O.S.Carvalho,P.M.Z.Coelho,andH.L.Lenzi,Schistosoma mansoni e Esquistossomose: Uma Visão Multidisciplinar, Editora Fiocruz, Rio de Janeiro, Brazil, 1st edition, 28. [33] M. G. Chiaramonte, A. W. Cheever, J. D. Malley, D. D. Donaldson, and T. A. Wynn, Studies of murine schistosomiasis reveal interleukin-13 blockade as a treatment for established and progressive liver fibrosis, Hepatology, vol. 34, no. 2, pp , 21. [34] M. G. Chiaramonte, D. D. Donaldson, A. W. Cheever, and T. A. Wynn, An IL-13 inhibitor blocks the development of hepatic fibrosis during a T-helper type 2-dominated inflammatory response, Journal of Clinical Investigation, vol. 14, no. 6, pp , 1999.

31 1 Journal of Parasitology Research [35] A. R. De Jesus, I. Araújo, O. Bacellar et al., Human immune responses to Schistosoma mansoni vaccine candidate antigens, Infection and Immunity, vol. 68, no. 5, pp , 2. [36] A. R. De Jesus, A. Magalhães, D. G. Miranda et al., Association of type 2 cytokines with hepatic fibrosis in human Schistosoma mansoni infection, Infection and Immunity,vol.72,no.6,pp , 24. [37] A. Magalhães, D. G. Miranda, R. G. Miranda et al., Cytokine profile associated with human chronic schistosomiasis mansoni, Memorias do Instituto Oswaldo Cruz, vol. 99, no. 5, supplement 1, pp , 24. [38] R. M. Reiman, R. W. Thompson, C. G. Feng et al., Interleukin-5 (IL-5) augments the progression of liver fibrosis by regulating IL-13 activity, Infection and Immunity, vol. 74, no. 3, pp , 26. [39] D. N. Silva-Teixeira, C. Contigli, J. R. Lambertucci, J. C. Serufo, and V. Rodrigues, Gender-related cytokine patterns in sera of schistosomiasis patients with symmers fibrosis, Clinical and Diagnostic Laboratory Immunology, vol. 11, no. 3, pp , 24. [4]C.N.L.DeMorais,J.R.DeSouza,W.G.DeMeloetal., Studies on the production and regulation of interleukin, IL- 13, IL-4 and interferon-γ in human schistosomiasis mansoni, Memorias do Instituto Oswaldo Cruz, vol. 97, supplement 1, pp , 22. [41] M. I. Araújo, A. R. De Jesus, O. Bacellar, E. Sabin, E. Pearce, and E. M. Carvalho, Evidence of a T helper type 2 activation in human schistosomiasis, European Journal of Immunology, vol. 26, no. 6, pp , [42] H. Tallima, M. Salah, F. R. Guirguis, and R. El Ridi, Transforming growth factor-β and Th17 responses in resistance to primary murine schistosomiasis mansoni, Cytokine, vol. 48, no. 3, pp , 29. [43] L. C. Borish and J. W. Steinke, 2. Cytokines and chemokines, Journal of Allergy and Clinical Immunology, vol. 111, no. 2, supplement, pp. S46 S475, 23. [44] K. F. Hoffmann, P. Caspar, A. W. Cheever, and T. A. Wynn, IFN-γ, IL-12, and TNF-α are required to maintain reduced liver pathology in mice vaccinated with Schistosoma mansoni eggs and IL-12, Journal of Immunology, vol. 161, no. 8, pp , [45]S.A.Shahat,M.A.El-Dhshan,S.S.Aissa,A.Dorra,and K. M. Metwally, Flowcytometric analysis of T-lymphocytes and serum tumour necrosis factor alpha (TNF-alpha) levels in Schistosoma mansoni patients, Journal of the Egyptian Society of Parasitology, vol. 37, no. 3, pp , 27. [46] M. O. Li, Y. Y. Wan, S. Sanjabi, A. K. L. Robertson, and R. A. Flavell, Transforming growth factor-β regulation of immune responses, Annual Review of Immunology, vol. 24, pp , 26. [47] J. Massague, J. Andres, L. Attisano et al., TGF-β receptors, Molecular Reproduction and Development, vol.32,no.2,pp , [48] R. R. El-Gamal, S. M. Nada, N. E. Moustafa et al., Relationship between serum cytokines profiles and hepatic fibrosis in schistosomiasis mansoni: an experimental study, Journal of the Egyptian Society of Parasitology, vol. 39, no. 3, pp , 29. [49] M. L. Burke, M. K. Jones, G. N. Gobert, Y. S. Li, M. K. Ellis, and D. P. McManus, Immunopathogenesis of human schistosomiasis, Parasite Immunology, vol. 31, no. 4, pp , 29. [5] A. L. S. Souza, E. Roffê, V. Pinho et al., Potential role of the chemokine macrophage inflammatory protein 1α in human and experimental schistosomiasis, Infection and Immunity, vol. 73, no. 4, pp , 25. [51] P. R. S. Souza, A. L. S. Souza, D. Negrão-Correa, A. L. Teixeira, and M. M. Teixeira, The role of chemokines in controlling granulomatous inflammation in Schistosoma mansoni infection, Acta Tropica, vol. 18, no. 2-3, pp , 28. [52] P. L. Falcão, R. Correa-Oliveira, L. A. O. Fraga et al., Plasma concentrations and role of macrophage inflammatory protein- 1α during chronic Schistosoma mansoni infection in humans, Journal of Infectious Diseases, vol. 186, no. 11, pp , 22. [53] D. M. Oliveira, D. N. Silva-Teixeira, S. Gustavson, S. M. P. Oliveira, and A. M. Goes, Nitric oxide interaction with IL- 1, MIP-1α, MCP-1 and RANTES over the in vitro granuloma formation against different Schistosoma mansoni antigenic preparations on human schistosomiasis, Parasitology, vol. 12, no. 4, pp , 2. [54] N. Berkman, V. L. Krishnan, T. Gilbey et al., Expression of RANTES mrna and protein in airways of patients with mild asthma, American Journal of Respiratory and Critical Care Medicine, vol. 154, no. 6, pp , [55] M. John, S. J. Hirst, P. J. Jose et al., Human Airway smooth muscle cells express and release RANTES in response to T helper 1 Cytokines: regulation by T helper 2 cytokines and corticosteroids, Journal of Immunology, vol. 158, no. 4, pp , [56] A. Marfaing-Koka, O. Devergne, G. Gorgone et al., Regulation of the production of the RANTES chemokine by endothelial cells: synergistic induction by IFN-γ plus TNF-α and inhibition by IL-4 and IL-13, Journal of Immunology, vol. 154, no. 4, pp , [57] S. W. Chensue, K. S. Warmington, E. J. Allenspach et al., Differential expression and cross-regulatory function of RANTES during mycobacterial (type 1) and schistosomal (type 2) antigen-elicited granulomatous inflammation, Journal of Immunology, vol. 163, no. 1, pp , 1999.

32 Hindawi Publishing Corporation Journal of Parasitology Research Volume 212, Article ID , 11 pages doi:1.1155/212/ Review Article New Frontiers inschistosoma Genomics and Transcriptomics Laila A. Nahum, 1, 2, 3 Marina M. Mourão, 1 and Guilherme Oliveira 1, 2 1 Genomics and Computational Biology Group, Centro de Pesquisas René Rachou (CPqRR), Instituto Nacional de Ciência e Tecnologia emdoenças Tropicais, Fundação Oswaldo Cruz (FIOCRUZ), Belo Horizonte, MG, Brazil 2 Centro de Excelência em Bioinformática (CEBio), Fundação Oswaldo Cruz (FIOCRUZ), Belo Horizonte, MG, Brazil 3 Faculdade Infórium de Tecnologia, Belo Horizonte, MG, Brazil Correspondence should be addressed to Guilherme Oliveira, oliveira@cpqrr.fiocruz.br Received 1 August 212; Accepted 16 October 212 Academic Editor: Sergio Costa Oliveira Copyright 212 Laila A. Nahum et al. 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. Schistosomes are digenean blood flukes of aves and mammals comprising 23 species. Some species are causative agents of human schistosomiasis, the second major neglected disease affecting over 23 million people worldwide. Modern technologies including the sequencing and characterization of nucleic acids and proteins have allowed large-scale analyses of parasites and hosts, opening new frontiers in biological research with potential biomedical and biotechnological applications. Nuclear genomes of the three most socioeconomically important species (S. haematobium, S. japonicum, and S. mansoni) have been sequenced and are under intense investigation. Mitochondrial genomes of six Schistosoma species have also been completely sequenced and analysed from an evolutionary perspective. Furthermore, DNA barcoding of mitochondrial sequences is used for biodiversity assessment of schistosomes. Despite the efforts in the characterization of Schistosoma genomes and transcriptomes, many questions regarding the biology and evolution of this important taxon remain unanswered. This paper aims to discuss some advances in the schistosome research with emphasis on genomics and transcriptomics. It also aims to discuss the main challenges of the current research and to point out some future directions in schistosome studies. 1. Introduction Schistosomatidae (Platyhelminthes: Digenea) includes several digenetic endoparasites with complex life cycles, whose developmental stages alternate between intermediate (freshwater gastropods) and definitive hosts (birds, reptiles, fishes, and mammals) [1]. These parasites differ from other blood flukes in having separate sexes. Another important feature is the increased longevity (over 5 years) of the Schistosoma species in the human host [1]. Schistosoma, the avian and mammalian blood flukes, is the best studied genus of Schistosomatidae [1]. Several species are described, six of which infect humans causing schistosomiasis: S. haematobium, S. intercalatum, S. japonicum, S. malayensis, S. mansoni, and S. mekongi. Other species are known to infect a broad range of mammals such as hippopotamus, rodents, carnivores, and ruminant animals like buffalo, cattle, goat, lechwe, and sheep. Some hybrid species are reported [1 4]. For example, S. mattheei, more commonly found in cattle, is believed to form hybrids between S. mattheei and S. haematobium thus increasing its snail and definitive host range [3]. Schistosomiasis, a chronic and debilitating disease, is considered by the World Health Organization as one of the most serious public health issues and the second most prevalent tropical disease with high morbidity in the world [5]. Schistosomiasis is endemic in 77 countries [6] and its transmission is dependent on the existence and distribution of intermediate hosts. It is estimated that 237 million people require treatment worldwide and that 6 to 779 million people live in endemic areas, under infection risk [6]. Yet, exacerbating this scenario, this disease is responsible for the loss of 1.7 to 4.5 millions of years of life in the world, measured by disability-adjusted life years (DALY) [7], one of the highest indexes among all the neglected tropical diseases. Control of schistosomiasis represents a great challenge and it is based on drug treatment (Praziquantel), snail control, improved sanitation, and health education [5].

33 2 Journal of Parasitology Research The development of powerful and scalable methods to analyse nucleic acids and proteins has changed the way biological data is surveyed. The application of such technologies, together with the development of powerful computational tools and methods, have expanded our perspective of schistosome biology and allowed a better understanding of processes such as host-parasite interaction [8 1]. This paper aims to discuss some advances in schistosome research with emphasis on genomics and transcriptomics. First, we summarize the current status of sequencing projects of nuclear and mitochondrial genomes. We also discuss some aspects of evolutionary genomics and biodiversity of schistosomes. Then, we present key findings in transcriptomic analyses. Finally, we point out the main challenges of the current research and suggest some future directions in Schistosoma genomic and transcriptomic studies. 2. Schistosoma Genomics In order to strengthen traditional methods and provide new information to improve success towards schistosomiasis control, the scientific community joined efforts to assess the Schistosoma genomic information, starting in 1994, as an initiative of WHO/TDR (UNICEF-UNDP-World Bank- WHO Special Programme for Research and Training in Tropical Diseases) [11, 12]. At that time, only a few hundred expressed sequence tags (ESTs) from S. mansoni were available [13]. The WHO/TDR support opened the possibility to generate data that could be translated into new tools for schistosomiasis diagnostics and treatment, representing the kickoff of Schistosoma nuclear genome studies Nuclear Genomes. The karyotype of known Schistosoma species comprises seven pairs of autosomes and one pair of sex chromosomes (female = ZW, male = ZZ), ranging in size from 18 to 73 megabases (Mb) [14, 15]. The nuclear genome of three Schistosoma species was sequenced (Table 1). The S. japonicum (Anhui isolate) and S. mansoni (PuertoRicoisolate)genomesweredecodedand simultaneously published as an initiative of the Wellcome Trust Sanger Center Institute in collaboration with the Institute for Genomic Research (TIGR) and the Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium [16, 17]. Recently, the S. haematobium (Egyptian isolate) genome was sequenced opening new possibilities in comparative genomics of schistosomes [18]. S. haematobium, S. japonicum, ands. mansoni carry a nuclear genome of 385, 397, and 363 Mb composed by approximately, 13,73, 13,469, and 1,852 protein-coding genes, respectively [16 19]. It is worth to mention that the number of predicted protein-coding genes does not reflect the genome structure per se, but the actual state of analyses of the different draft genome data. The genetic linkage map of S. mansoni was obtained and allowed the refinement of the genome sequencing data providing a means for gene discovery and gene function analysis [2, 21]. More importantly, it has allowed the identification of genetic loci that determine important traits such 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. haematobium and S. japonicum are still considered drafts [16 19]. Their genome sequences are distributed in a large number of contigs; therefore extensive work is required. Constant data refinement is necessary to provide a reliable comprehension of the genome and gene models and to provide a curated annotation. The S. mansoni genome annotation has been improved by combining different sequencing strategies [19]. Such strategies include new assembly of existing capillary reads supplemented with an additional 9, fosmid and BAC end sequences, deep sequencing of clonal DNA (NMRI strain, Puerto Rican origin) using 18-based paired reads on Illumina Genome Analyzer IIx platform, and RNA-seq data of different parasite life stages. Progress on the knowledge of S. mansoni genome features is still expanding, especially with the application of the nextgeneration sequencing platforms and single nucleotide polymorphism typing methods. For instance, 14 new microsatellite markers were recently identified by de novo assembly of massive sequencing reads; these new features can be employed for pedigree studies [22]. Yet, newer sequencing technologies will allow for the development of population genomics studies of field, laboratory, and clinical isolates. As a result of the availability of the Schistosoma genome sequence data, comparative analyses across different species became possible bringing together different areas of research [21, 23, 24]. Comparative genomics of three Schistosoma species revealed several conserved features such as the overall GC content, sequence identity, presence of repetitive elements, and synteny [18]. This genome-wide analysis corroborates previous genetic studies and supports the evolutionary hypothesis of S. haematobium and S. mansoni as the most related species, followed by S. japonicum, to the exclusion of other Trematoda such as Clonorchis, Fasciola, and Schmidtea Mitochondrial Genomes. Mitochondrial genes have been used as molecular markers for species and strain identification, 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 relationships among taxa, mitochondrial genes have been successfully applied into epidemiological studies, monitoring, and control of microbes, parasites, and vectors of medical and socioeconomic importance. The analysis of the whole mitochondrial genome of diverse taxa has changed the perspective of such studies. The mitochondrial genome of six Schistosoma species was sequenced (Table 1) including S. haematobium (NC 874), S. japonicum (NC 2544), S. mansoni (NC 2545), S. malayensis, S. mekongi (NC 2529), and S. spindale (NC 867) [27, 3 32]. These genomes range in size from 13,53 to 16,91 bp and encode 36 genes

34 Journal of Parasitology Research 3 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 Schistosoma edwardiense No No No Yes Schistosoma guineensis No No No Yes 6185 Schistosoma haematobium Yes Yes Yes Yes Schistosoma hippopotami No No No Yes Schistosoma incognitum No No No Yes Schistosoma indicum No No No Yes 6187 Schistosoma intercalatum No No No Yes 6182 Schistosoma japonicum Yes Yes Yes Yes Schistosoma kisumuensis ( ) No No No Yes Schistosoma leiperi No No No Yes Schistosoma malayensis No Yes No Yes 6183 Schistosoma mansoni Yes Yes Yes Yes Schistosoma margrebowiei No No No Yes Schistosoma mattheei No No No Yes Schistosoma mekongi No Yes No Yes Schistosoma nasale No No No Yes 6188 Schistosoma rodhaini No No No Yes Schistosoma sinensium No No No Yes Schistosoma sp. Uganda-JATM-23 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 Genome Resources). ESTs: data from NCBI dbest (release 1271, July 1, 212). Barcoding: DNA barcoding used on cox1 sequences (BOLD Systems). Schistosoma sp. BH-29. comprising two ribosomal genes (large and small subunit rrna genes), and 22 transfer RNA (trna) genes, as well as 12 protein-encoding genes (atp6, cob, cox1 3, nad1 6, and nad4l). The atp8 gene, commonly found in other phyla, is absent from Platyhelminthes, whose mitochondrial genomes have been analysed [31]. Moreover, each Schistosoma mitochondrial genome contains a long noncoding region that is divided into two parts by one or more trna genes, which is found in other Platyhelminthes and vary in length according to each taxon. Comparative mitogenomics have revealed a highly conserved gene order among distinct Platyhelminthes with the exception of some Schistosoma species [25, 3 33]. The mitochondrial genome of the Asian schistosomes, S. japonicum and S. mekongi, displays the same gene order as other Digenea and Cestoda [25, 32]. In contrast, the gene order in the mitochondrial genome of S. haematobium, S. mansoni, and S. spindale is strikingly different from other taxa [25, 3]. The unique gene order rearrangements identified in these species constitute valuable information to the reconstruction of the phylogenetic relationships of Schistosoma and other Platyhelminthes (Section 3.1). 3. Evolutionary Genomics and Biodiversity 3.1. Evolutionary Genomics of Schistosoma. The use of genomic data is valuable to the understanding of the evolutionary history of Schistosoma especially due to the absence of fossil records. Molecular markers have been used to test hypotheses in a variety of Schistosoma phylogenetic, phylogeographic and epidemiological studies [4, 25, 34 36]. Traditionally, these markers include the nuclear ribosomal RNA genes (18S, 5.8S, and 28S) and the internally transcribed spacer (ITS) region besides a number of mitochondrial genes (cob, cox1 3, nad1 6, etc.). Based on mitochondrial data from different studies, a standard phylogeny of 23 Schistosoma species was proposed [25]. Six clades were identified that correlate with the geographic distribution of the species analysed. These clades include (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, andeasterneurope; (4) the S. mansoni clade (S. mansoni and S. 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 parts of Africa; (6) the S. indicum clade (S. indicum, S. nasale,and S. spindale) found in India and South Asia. There are divergent opinions concerning the origin of the genus Schistosoma and its intermediate snail host [25, 37 39]. The out of Asia hypothesis is the most generally

35 4 Journal of Parasitology Research accepted, indicating a migration followed by dispersion of these parasites from Asia to Africa [25]. In this regard, studies of molecular phylogeny suggested that the genus Schistosoma had its origin in Asia and (at least two descendants) colonized the African continent independently, where they easily radiated, becoming exclusive parasites of planorbid snails. Returning to Asia, the parasites diversified into groups of species, characterized by the position of the spike in the eggs [38]. The molecular phylogeny of seven Schistosoma species suggested that schistosomes were endoparasites of rodents and ruminants and were transmitted to the first hominids in Africa as a migration to savanna areas occurred [4]. However,inordertobetterdefinetheSchistosoma origin, there is a need to sample a broader range of species. Most of the widely accepted Schistosoma phylogenies are primarily based on sequence alignments of mitochondrial genes such as cox1 3, and nad1 6, [25, 3]. Other studies use changes in the mitochondrial gene order as phylogenetic markers. Indeed, these gene rearrangements, considered to be rare evolutionary events, have been applied to an increasing number of studies (cf. [41]). As mentioned before, major changes in the mitochondrial 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 Asian ancestry of schistosomes and corroborates the concept of the reinvasion of Asia by members of the S. indicum group from Africa [27, 3]. Besides reconstructing the phylogenetic relationships of diverse taxa, the evolutionary framework has been applied to improve functional annotation of genes and gene products, as well as to study gene/protein family evolution [41]. Phylogenomics has allowed the identification and characterization of all eukaryotic protein kinases encoded in the S. mansoni genome and improved the functional annotation of over 4% of them, highlighting the molecular diversity of these enzymes [42, 43]. A phylogenetic analysis was also employed in the classification of S. mansoni histone proteins, which play a key role in epigenetic modifications that might reflect the parasite complex lifestyle [44]. By applying an evolutionary framework to analyse a large sequence dataset from a broad range of organisms, functional annotation of the S. mansoni histone proteins was improved. Moreover, phylogenomics has unraveled the distinct evolutionary histories of three expanded endopeptidase families in S. mansoni [45]. This analysis included members of the metallopeptidase M8, serine peptidase S1, and aspartic peptidase A1 families, which were originated from successive events of gene duplication followed by divergence in the parasite lineage after its diversification from other metazoans. Other protein families such as nuclear receptors and antioxidants have been characterized in S. mansoni by applying evolutionary and functional approaches [46, 47]. The identification of SmNR4A, a member of the nuclear receptor subfamily 4, and its relatedness to the human homologs were corroborated by phylogenetic analysis [46]. This study also demonstrated that SmNR4A expression was regulated throughout parasite development. Thioredoxin and glutathione systems, involved in a variety of cellular processes including antioxidant defense, differ in parasitic and free-living Platyhelminthes [47]. By applying different phylogenetic methods and experimental testing, it has been shown that the canonical enzymes of such systems were lost in the parasitic lineages DNA Barcoding for Biodiversity Assessment. The availability of sequencing data from mitochondrial genomes from closely related taxa to Schistosoma has provided a basis for studies ranging from phylogenetic systematics to molecular ecology and so forth [27, 29, 48, 49]. Importantly, it allowed the assessment of molecular markers prior to broad scale sampling by comparing data from individual genes versus completely sequenced mitochondrial genomes. Data from African and Asian schistosomes were compared to an S. mansoni intraspecific dataset showing a positive correlation between polymorphism and species divergence [27]. A positive correlation rather than random distribution was identified for all mitochondrial genes with the exception of cox1 and nad1. Furthermore, partial sequences of cox1 have been shown not to be the ideal marker for either species identification or population studies of Schistosoma species. Instead, authors have suggested the use of cox3 and nad5 sequences for both phylogenetic and population studies of such species [27]. DNA barcoding was performed for samples of S. haematobium using cox1 and nad1 as molecular markers [49]. The results supported the identification of group 1 and 2 haplotypes based on phylogenetic analysis. Moreover, no change in genetic diversity was detected across samples collected over different time points. This approach allowed the development of new assays based on group-specific PCR primers and SNaPshot probes to assist genetic screening of schistosome isolates. Another study employing mitochondrial sequences was performed in Schistosoma cercariae harvested from Biomphalaria choanomphala as part of parasitological and malacological surveys for S. mansoni across Lake Victoria [48]. DNA barcoding of cercarial samples revealed the presence of hybrid species between S. mansoni and S. rodhaini, which is typically found in small mammals. Moreover, the phylogenetic analysis identified a new sublineage within S. rodhaini. DNA barcoding studies are underway for an increasing number of Schistosoma species (Table 1), whose data is made available through the Barcode of Life Database (BOLD) system ( This initiative includes contributors from the Biodiversity Institute of Ontario, Canada, and the University of New Mexico, USA, among others. Other Schistosomatidae genera are subject to DNA barcoding analysis such as Allobilharzia, Austrobilharzia, Bilharziella, Bivitellobilharzia, Dendritobilharzia, Gigantobilharzia, Griphobilharzia, Heterobilharzia, Macrobilharzia, Orientobilharzia, Ornithobilharzia, Schistosomatium, and Trichobilharzia.

36 Journal of Parasitology Research 5 Advances in DNA barcoding of a broader range of Schistosomatidae will create a framework for species identification from environmental and clinical samples as well as specimens deposited in biological collections. Moreover, DNA barcoding analysis will help reconstructing the evolutionary relationships across different Platyhelminthes and ultimately improve our understanding of parasite biology and evolution. 4. Schistosoma Transcriptomics Transcriptomic analyses are valuable not only for studies of differential regulation in gene expression, but also for the identification of splicing variants. These approaches have proved to be essential in the identification of gene-coding regions during the process of genome annotation. In this section, we aim to review the achievements in Schistosoma transcriptomic studies that range from the whole organism and tissue analyses to those performed at the cellular level. Transcriptomics in physiological conditions and in response to a variety of treatments are investigated with respect to the interaction with the host as well as other environments. This section summarizes the accomplishments of gene function studies and the diverse techniques that are being employed Schistosoma Transcriptomics. A fine tuning in gene expression of parasites must occur to enable them to survive in such a complex and extremely diverse milieu, adapting to diverse environments as evading the immune system and, at the same time, exploiting different hosts. Transcriptomic studies came along allowing a temporal and quantitative analyses of gene expression, which are crucial to completely exploit different datasets and assess parasite biology at the molecular level. Aiming to elucidate the differential regulation on the Schistosoma transcriptomics, a large number of projects employed diverse strategies, such as the ones using expressed sequence tags (ESTs) [5], open reading frame expressed sequence tags (ORESTEs) [51], and serial analysis of gene expression (SAGE) [52, 53]. The increasing number of projects resulted in the production of a vast amount of sequence data and their availability in public databases. It is noteworthy that only S. japonicum, S. mansoni and, not until very recently, S. haematobium had some of their transcriptome explored (Table 1) Exploring the Transcriptome. The large number of transcriptome projects accelerated the studies in this area by providing means to the use of complementary approaches, especially microarray analysis. Together, these approaches have been used to answer a wide range of questions. In the early years, microarray datasets conceived the profile of gene expression between genders of Schistosoma species such as S. japonicum and S. mansoni [54 56]. This greatly extended the number of known sex-associated genes and also provided new insights into changes in gene expression promoted by parasite pairing. In addition, gender biased alternative splicing patterns were observed, which are perhaps involved in the generation and maintenance of the sexual dimorphism [57]. Later, studies profiled the S. mansoni gene expression in different life stages broadening our understanding of factors controlling schistosome development [8, 53, 58]. While comparing different parasite life stages, information acquired from microarray datasets highlighted putative antischistosome candidate molecules to be used as vaccine targets including Dyp-type peroxidases, fucosyltransferases, G-protein coupled receptors, leishmanolysins, tetraspanins, and the netrin/netrin receptor complex [59]. Some of these candidates (fucosyltransferases, leishmanolysins, and tetraspanins) are members of protein families, which are expanded in the S. mansoni predicted proteome in comparison with other metazoans as inferred by phylogenomic analysis ( Leishmanolysins, which are members of the metallopeptidase M8 family, were analysed in detail corroborating the potential of these enzymes as future therapeutic targets [45]. Another study identified upregulated genes during the first five days of schistosomula development in vitro that are involved in blood feeding, tegument and cytoskeletal development, cell adhesion, and stress responses [8]. The highly upregulated genes included a tegument tetraspanin Sm-tsp-3, a protein kinase, a novel serine protease and serine protease inhibitor, and intestinal proteases belonging to distinct mechanistic classes. Other groups have analysed immunity towards Schistosoma using protein microarrays as a vaccine discovery tool. They selected a subset of proteins from genomic, transcriptomic, and proteomic data to perform experimental validation aiming at identifying novel antischistosome vaccine candidates [6, 61]. Recently, an elegant approach to investigate specific tissuessuchasgastrodermisandbothgendersreproductive tissues was performed by applying laser microdissection microscopy combined to microarray. These studies brought the perspective of expediting tissue-specific profiling [62, 63]. Additionally, expression profiles of S. japonicum were explored in studies comparing two Chinese isolates (Anhui and Zhejiang), as well as the Anhui Chinese and Philippines isolates [55, 64]. Later, an interspecies study compared the S. japonicum and S. mansoni transcriptomes. Surprisingly, despite their large phenotypic differences, there was a reduced number of differentially expressed genes based on the expression profile, which might reflect a limitation of the technique (preferential hybridization related to species polymorphisms) [8]. Bypassing these pitfalls, the next generation sequencing platforms allow exploiting expression data (RNA-seq) of any organism without previous knowledge of the genome or transcriptome and, further, will reveal new features of the parasite transcriptional landscape. The most recent achievement in this area was the refinement of the genomics and transcriptomics data using a combination of Sanger capillary sequencing, next generation DNA sequencing, genetic markers, and RNA-seq data from several S. mansoni life stages [19]. A previous study, explored

37 6 Journal of Parasitology Research the transcriptome of S. mansoni adult males obtaining 11 novel microexon genes (MEGs), as well as mapping 989 and 1196 contigs to intergenic and intronic genomic regions, respectively. This data could represent new protein-coding genes or noncoding RNAs (ncrnas) [65]. Together, these approaches have significantly advanced our understanding of the dynamics parasite transcriptomes [9, 65]. However, there is a need to polish and translate sequence data and gene expression profiles into functional information by computational predictions and experimental characterization. The vast amount of available data opened a myriad of possibilities towards the elucidation of gene functions. It is time for data integration Posttranscriptional Mechanisms. Transcriptomics is also a key approach to study posttranscriptional regulatory mechanisms such as alternative and transsplicing [57, 66 68]. It is known that schistosomes use such mechanisms to keep their intricate life cycle. Alternative splicing of transcripts of secreted proteins, like MEGs and polymorphic mucin isoforms, has been largely studied in schistosomes [51, 66]. This posttranscriptional processing has been proposed as a distinct genetic system to generate protein variation that would allow parasite adaptation to the immune response of different hosts. Additionally, alternative splicing has been shown to generate protein variation involved in transcriptional control and splicing [57]. In the case of SmCA15, a spliceosome interacting transcriptional cofactor, it could impact many other transcripts leading to several effects in the parasite, especially in sexual dimorphism. Spliced leader (SL) transsplicing has been shown to be an important posttranscriptional regulation mechanism for S. mansoni [69]. At least 11% of all transcripts has been described as undergoing transsplicing in this organism [19]. SL-RNAs are small ncrnas, products of small intronless genes, transcribed by DNA polymerase II and tandemly repeated in the genome [67]. Differently from nematodes, S. mansoni carries a single SL sequence, which is composed by 36 nucleotides with an 3 -terminal AUG codon. Although the role of transsplicing is poorly understood, there is an evidence suggesting that SL transsplicing would function providing an initiator methionine for translation initiation of some recipient mrnas in Platyhelminthes [68]. This mechanism is also involved in the resolution of polycistronic operons in monocistronic transcripts in S. mansoni [19, 7] Transcriptome Data and Gene Function. Despite the great potential revealed by the increasing number of genome and transcriptome studies in schistosomes, there are still several Schistosoma species that remain neglected. In contrast to the vast amount of data available in public databases, limited data has been decoded in functional studies and/or in 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 schistosomes life cycle, the complex interaction with their hosts, time and labor-demand functional studies, the difficulties to nurture the complete life cycle in vitro, and the absence of immortalized cell lines, which has delayed the development of transgenesis tools and models [71]. Lately, the posttranscriptional suppression of genes through RNA interference (RNAi) techniques has been a promise to fetch progress on new interventions for schistosomiasis and other helminthiases. In 23, two pioneer investigations marked the use of this technique in S. mansoni research. Simultaneously gene knockdown of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a glucose transporter (SGTP1) in the larval stage (sporocysts) [72] and cathepsin B in adult worms [73]ofS. mansoni were reported. Currently, this powerful technique is widely used in schistosomes and has been employed in two studies of functional screening. The first study encompassed the exposure of sporocysts by soaking parasites to dsrna targeting 33 genes, 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 11 different genes, it showed that, in S. mansoni, there is a geneto-gene variability and exposure to dsrna could trigger gene specific upregulation. Later, in a different study using electroporation to expose schistosomula to dsrna of 11 target genes (expressed in distinct tissues), the efficiency of RNAi in a target specific fashion was observed [75]. Other issues with the use of RNAi have been widely discussed, not only in schistosomes, but also in other helminth parasites, showing that care should be taken when conducting and evaluating such experiments [76]. Despite the difficulties in exploring gene function by RNAi in schistosomes, many interesting advances have been achieved. Many proteases (cathepsins and aminopeptidases) [77 79], kinases [8], multidrug resistance transporters [81], and type V collagen [82], among other proteins have been studied in diverse life stages of S. japonicum and S. mansoni. Recently, this tool has been described targeting tetraspanin 2 in diverse developmental stages of S. haematobium indicating the RNAi feasibility in this species [83]. Tetraspanins belong to a family of integral membrane proteins present in the outer surface membranes of the parasite tegument. Studies have shown that tetraspanins play important roles in the tegument development, maturation, or stability [84]. Importantly, the knockdown of peroxiredoxin [85] and thioredoxin glutathione reductase [86], antioxidant enzymes, were amongst the first reports of lethality after gene silencing in the parasite, demonstrating the potential of RNAi on drug target discovery. Moreover, enzymes such as glutathione-s-transferases (GST26 and GST28), peroxiredoxins (Prx1 and Prx2), and superoxide dismutase have been shown to play important roles in the protection of S. mansoni sporocysts in the host-parasite interplay. Knockdown of those targets increased parasite susceptibility to oxidative stress and to intermediate host immune cells as described elsewhere [87]. Recently, significant progress has been made in the area of schistosome transgenesis. Researchers are developing new

38 Journal of Parasitology Research 7 tools for the introduction and expression of homologous or heterologous gene constructs via lentiviruses, replicationdefective retroviruses, transposons, retrotransposons, and DNA plasmid vectors [9]. Sophisticated systems applied to assess gene function in the parasite are being achieved [88 91]. To date, eggs and miracidia are indicated as the preferred life stages for successful transformation approaches in order to enter the germline [92]. In summary, results from such independent studies have shown that schistosome transgenesis works in all the developmental stages tested and that some success has been achieved in integrating genes into the germline [88 92]. Thus, genetic knockdown experiments in schistosomes are possible. 5. Genomic and Transcriptomic Databases Altogether, the increasing information on schistosome genomes and transcriptomes is now raising the possibilities to better explore molecular techniques in the field of parasitology, genomics, and computational biology allowing the development of approaches to unravel mechanisms of parasite infection, host-parasite interaction, and so forth [21, 23, 24]. In order to accelerate both parasite research and development of effective tools for schistosomiasis diagnostics and treatment, user-friendly databases have been created and made publicly available to the scientific community [9, 93 95]. Here, we highlight some of these major resources. SchistoDB integrates genome and proteome sequence data along with functional annotation of genes and gene products of S. haematobium, S. japonicum, and S. mansoni [93]. This resource also covers results from large-scale analysis including ESTs, metabolic pathways, and candidate drug targets. HelmCoP (Helminth Control and Prevention) integrates functional, structural, and comparative genomics data of helminths from different taxonomic groups and distinct hosts (plant, animal, and human) [94]. HelmCoP offers a comprehensive suite of structural and functional annotations to support comparative analyses and host-parasite interaction studies. GeneDB is an annotation database that brings together data from a broad range of pathogens and closely related organisms [95]. GeneDB offers database-driven annotation tools and pipelines and includes manually curated information maintained by computational and biological scientists. Collectively, these computational resources support biological and biomedical research and open new frontiers in evolutionary and functional genomics, transcriptomics, and proteomics of Schistosoma species and other parasites. Besides biodiversity assessment at the molecular level, these resources provide a framework for the identification and prioritization of vaccine and drug targets against human diseases. As the scientific community progresses in data integration and interpretation, we will meet the goals of current 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 gaps of long-established approaches, enabling the use of a broad range of experimental and computational methods and contributing to dissect the biological basis of host-parasite interaction, diagnostics improvement, and the discovery of new drug and vaccine targets. Nuclear and mitochondrial genomes of some Schistosoma species were completely sequenced opening new frontiers for comparative analyses, which will improve our understanding of parasite biology and evolution. Other genomes to be analysed include Schistosoma species from each of the six clades currently recognized, and other representatives of the Schistosomatidae. Improving accuracy/quality of genome assembly and annotation will be crucial for such analyses. Despite the efforts in the characterization of genomes and transcriptomes of distinct Schistosoma species, many questions regarding the biology and evolution of this important taxon remain unanswered. These difficulties might be due to the size of schistosome genomes ( 1-times larger compared to protozoan parasites), the complexity of its life cycle, the presence of rare features in posttranscriptional regulation (e.g., transsplicing), and little integration of the current information available, among other reasons. There is an urgent need for data integration to help interpreting the massive amount of data generated over the years in this and other areas or research. The need for biocuration is essential to guide this process. We envision that, in the future, more transcriptomic and proteomic data will be used to improve the understanding of the origin and evolution of Schistosomatidae. The analysis of structural data generated by experimental or computational approaches will also shed light in the biodiversity of schistosomes at the molecular level. In conclusion, the availability of schistosome genome and transcriptome data has provided an unprecedented resource for many research areas. Limitations do exist and should be addressed to allow advances in understanding schistosome biology and evolution and ultimately support vaccine and drug development against schistosomiasis. List of Abbreviations atp6: ATP synthase F subunit6 atp8: ATP synthase F subunit 8 bp: Base pairs cob: Apocytochrome b cox1: Cytochrome c oxidase subunit 1 cox1 3: Cytochrome c oxidase subunit 1 to 3 DALY: Disability-adjusted life years dbest: Database of expressed sequence tags dsrna: Double strand DNA ESTs: Expressed sequence tags MEGs: Microexon genes nad1 6: NADH dehydrogenase subunit 1 to 6 nad4l: NADH dehydrogenase subunit 4L ncrnas: Noncoding RNAs ORESTEs: Open reading frame expressed sequence tags

39 8 Journal of Parasitology Research RNAi: RNA interference RNA-seq: RNA sequencing SAGE: Serial analysis of gene expression SL: Spliced leader TDR: Special Programme for Research and Training in Tropical Diseases UNDP: United Nations Development Programme UNICEF: United Nations Children s Fund WHO: World Health Organization Acknowledgments The research leading to these results has received funding from the European Community s Seventh Framework Programme (FP7/ FP7/27 211) under Grant agreement number This work was also partially supported by the National Institutes of Health- NIH/Fogarty International Center (TW712 to GO), the National Council for Research and Development, CNPq (CNPq Research Fellowship 36879/29-3 to GO, INCT- DT /28-5 to GO, CNPq-Universal 47636/21- to LAN, and 48576/21-6 to MMM), and the Research Foundation of the State of Minas Gerais (FAPEMIG) (CBB- 1181/8 and PPM to GO and CBB-APQ-1715/11 to MMM). The authors are very grateful to Larissa Lopes Silva and two anonymous reviewers for their constructive feedback and valuable suggestions, which greatly improved this paper. References [1] B. Gryseels, Schistosomiasis, Infectious Disease Clinics of North America, vol. 26, no. 2, pp , 212. [2] T. Huyse, B. L. Webster, S. Geldof et al., Bidirectional introgressive hybridization between a cattle and human schistosome species, PLoS Pathogens, vol. 5, no. 9, Article ID e1571, 29. [3] F. J. Kruger and A. C. Evans, Do all human urinary infections with Schistosoma mattheei represent hybridization between S. haematobium and S. mattheei? Journal of Helminthology, vol. 64, no. 4, pp , 199. [4] M. L. Steinauer, M. S. Blouin, and C. D. Criscione, Applying evolutionary genetics to schistosome epidemiology, Infection, Genetics and Evolution, vol. 1, no. 4, pp , 21. [5] A. Fenwick and J. P. Webster, Schistosomiasis: challenges for control, treatment and drug resistance, Current Opinion in Infectious Diseases, vol. 19, no. 6, pp , 26. [6] World Health Organization, Weekly Epidemiological Record, WHO, 87th edition, 212. [7] World Health Organization, 212, info/global burden disease/estimates regional 22 original/ en/. [8] G. N. Gobert, Applications for profiling the schistosome transcriptome, Trends in Parasitology, vol. 26, no. 9, pp , 21. [9] M. M. Mourão, C. Grunau, P. T. Loverde, M. K. Jones, and G. Oliveira, Recent advances in Schistosoma genomics, Parasite Immunology, vol. 34, no. 2-3, pp , 212. [1] M. T. Swain, D. M. Larkin, C. R. Caffrey et al., Schistosoma comparative genomics: integrating genome structure, parasite biology and anthelmintic discovery, Trends in Parasitology, vol. 27, no. 12, pp , 211. [11] G. Oliveira, G. Franco, and S. Verjovski-Almeida, The Brazilian contribution to the study of the Schistosoma mansoni transcriptome, Acta Tropica, vol. 18, no. 2-3, pp , 28. [12]P.T.LoVerde,H.Hirai,J.M.Merrick,N.H.Lee,andN. El-Sayed, Schistosoma mansoni genome project: an update, Parasitology International, vol. 53, no. 2, pp , 24. [13] G.R.Franco,M.D.Adams,M.B.Soares,A.J.G.Simpson,J. C. Venter, and S. D. J. Pena, Identification of new Schistosoma mansoni genes by the EST strategy using a directional cdna library, Gene, vol. 152, no. 2, pp , [14] A. J. G. Simpson, A. Sher, and T. F. McCutchan, The genome of Schistosoma mansoni: isolation of DNA, its size, bases and repetitive sequences, Molecular and Biochemical Parasitology, vol. 6, no. 2, pp , [15] A. I. Grossman, R. B. Short, and G. D. Cain, Karyotype evolution and sex chromosome differentiation in schistosomes (trematoda, schistosomatidae), Chromosoma, vol. 84, no. 3, pp , [16]M.Berriman,B.J.Haas,P.T.Loverdeetal., Thegenome of the blood fluke Schistosoma mansoni, Nature, vol. 46, no. 7253, pp , 29. [17] Y. Zhou, H. Zheng, Y. Chen et al., The Schistosoma japonicum genome reveals features of host-parasite interplay, Nature, vol. 46, no. 7253, pp , 29. [18] N. D. Young, A. R. Jex, B. Li et al., Whole-genome sequence of Schistosoma haematobium, Nature Genetics, vol. 44, no. 2, pp , 212. [19] A. V. Protasio, I. J. Tsai, A. Babbage et al., A systematically improved high quality genome and transcriptome of the human blood fluke Schistosoma mansoni, PLoS Neglected Tropical Diseases, vol. 6, no. 1, Article ID e1455, 212. [2] C. D. Criscione, C. L. L. Valentim, H. Hirai, P. T. LoVerde, and T. J. C. Anderson, Genomic linkage map of the human blood fluke Schistosoma mansoni, Genome Biology, vol. 1, no. 6, article R71, 29. [21] A. Tait, Genetics and genomics converge on the human blood fluke, Genome Biology, vol. 1, no. 6, article 225, 29. [22] J. M. J. Lepesant, D. Roquis, R. Emans et al., Combination of de novo assembly of massive sequencing reads with classical repeat prediction improves identification of repetitive sequences in Schistosoma mansoni, ExperimentalParasitology, vol. 13, no. 4, pp , 212. [23] R. A. Wilson, P. D. Ashton, S. Braschi, G. P. Dillon, M. Berriman, and A. Ivens, Oming in on schistosomes: prospects and limitations for post-genomics, Trends in Parasitology, vol. 23, no. 1, pp. 14 2, 27. [24] P. J. Brindley, M. Mitreva, E. Ghedin, and S. Lustigman, Helminth genomics: the implications for human health, PLoS Neglected Tropical Diseases, vol. 3, no. 1, article e538, 29. [25] S. P. Lawton, H. Hirai, J. E. Ironside, D. A. Johnston, and D. Rollinson, Genomes and geography: genomic insights into the evolution and phylogeography of the genus Schistosoma, Parasites & Vectors, vol. 4, article 131, 211. [26] A. Waeschenbach, B. L. Webster, and D. T. J. Littlewood, Adding resolution to ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with large fragments of mtdna, Molecular Phylogenetics and Evolution, vol. 63, no. 3, pp , 212. [27] M. Z. Zarowiecki, T. Huyse, and D. T. J. Littlewood, Making the most of mitochondrial genomes markers for phylogeny,

40 Journal of Parasitology Research 9 molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea), International Journal for Parasitology, vol. 37, no. 12, pp , 27. [28] G.-H. Zhao, J. Li, H.-Q. Song et al., A specific PCR assay for the identification and differentiation of Schistosoma japonicum geographical isolates in mainland China based on analysis of mitochondrial genome sequences, Infection, Genetics and Evolution, vol. 12, no. 5, pp , 212. [29] J. R. Stothard, H. Ameri, I. S. Khamis, L. Blair, U. S. Nyandindi, R. A. Kane et al., Parasitological and malacological surveys reveal urogenital Schistosomiasis on Mafia Island, Tanzania to be an imported infection, Acta Tropica. In press. [3] D.T.J.Littlewood,A.E.Lockyer,B.L.Webster,D.A.Johnston, and T. H. Le, The complete mitochondrial genomes of Schistosoma haematobium and Schistosoma spindale and the evolutionary history of mitochondrial genome changes among parasitic flatworms, Molecular Phylogenetics and Evolution, vol. 39, no. 2, pp , 26. [31] T. H. Le, D. Blair, and D. P. McManus, Mitochondrial genomes of parasitic flatworms, Trends in Parasitology, vol. 18, no. 5, pp , 22. [32]T.H.Le,P.F.Humair,D.Blair,T.Agatsuma,D.T.J.Littlewood, and D. P. McManus, Mitochondrial gene content, arrangement and composition compared in African and Asian schistosomes, Molecular and Biochemical Parasitology, vol. 117, no. 1, pp , 21. [33] Y. Vallès and J. L. Boore, Lophotrochozoan mitochondrial genomes, Integrative and Comparative Biology, vol. 46, no. 4, pp , 26. [34] S. W. Attwood, Studies on the parasitology, phylogeography and the evolution of host-parasite interactions for the snail intermediate hosts of medically important trematode genera in Southeast Asia, Advances in Parasitology, vol. 73, pp , 21. [35] B. L. Webster, V. R. Southgate, D. Timothy, and J. Littlewood, A revision of the interrelationships of Schistosoma including the recently described Schistosoma guineensis, International Journal for Parasitology, vol. 36, no. 8, pp , 26. [36] M. L. Steinauer, I. N. Mwangi, G. M. Maina et al., Interactions between natural populations of human and rodent schistosomes in the lake victoria region of Kenya: a molecular epidemiological approach, PLoS Neglected Tropical Diseases, vol. 2, no. 4, article e222, 28. [37] T. Agatsuma, Origin and evolution of Schistosoma japonicum, Parasitology International, vol. 52, no. 4, pp , 23. [38]J.A.T.Morgan,R.J.Dejong,S.D.Snyder,G.M.Mkoji, and E.S.Loker, Schistosoma mansoni and Biomphalaria: past history and future trends, Parasitology, vol. 123, supplement, pp. S211 S228, 21. [39] D. Rollinson, A. Kaukas, D. A. Johnston, A. J. G. Simpson, and M. Tanaka, Some molecular insights into schistosome evolution, International Journal for Parasitology, vol. 27, no. 1, pp , [4] L. Despres, D. Imbert-Establet, C. Combes, and F. Bonhomme, Molecular evidence linking hominid evolution to recent radiation of schistosomes (Platyhelminthes: Trematoda), Molecular Phylogenetics and Evolution, vol. 1, no. 4, pp , [41] L. A. Nahum and S. L. Pereira, Phylogenomics, protein family evolution, and the tree of life: an integrated approach between molecular evolution and computational intelligence, Studies in Computational Intelligence, vol. 122, pp , 28. [42] L. F. Andrade, L. A. Nahum, L. G. A. Avelar et al., Eukaryotic Protein Kinases (epks) of the Helminth Parasite Schistosoma mansoni, BMC Genomics, vol. 12, article 215, 211. [43] L. G. Avelar, L. A. Nahum, L. F. Andrade, and G. Oliveira, Functional diversity of the Schistosoma mansoni tyrosine kinases, Journal of Signal Transduction, vol. 211, Article ID 6329, 11 pages, 211. [44]L.Anderson,R.J.Pierce,andS.Verjovski-Almeida, Schistosoma mansoni histones: from transcription to chromatin regulation; An in silico analysis, Molecular and Biochemical Parasitology, vol. 183, no. 2, pp , 212. [45] L. L. Silva, M. Marcet-Houben, A. Zerlotini, T. Gabaldón, G. Oliveira, and L. A. Nahum, Evolutionary histories of expanded peptidase families in Schistosoma mansoni, Memorias do Instituto Oswaldo Cruz, vol. 16, no. 7, pp , 211. [46]W.Wu,E.G.Niles,H.Hirai,andP.T.LoVerde, Evolution of a novel subfamily of nuclear receptors with members that each contain two DNA binding domains, BMC Evolutionary Biology, vol. 7, article 27, 27. [47] L. Otero, M. Bonilla, A. V. Protasio, C. Fernández, V. N. Gladyshev, and G. Salinas, Thioredoxin and glutathione systems differ in parasitic and free-living platyhelminths, BMC Genomics, vol. 11, no. 1, article 237, 21. [48] C. J. Standley and J. R. Stothard, DNA barcoding of Schistosoma cercariae reveals a novel sub-lineage within Schistosoma rodhaini from Ngamba Island Chimpanzee Sanctuary, Lake Victoria, Journal of Parasitology, vol. 98, no. 5, pp , 212. [49] B. L. Webster, C. L. Culverwell, I. S. Khamis, K. A. Mohammed, D. Rollinson, and J. R. Stothard, DNA barcoding of Schistosoma haematobium on Zanzibar reveals substantial genetic diversity and two major phylogenetic groups, Acta Tropica. In press. [5] G. R. Franco, A. F. Valadão, V. Azevedo, and E. M. L. Rabelo, The Schistosoma gene discovery program: state of the art, International Journal for Parasitology, vol. 3, no. 4, pp , 2. [51] S. Verjovski-Almeida, R. DeMarco, E. A. L. Martins et al., Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni, Nature Genetics, vol. 35, no. 2, pp , 23. [52] E. P. B. Ojopi, P. S. L. Oliveira, D. N. Nunes et al., A quantitative view of the transcriptome of Schistosoma mansoni adult-worms using SAGE, BMC Genomics, vol. 8, article 186, 27. [53] D. L. Williams, A. A. Sayed, J. Bernier et al., Profiling Schistosoma mansoni development using serial analysis of gene expression (SAGE), Experimental Parasitology, vol. 117, no. 3, pp , 27. [54] J. M. Fitzpatrick and K. F. Hoffmann, Dioecious Schistosoma mansoni express divergent gene repertoires regulated by pairing, International Journal for Parasitology, vol. 36, no. 1-11, pp , 26. [55] L. Moertel, D. P. McManus, T. J. Piva, L. Young, R. L. McInnes, and G. N. Gobert, Oligonucleotide microarray analysis of strain- and gender-associated gene expression in the human blood fluke, Schistosoma japonicum, Molecular and Cellular Probes, vol. 2, no. 5, pp , 26. [56] M. Waisberg, F. P. Lobo, G. C. Cerqueira et al., Schistosoma mansoni: microarray analysis of gene expression induced by host sex, Experimental Parasitology, vol. 12, no. 4, pp , 28.

41 1 Journal of Parasitology Research [57] R. DeMarco, K. C. Oliveira, T. M. Venancio, and S. Verjovski- Almeida, Gender biased differential alternative splicing patterns of the transcriptional cofactor CA15 gene in Schistosoma mansoni, Molecular and Biochemical Parasitology, vol. 15, no. 2, pp , 26. [58] E. R. Jolly, C. S. Chin, S. Miller et al., Gene expression patterns during adaptation of a helminth parasite to different environmental niches, Genome Biology, vol. 8, no. 4, article R65, 27. [59] J. M. Fitzpatrick, E. Peak, S. Perally et al., Anti-schistosomal intervention targets identified by lifecycle transcriptomic analyses, PLoS Neglected Tropical Diseases, vol. 3, no. 11, article e543, 29. [6] P. Driguez, D. L. Doolan, A. Loukas, P. L. Felgner, and D. P. McManus, Schistosomiasis vaccine discovery using immunomics, Parasites and Vectors, vol. 3, no. 1, article 4, 21. [61] H. E. G. McWilliam, P. Driguez, D. Piedrafita, D. P. McManus, and E. N. T. Meeusen, Novel immunomic technologies for schistosome vaccine development, Parasite Immunology, vol. 34, no. 5, pp , 212. [62]G.N.Gobert,D.P.McManus,S.Nawaratna,L.Moertel, J. Mulvenna, and M. K. Jones, Tissue specific profiling of females of Schistosoma japonicum by integrated laser microdissection microscopy and microarray analysis, PLoS Neglected Tropical Diseases, vol. 3, no. 6, article e469, 29. [63] S. S. K. Nawaratna, D. P. McManus, L. Moertel, G. N. Gobert, and M. K. Jones, Gene atlasing of digestive and reproductive tissues in Schistosoma mansoni, PLoS Neglected Tropical Diseases, vol. 5, no. 4, Article ID e143, 211. [64] M. Hope, M. Duke, and D. P. McManus, A biological and immunological comparison of Chinese and Philippine Schistosoma japonicum, International Journal for Parasitology, vol. 26, no. 3, pp , [65] G. T. Almeida, M. S. Amaral, F. C. F. Beckedorff, J. P. Kitajima, R. DeMarco, and S. Verjovski-Almeida, Exploring the Schistosoma mansoni adult male transcriptome using RNA-seq, Experimental Parasitology, vol. 132, no. 1, pp , 211. [66] S. Verjovski-Almeida and R. DeMarco, Gene structure and splicing in schistosomes, Journal of Proteomics, vol.74,no.9, pp , 211. [67] K. E. M. Hastings, SL trans-splicing: easy come or easy go? Trends in Genetics, vol. 21, no. 4, pp , 25. [68] G. Cheng, L. Cohen, D. Ndegwa, and R. E. Davis, The flatworm spliced leader 3 -terminal AUG as a translation initiator methionine, The Journal of Biological Chemistry, vol. 281, no. 2, pp , 26. [69] R. E. Davis, C. Hardwick, P. Tavernier, S. Hodgson, and H. Singh, RNA trans-splicing in flatworms. Analysis of transspliced mrnas and genes in the human parasite, Schistosoma mansoni, The Journal of Biological Chemistry, vol. 27, no. 37, pp , [7] R. E. Davis and S. Hodgson, Gene linkage and steady state RNAs suggest trans-splicing may be associated with a polycistronic transcript in Schistosoma mansoni, Molecular and Biochemical Parasitology, vol. 89, no. 1, pp , [71] P. J. Brindley and E. J. Pearce, Genetic manipulation of schistosomes, International Journal for Parasitology, vol. 37, no. 5, pp , 27. [72] J. P. Boyle, X. J. Wu, C. B. Shoemaker, and T. P. Yoshino, Using RNA interference to manipulate endogenous gene expression in Schistosoma mansoni sporocysts, Molecular and Biochemical Parasitology, vol. 128, no. 2, pp , 23. [73] P. J. Skelly, A. Da dara, and D. A. Harn, Suppression of cathepsin B expression in Schistosoma mansoni by RNA interference, International Journal for Parasitology, vol. 33, no. 4, pp , 23. [74] M. M. Mourão, N. Dinguirard, G. R. Franco, and T. P. Yoshino, Phenotypic screen of early-developing larvae of the blood fluke, Schistosoma mansoni, using RNA interference, PLoS Neglected Tropical Diseases, vol. 3, no. 8, article e52, 29. [75] S. Štefanic, J. Dvořák, M. Horn et al., RNA interference in Schistosoma mansoni schistosomula: selectivity, sensitivity and operation for larger-scale screening, PLoS Neglected Tropical Diseases, vol. 4, no. 1, article e85, 21. [76] P. Geldhof, A. Visser, D. Clark et al., RNA interference in parasitic helminths: current situation, potential pitfalls and future prospects, Parasitology, vol. 134, no. 5, pp , 27. [77]J.M.Correnti,P.J.Brindley,andE.J.Pearce, Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth, Molecular and Biochemical Parasitology, vol. 143, no. 2, pp , 25. [78] M. E. Morales, G. Rinaldi, G. N. Gobert, K. J. Kines, J. F. Tort, and P. J. Brindley, RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade, Molecular and Biochemical Parasitology, vol. 157, no. 2, pp , 28. [79] G. Rinaldi, M. E. Morales, Y. N. Alrefaei et al., RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs, Molecular and Biochemical Parasitology, vol. 167, no. 2, pp , 29. [8] B. E. Swierczewski and S. J. Davies, A schistosome campdependent protein kinase catalytic subunit is essential for parasite viability, PLoS Neglected Tropical Diseases, vol. 3, no. 8, article e55, 29. [81] R.S.Kasinathan,W.M.Morgan,andR.M.Greenberg, Genetic knockdown and pharmacological inhibition of parasite multidrug resistance transporters disrupts EGG production in Schistosoma mansoni, PLoS NeglectedTropical Diseases, vol. 5, no. 12, Article ID e1425, 211. [82] Y. Yang, Y. Jin, P. Liu et al., RNAi silencing of type v collagen in Schistosoma japonicum affects parasite morphology, spawning, and hatching, Parasitology Research, vol. 111, no. 3, pp , 212. [83] G. Rinaldi, T. I. Okatcha, A. Popratiloff et al., Genetic manipulation of Schistosoma haematobium, the neglected Schistosome, PLoS Neglected Tropical Diseases, vol. 5, no. 1, Article ID e1348, 211. [84]M.H.Tran,T.C.Freitas,L.Cooperetal., Suppression of mrnas encoding tegument tetraspanins from Schistosoma mansoni results in impaired tegument turnover, PLoS Pathogens, vol. 6, no. 4, Article ID e184, 21. [85] A. A. Sayed, S. K. Cook, and D. L. Williams, Redox balance mechanisms in Schistosoma mansoni rely on peroxiredoxins and albumin and implicate peroxiredoxins as novel drug targets, The Journal of Biological Chemistry, vol. 281, no. 25, pp , 26. [86] A. N. Kuntz, E. Davioud-Charvet, A. A. Sayed et al., Thioredoxin glutathione reductase from Schistosoma mansoni: an essential parasite enzyme and a key drug target, PLoS Medicine, vol. 4, no. 6, Article ID e26, 27. [87] M. D. M. Mourão, N. Dinguirard, G. R. Franco, and T. P. Yoshino, Role of the endogenous antioxidant system in the protection of Schistosoma mansoni primary sporocysts against exogenous oxidative stress, PLoS Neglected Tropical Diseases, vol. 3, no. 11, article e55, 29.

42 Journal of Parasitology Research 11 [88] M. A. Ayuk, S. Suttiprapa, G. Rinaldi, V. H. Mann, C. M. Lee, and P. J. Brindley, Schistosoma mansoni U6 gene promoterdriven short hairpin RNA induces RNA interference in human fibrosarcoma cells and schistosomules, International Journal for Parasitology, vol. 41, no. 7, pp , 211. [89] R. Duvoisin, M. A. Ayuk, G. Rinaldi et al., Human U6 promoter drives stronger shrna activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells, Transgenic Research, vol. 21, no. 3, pp , 212. [9] G. Rinaldi, S. Suttiprapa, J. F. Tort, A. E. Folley, D. E. Skinner, and P. J. Brindley, An antibiotic selection marker for schistosome transgenesis, International Journal for Parasitology, vol. 42, no. 1, pp , 212. [91] S. Beckmann and C. G. Grevelding, Paving the way for transgenic schistosomes, Parasitology, vol. 139, no. 5, pp , 212. [92] G. Rinaldi, S. E. Eckert, I. J. Tsai et al., Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus, PLoS Pathogens, vol. 8, no. 7, Article ID e1282, 16 pages, 212. [93] A. Zerlotini, M. Heiges, H. Wang et al., SchistoDB: a Schistosoma mansoni genome resource, Nucleic Acids Research, vol. 37, no. 1, pp. D579 D582, 29. [94] S. Abubucker, J. Martin, C. M. Taylor, and M. Mitreva, Helm- CoP: an online resource for Helminth functional genomics and drug and vaccine targets prioritization, PLoS ONE, vol. 6, no. 7, Article ID e21832, 211. [95] F. J. Logan-Klumpler, N. De Silva, U. Boehme et al., GeneDBan annotation database for pathogens, Nucleic Acids Research, vol. 4, no. D1, pp. D98 D18, 212.

43 Hindawi Publishing Corporation Journal of Parasitology Research Volume 212, Article ID 5238, 1 pages doi:1.1155/212/5238 Clinical Study Changes in T-Cell and Monocyte Phenotypes In Vitro by Schistosoma mansoni Antigens in Cutaneous Leishmaniasis Patients Aline Michelle Barbosa Bafica, 1 Luciana Santos Cardoso, 1, 2 Sérgio Costa Oliveira, 3, 4 Alex Loukas, 5 Alfredo Góes, 3 RicardoRiccioOliveira, 1 Edgar M. Carvalho, 1, 4, 6 and Maria Ilma Araujo1, 4, 6 1 Serviço de Imunologia, Complexo Hospitalar Universitário Professor Edgard Santos, Universidade Federal da Bahia, 5RuaJoão das Botas s/n, Canela, Salvador, BA, Brazil 2 Departamento de Ciências da Vida, Universidade do Estado da Bahia, 2555 Rua Silveira Martins, Cabula, Salvador, BA, Brazil 3 Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 6627 Avenida Antônio Carlos, Pampulha, Belo Horizonte, MG, Brazil 4 Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais (INCT-DT-CNPQ/MCT), Rua João das Botas s/n, Canela, 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 Saúde Pública, No. 275 Avenida Dom João VI, Brotas, 429- Salvador, BA, Brazil Correspondence should be addressed to Aline Michelle Barbosa Bafica, alinebafica@gmail.com Received 13 July 212; Revised 24 September 212; Accepted 8 October 212 Academic Editor: Andrea Teixeira-Carvalho Copyright 212 Aline Michelle Barbosa Bafica et al. 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. 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. In the 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 the S. mansoni antigens rsm29, rsmtsp-2- and PIII. Cells were stained with fluorochrome conjugated antibodies and analyzed by flow 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 CD8 expression in nonclassical and CD86 expression in intermediate monocytes, respectively. These two antigens increased the expression of CTLA-4 in CD4 + T cells and they also expanded the frequency of CD4 + CD25 high Foxp3 + 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 of modulatory molecules in T lymphocytes. 1. Introduction American tegumentary leishmaniasis is a disease caused by parasites of the genus Leishmania. This disease represents a significative public health problem worldwide with an incidence of 1.5 million new cases in recent years [1]. Tegumentary leishmaniasis presents with a wide spectrum of clinical manifestations ranging from localized skin to widespread mucocutaneous lesions depending on the parasite species [2] and the host immune response [3, 4]. T cell-mediated immunity is crucial to host protection against Leishmania sp. infection; however skin and mucosal lesions

44 2 Journal of Parasitology Research occurs due to a deregulated T helper 1 (Th1) cell response with high production of proinflammatory cytokines such as IFN-γ and TNF [3, 4]. Experimental studies have shown that Schistosoma mansoni infection or parasite products, by inducing regulatory cells and cytokines, are able to prevent some Th1-mediated autoimmune 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 downmodulated the production of IFN-γ and TNF in a group of cutaneous leishmaniasis (CL) patients [8]. In the present study, these antigens were tested regarding their ability to alter the monocyte and lymphocyte profiles. Studies have shown that Sm29 and SmTSP-2 antigens are secreted by the membrane and/or tegument of the S. mansoni adult worm. Proteins secreted or localized on the surface of Schistosoma spp., which are in intimate contact with host tissues, might be more effective in triggering immunoregulatory processes [9]. The Sm29 is a membrane-bound glycoprotein located on the tegument of the adult worm and lung stage schistosomula [1]. SmTSP-2 is a recombinant protein (tetraspanin) from S. mansoni tegument. In a mice model, immunization with SmTSP-2 resulted in a 57% reduction in adult worm burdens and a 64% reduction in liver egg burdens compared with control animals [11]. PIII is a multivalent antigen obtained from S. mansoni adult worms that modulates granuloma size in mice infected with the parasite [12, 13]. These antigens have been evaluated by our group regarding their potential to induce IL-1 production and suppress Th2 response in vitro in cells of asthmatic individuals [14]. Together with the Th1 immune response, monocytes and macrophage are key cells in controlling Leishmania sp. infection. Monocytes have been classifying into different subpopulations in mice models and also in humans. A new nomenclature was recently published defining human monocytes into three subtypes. The major population of human monocytes (9%) presents high expression of CD14 and lack of expression of CD16 (CD14 ++ CD16 ). They are referred to as classical monocytes. Intermediate monocytes are those which express CD14 and low CD16 expression (CD14 ++ CD16 + ), while the nonclassical monocytes express CD16 and relatively low CD14 (CD14 + CD16 ++ )[15]. In a study conducted by Wong et al. [16] the characteristics of classical, intermediate and, nonclassical monocytes were evaluated through the gene expression profiling. They observed that classical monocytes express genes involved in angiogenesis, wound healing, and coagulation, being involved in tissue repair functions in addition to high expression of proinflammatory genes [16]; intermediate monocytes highly express MHC class II indicating they have antigen presenting cell function and T cell stimulatory properties. Nonclassical monocytes express genes involved in cytoskeleton rearrangement, which may be responsible for its high motility observed in vivo [17]. A high frequency of CD16 + monocyte subsets have been demonstrated in Mycobacterium tuberculosis infection and in viral infections such as hepatitis B (HBV) [18], hepatitis C (HCV) [19], HIV [2], and Dengue [21]. In CL patients, the frequency of CD14 + CD16 + monocytes was significantly higher compared to healthy controls and they were positively correlated with the lesion size [22]. This subtype of monocytes is considered as antigen presenting cell [16], which produces cytokines and might be the most important subtype in activating T cells response. The activation of T cells requires the antigen recognition in the MHC context and also signaling given by costimulatory molecules which interact with corresponding ligands on antigen presenting cells (APC). One of the most important co-stimulatory molecules on T cells is CD28, which is constitutively expressed and binds to CD8 and CD86 on the APC. CD86 is constitutively expressed at low levels and it is rapidly upregulated after primary antigen recognition, whereas CD8 exhibits delayed expression kinetics [23]. Other ligand for CD8 and CD86 is CTLA-4. The expression of this molecule is rapidly upregulated after T cell activation and provides a negative signal limiting the immune response [24 26]. In vitro study has shown that the addition of CTLA-4-Ig to block CD28-B7 interaction in CL patients PBMC cultures stimulated with Leishmania antigen led to a downmodulation of IFN-γ and TNF secretion [27]. Other similar study showed that blocking the co-stimulatory molecules CD8 and CD86 in human macrophages from Leishmania-naive donors infected with L. major resulted in a significant reduction in IFN-γ, IL-5, and IL-12 production [28]. In this study we evaluated whether S. mansoni antigens alter the expression of CD8 and CD86 on monocytes of CL patients and also the expression of CTLA-4 in T lymphocytes. Additionally, we accessed the frequency of regulatory T cells induced by S. mansoni antigens when they were added to the cell cultures stimulated with Leishmania antigens. The CD4 + CD25 + regulatorytcellshavebeenextensively studied due to their critical function in maintaining selftolerance. These cells can express both low and high CD25 levels in mice models; however, only the CD4 + CD25 high population exhibits a strong regulatory function in humans. These CD4 + CD25 high T cells also express FOXP3, a molecule associated with regulatory functions [29]. This T cell subset in humans comprises 1.5 3% of circulating CD4 + T cells. They inhibit proliferation and cytokine secretion induced by TCR cross-linking of CD4 + CD25 responder T cells in a contact-dependent manner [3] and completely abrogate IL-2-dependent proliferation of NK cells [31]. Moreover, regulatory T cells are able to downregulate the intensity and duration of both Th1 and Th2 immune responses in infectious diseases limiting damage to self-tissue [32, 33]. Our hypothesis in the present study is that the pathology of cutaneous leishmaniasis results from monocyte and T cell hypersensitivity due to impaired regulatory mechanisms. In this context, the use of S. mansoni antigens which induce regulatory cells and molecules would prevent the inflammatory process. To test this hypothesis we evaluated whether the addition of S. mansoni antigens to cell cultures of leishmaniasis patients would modify the phenotype and activation status of monocytes and lymphocytes. Specifically, we evaluated the expression of HLA-DR, CD8, and CD86 on classical, intermediate, and nonclassical monocytes and

45 Journal of Parasitology Research 3 CD28, CTLA-4, CD25, and Foxp3 in T lymphocytes from CL patients in response to the soluble Leishmania braziliensis antigen (SLA) in the presence or absence of the S. mansoni antigens rsm29, rsmtsp-2, and PIII. 2. Materials and Methods 2.1. Patients and the Endemic Area. The study included the first 3 cutaneous leishmaniasis patients living in the endemic area of tegumentary leishmaniasis, Corte de Pedra, Bahia, Brazil, who attended the local Health Post from March 21 to March 212 and agreed in participate. Corte de Pedra is located in the southeast region of the State of Bahia, Brazil which is well known for its high rate of L. braziliensis transmission. The diagnostic of cutaneous leishmaniasis was made considering a clinical picture characteristic of CL, parasite isolation or a positive delayed-type hypersensitivity (DTH) response to Leishmania antigen, and a histological feature of CL. The inclusion criteria to this study were patients with 5 to 6 years of age diagnosis of CL with the presence of active skin lesions. The exclusion criteria were pregnancy, chronic diseases such as diabetes and asthma, and also HIV and HTLV-1 infection. Immunological analyses were performed prior to the specific therapy to leishmaniasis for all patients. There were not enough cells to perform the whole experiment every time since they require a larger number of cells more than what could be obtained from some patients. The frequency of helminth infection in the leishmaniasis endemic area of Corte de Pedra is 88.3% and S. mansoni infection presents in 16.7% of cutaneous leishmaniasis patients. The Ethical Committee of the Maternidade Climério de Oliveira, Federal University of Bahia approved the present study, and an informed consent was obtained from all participants or their legal guardians Antigen Preparation. The soluble Leishmania antigen used in this study was obtained from a Leishmania lysate (crude antigen). It was prepared from a L. braziliensis strain (MHOM/BR/21) as previously described [34]. The S. mansoni antigens used in this study included Sm29, a Schistosoma mansoni recombinant protein [1]; SmTSP-2, a recombinant protein (tetraspanin) from S. mansonitegument [11];afractionofS. mansoni soluble adult worm antigen (SWAP) obtained by anionic chromatography (FPLC), called PIII. The Sm29 was cloned in E. coli and tested for lipopolysaccharide (LPS) contamination using a commercially available LAL Chromogenic Kit (CAMBREX). ThelevelsofLPSinSm29werebelow.25ng/mL.However in order to neutralize potential effects of LPS presented in lowlevelsinthes. mansoni recombinant antigen, Polymyxin B was added to cell cultures every 12 hours according to the established protocol [35]. The SmTSP-2 used in this study was produced in Pichia pastoris fermentation cultures and it has been kindly provided by Dr. Alex Loukas [11, 36]. The proteins rsm29 and PIII were provided by the Institute of Biological Science, Department of Biochemistry and Immunology, UFMG, Brazil Cell Culture and Intracellular and Surface Staining. Intracellular and surface molecules were evaluated by flow cytometry (FACSort, BD Biosciences, San Jose, CA). In order to perform the intracellular staining, peripheral blood mononuclear cells (PBMC, ) obtained through the Ficoll-Hypaque gradient were cultured in vitro with SLA (5μg/mL) in the presence or absence of the recombinant S. mansoni antigens Sm29, SmTSP-2, and PIII (5 μg/ml) for 2 h, 37 C, and 5% of CO 2. During the last 4 h of culture, Brefeldin A (1 μg/ml; Sigma, St. Louis, MO), which impairs protein secretion by the Golgi complex, was added to the cultures. Afterwards, the cells were washed in PBS and fixed in 4% formaldehyde for 2 min at room temperature. Specific staining was performed with cychrome (CY)-labeled antibody conjugated with anti-ctla-4 mabs in saponin buffer (PBS, supplemented with.5% BSA and.5% saponin) and phycoerythrin (PE)-labeled antibody conjugated with anti-foxp3 in Foxp3 staining buffer set (ebioscience). For double or triple staining, mabs to human CD4, CD8, and/or CD25 conjugated with FITC or CY were used. The evaluation of co-stimulatory molecule expression was performed in PBMCs stimulated for 6 h with the same antigens aforementioned. Cells were incubated with fluorescein isothiocyanate (FITC)-, PE-, or CY-labeled antibody solutions for 2 min at 4 Cinavolumeof2μL in PBS. After staining, preparations were washed with.1% sodium azide PBS, fixed with 2 μl of 4% formaldehyde in PBS and kept at 4 C. The antibodies used for the staining were immunoglobulin isotype controls-fitc, PE and CY, anti-cd4-fitc, anti-cd8-fitc, anti-cd25-fitc, anti-cd14-fitc, CD16-CY, anti-cd4-cy and anti-cd8- CY, anti-cd8-pe, anti-cd86-pe, anti-hla-dr-pe, anti- CD28-PE (all from BD Biosciences Pharmingen). A total of 5, and 1, events were acquired for surface and intracellular experiments, respectively Analysis of FACS Data. The frequency of positive cells was analyzed by flow cytometry using the program flowjo in two regions. The lymphocyte region was determined using granularity (SSC) size (FSC) plot. Monocytes were selected based on their granularity and expression of CD14 and CD16. Limits for the quadrant markers were always set based on negative populations and isotype controls. A representative density graph of one experiment showing lymphocyte and monocyte regions is shown in Figures 1(a) and 1(b),respectively Statistical Analyses. Statistical analyses were performed using the software GraphPad Prism (GraphPad Software, San Diego, CA). The frequency of positive cells was expressed as percentages and the intensity of expression as mean intensity fluorescence (MFI). The differences between means

46 4 Journal of Parasitology Research 1K 1 4 Nonclassical SSC-H:: SSC-height FL3-H:: CD16PE-Cy Intermediate K FSC-H:: FSC-height (a) Classical FL1-H:: CD14 FITC (b) Figure 1: Strategy for T cell and monocytes evaluation by flow cytometry. The cell populations were defined by nonspecific fluorescence from the forward (FSC) and side scatter (SSC) as parameters of cell size and granularity, respectively. Lymphocytes region was determined using SSC FSC plot (G1) and monocytes region (G2) were selected based on their granularity and expression of CD14 (a). (b) represents the strategy for monocytes subsets classification through the expression of CD14 and CD16. Representative graph of one experiment. Table 1: Demographic characteristics of the cutaneous leishmaniasis patients included in the study. Characteristics (n = 3) Values Age, mean ± SD, years 29.1 ± 11.8 Sex, female/male 13/17 N of lesion 1, no. (%) 24 (8.) 2, no. (%) 6 (2.) Size of lesion, median mm 2 (IQR) 13 ( ) IQR: interquartile range; SD: standard deviation. were assessed using Wilcoxon matched pairs test. Statistical significance was established at the 95% confidence interval. 3. Results A total of 3 patients with cutaneous leishmaniasis were enrolled 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 (8%) and the median lesions size was 13 mm 2 (IQR, ; Table 1) The Effect of the Addition of S. mansoni Antigens on Monocyte Phenotype and Expression of Co-Stimulatory Molecules. We evaluated the frequency of different monocyte subsets (classical, intermediate, and nonclassical) and also the expression of co-stimulatory molecules in these monocytes after in vitro stimulation with SLA in the presence or absence of S. mansoni antigens. The addition of the antigens rsm29 and PIII to the cultures expanded the frequency of nonclassical (CD14 + CD16 ++ )(mean± SEM = 7.4 ± 1.2% and 8.1 ± 1.9%, resp.) compared to cultures stimulated with SLA alone (5.7 ±.9%; P <.5. Figure 2(A)). We also observed that the frequency of classical monocytes was higher in cultures without S. mansoni antigens (Figure 2(A)). Moreover, in the presence of PIII there was a reduction in the expression of HLA-DR in classical (CD14 ++ CD16 )and intermediate (CD14 ++ CD16 + ) monocytes (496±72 MFI and 544 ± 78.6MFI,resp.)comparedtoculturesstimulatedwith SLA alone (611 ± 91 MFI and 771 ± 128 MFI, respectively; P <.5. Figure 2(B)). There was also a reduction in HLA-DR expression on CD14 + CD16 ++ monocytes in the presence of rsm29 (547 ± 14.5 MFI) in comparison to SLA alone (718 ± MFI; P <.5. Figure 2(B)). Additionally, the expression of HLA-DR was higher in classical and intermediate monocytes in the presence of SLA or SLA plus S. mansoni antigens compared to nonstimulated cultures. The expression of CD8 was also reduced in nonclassical monocytes compared to cultures stimulated with SLA. The addition of PIII to the cultures also resulted in reduced expression of the co-stimulatory molecule CD8 on nonclassical monocytes (61.2 ± 19.5 MFI)comparedtocultures without S. mansoni antigens (82.3±23.3 MFI;P<.5. Figure 2(C)). Also a decrease in the expression of CD86 on intermediate monocytes from 562 ± MFI to ± MFI was observed when rsmtsp-2 was added to the cultures (P <.5; Figure 2(D)). There was no significant difference in the levels of CD86 expression on monocytes in culture stimulated with SLA after the addition of rsm29 and PIII (Figure 2(D)).

47 Journal of Parasitology Research 5 Frequency of monocyte (%) a a a b b HLA-DR expression in monocytes (MIF) a a a a b a a a b b Classical Intermediate Nonclassical (A) Classical Intermediate Nonclassical (B) 16 1 CD8 expression in monocytes (MIF) a b CD86 expression in monocytes (MIF) b Classical Intermediate Nonclassical Classical Intermediate Nonclassical WS SLA SLA+rSm29 SLA+rSmTSP-2 SLA+PIII WS SLA SLA+rSm29 SLA+rSmTSP-2 SLA+PIII (C) (D) Figure 2: Subsets of monocytes and co-stimulatory molecules expression in monocytes of CL patients (n = 18) stimulated in vitro with SLA in the presence or absence of S. mansoni antigens rsm29 and rsmtsp-2 or with a fraction of S. mansoni soluble adult worm antigen (SWAP) PIII. Frequency of classical (CD14 ++ CD16 ), intermediate (CD14 ++ CD16 + ) and nonclassical (CD14+CD16 ++ ), monocytes (A), and expression of HLA-DR, CD8, and CD86 in classical, intermediate and nonclassical monocytes of cutaneous leishmaniasis patients (B D). Cells were stained for surface expression of CD14, CD16, HLA-DR, CD8, and CD86 using flow cytometry. a P<.5 cultures without stimulation (WS) versus SLA + S. mansoni antigens; b P<.5 SLA versus SLA + S. mansoni antigens (Wilcoxon matched pairs test) The Effect of the Addition of S. mansoni Antigens on T Cell Phenotype and Expression of Co-Stimulatory Molecules. PBMC of CL patients were incubated with SLA in the presence or absence of S. mansoni antigens and the phenotype of T cells were evaluated. The addition of rsm29 antigen to the cultures increased the frequency of CD4 + T cells (mean ± SEM = 4.8 ± 2.8%) in comparison to cultures with SLA alone (34.8 ± 2.8%, P<.5; Figure 3(A)). There was no significant difference in the frequency of CD4 + T cells after the addition of rsmtsp-2 or PIII antigens to the cultures (37.8 ± 2.7% and 35.8 ± 2.6%, resp.) compared to SLA alone (34.8 ± 2.8%; Figure 3(A)). Likewise, there was no significant variation in the frequency of CD8 + T cells by the presence of rsm29, rsmtsp-2, and PIII antigens (5.9 ± 1.2%, 4.6±.8%, and 6.2±1.1%, resp.) to the cultures in relation to the cultures stimulated with SLA without S. mansoni antigens (5.7 ± 1.2%; Figure 3(A)). The frequency of CD4 + T cells was diminished by the presence of SLA and SLA plus rsmtsp-2 or PIII, while the frequency of CD8 + T cells was higher in the presence of SLA and SLA plus S. mansoni antigens comparedto nonstimulated cultures (Figure 3(A)). We also evaluated the expression of CD28 + on lymphocytes in cultures stimulated with SLA with or without the addition of S. mansoni antigens. The expression of CD28 + on CD8 + T cells was higher in cultures stimulated with rsm29 (91 ± 14 MFI, resp.) compared with SLA alone (84 ± 14 MFI; P<.5. Figure 3(B)). The addition of rsmtsp-2 and PIII antigens to the cultures did not alter significantly the expression of CD28 on CD4 + and CD8 + T cells (Figure 3(B)). Regarding the expression of CTLA-4, rsmtsp-2 and PIII antigens were able to increase the expression of this molecule in CD4 + T cells (58 ± 5MFIand53± 4.6MFI,resp.)incomparison with SLA alone (49.6 ± 4MFI,respectively;P<.5. Figure 3(C)). The expression of CTLA-4 was also higher in CD4 + T cells and CD8 + T cells in cultures stimulated with SLA plus rsmtsp-2 or PIII compared to nonstimulated cultures (Figure 3(C)). Moreover, the addition of rsmtsp-2 and PIII antigens to the cultures expanded the frequency of

48 6 Journal of Parasitology Research 6 16 Frequency of lymphocyte (%) 4 2 a b a a a a a a CD28 expression in lymphocyte (MFI) b TCD4 + TCD8 + TCD4 + TCD8 + (A) (B) CTLA-4 expression in lymphocyte (MIF) a b a b a a CD4 + CD25 high Foxp3 + cells (%) b a b TCD4 + TCD8 + WS SLA SLA+rSm29 SLA+rSmTSP-2 SLA+PIII WS SLA SLA+rSm29 SLA+rSmTSP-2 SLA+PIII (C) (D) Figure 3: Phenotype of T cells and co-stimulatory molecules expression on T lymphocytes of CL patients (n = 17) stimulated in vitro with 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 the frequency of CD4 + CD25 high Foxp3 + Tcells(n = 13) (D). Cells were stained for surface expression of CD4, CD8, CD25 and CD28, while the expression of CTLA-4 and Foxp3 were evaluated intracellularly using flow cytometry. a P<.5 cultures without stimulation (WS) versus SLA + S. mansoni antigens; b P<.5 SLA versus SLA + S. mansoni antigens (Wilcoxon matched pairs test). CD4 + CD25 high Foxp3 + T cells (1 ± 2.4% and 1.3 ± 1.9%, resp.) compared to cultures with SLA in the absence of S. mansoni antigens (8.1 ± 1.8%; P <.5. Figure 3(D)). The frequency of CD4 + CD25 high Foxp3 + T cells was also higher in cultures stimulated with SLA plus PIII compared to nonstimulated cultures (Figure 3(D)). 4. Discussion The Th1 immune response associated with macrophage activation and killing of Leishmania sp. is paradoxically related to the development of cutaneous and mucosal leishmaniasis. In an attempt to control parasite growth, activated macrophages release high levels of proinflammatory cytokines and nitrogen and oxygen reactive intermediates, leading to a tissue lesion. A balance between the proinflammatory response characterized by the production of IFN-γ and TNF, and the regulatory response with the production of IL-1 has been observed in individuals exposed to the Leishmania braziliensis in endemic areas of leishmaniasis in Brazil. Those individuals do not develop the disease and they are designated as subclinical subjects [37]. The balanced immune response described in these individuals may be the key to achieving a harmless host-parasite relationship. There are other evidences that IL-1 is capable of downmodulating the inflammatory response associated with

49 Journal of Parasitology Research 7 human tegumentary leishmaniasis [4, 38 4]. However, mononuclear cells of mucosal leishmaniasis (ML) patients have a decreased ability to produce this cytokine and to respond to IL-1 after in vitro restimulation with L. braziliensis antigen [3]. Recently it has been shown that during chronic Schistosoma mansoni infection, cells from the innate immune response, such as monocytes and regulatory T cells produce high levels of IL-1 [24, 41 43]. Our group has performed studies in an attempt to identify S. mansoni antigens with regulatory properties that enable them to downregulate the inflammatory process associated with certain immunemediated diseases. For instance, we have shown that the S. mansoni antigens rsm29, rsmtsp-2, and PIII induce IL-1 and IL-5 production by PBMC of cutaneous leishmaniasis patients and that they are able to control the in vitro inflammatory response in a group of patients, independent of the clinical features, such as number and size of lesions [8]. In this current study we extended the assessment of the potential of the antigens rsm29, rsmtsp-2, and PIII in modifying the immune response during cutaneous leishmaniasis. Specifically, we evaluated the impact of the addition of these antigens to the cell culture stimulated with Leishmania antigens on monocyte and lymphocyte profile and activation status. CD4 + T lymphocytes and monocytes are key cells in the protection against leishmaniasis; however they have been associated to the inflammatory process and tissue lesion in the cutaneous form of disease [2, 44 46]. In leishmaniasis and also in some others diseases the role of different subsets of monocytes in the pathology has been described [47]. For more than two decades, monocytes have been classified into classical (CD14 + CD16 ) and inflammatory, those which express the molecule CD16 and produce high levels of TNFα [48]. The frequency of CD14 + CD16 + in CL patients was found to be significantly higher compared to healthy controls and they were positively correlated with the lesion size [22]. Recently, a new nomenclature has been used to classify human blood monocytes into three subsets: classical (CD14 ++ CD16 ), intermediate (CD14 ++ CD16 + ), and nonclassical (CD14 + CD16 ++ )[15]. The classical monocytes account for about 85% of the total monocytes and are characterized by the expression of IL-13Rα1, IL-1, and RANTES and by the expression of genes associated with antiapoptosis and wound healing properties. The intermediate monocytes represent 5% of monocytes, express high levels of HLA-DR and are associated with antigen processing and presentation to T cell and T cell activation. Nonclassical subset are characterized by a low expression of CD14 and high expression of CD16 (CD14 + CD16 ++ ). They represent 1% of human monocytes and studies have shown that they express genes associated with cytoskeletal rearrangement which account for their highly mobility and for the patrolling behavior in vivo [16, 47]. Since the roles of the different monocyte subsets in cutaneous leishmaniasis remain unclear, we decided to evaluate not only their frequency in leishmaniasis patients, but also whether the addition of S. mansoni antigens would alter the profile of these cells in an in vitro study. The addition of the antigens rsm29 and PIII to the PBMC cultures stimulated with SLA increased the frequency of nonclassical monocytes. This was not expected as a cell with high mobility and migratory capacity [16, 47], they may contribute to Leishmania metastasis and consequent development of more severe forms of the disease, such as the mucosal and disseminated forms. We found, however, that the S. mansoni antigens used in this study, particularly PIII, diminished the expression of HLA-DR as well as the expression of the co-stimulatory molecules B7.1 and B7.2 on different monocyte subsets. A downmodulation of monocyte activation is desirable in leishmaniasis and may result in a reduced inflammatory process. In this study, we also evaluated the expression of costimulatory molecules in T cells. We observed a significant increase in the frequency of CD4 + T cells by the presence of the rsm29 antigen in the cultures. The addition of rsmtsp-2 and PIII antigens to the cultures stimulated with SLA increased the expression of CTLA-4 in CD4 + T cells and also expanded the frequency of CD4 + CD25 high Foxp3 + T cells. We previously showed that S. mansoni infection expands CD4 + T cell population of the regulatory profile in S. mansoniinfected asthmatic individuals [24]. In such allergic disease, S. mansoni antigens downmodulate the Th2 exacerbated inflammatory response in vitro by inducing the production of IL-1 and the expression of the regulatory molecules, CTLA-4 in T lymphocytes [9, 24, 43]. In a murine model of ovalbumin-induced asthma, inhibition of lung inflammatory process, by S. mansoni eggs or by the S. mansoni antigens, Sm22.6, PIII, and rsm29 were associated with an increase in the number of CD4 + CD25 + Foxp3 + T cells and high levels of IL-1 production [49, 5]. T cell-mediated immunity is fundamental to host protection against Leishmania sp. and the activation of T cells depends upon signals from the interaction between the costimulatory molecules CD28 to its ligands B7.1 and B7.2 on antigen presenting cells (APCs) [23]. We evaluated the expression of CD28 on lymphocytes in cultures stimulated with SLA with or without the addition of S. mansoni antigens and found that the expression of this molecule on CD8 + T cells was higher in cultures stimulated with rsm29 compared with SLA alone. These results suggest that the CD8 + T cells became more activated in the presence of rsm29. There was, however, an increased expression of CTLA-4 in CD4 + T cells, a molecule which is expressed to inhibit the T cell activation when rsmtsp-2 and PIII were added to the cultures. In vitro study has shown that the addition of CTLA-4-Ig to block CD28-B7 interaction in CL patients, PBMC cultures stimulated with Leishmania antigen led to a downmodulation of IFN-γ, IL-1, and TNF secretion [27]. Our findings suggest that the CD4 + Tcells were downmodulated by the presence of the rsmtsp-2 and PIII. These antigens were also able to increase the frequency of the CD4 + CD25 high Foxp3 + regulatory T cells. In a survey performed by O Neal et al. [51], the prevalence of S. mansoni infection in cutaneous leishmaniasis patients from the endemic area of Corte de Pedra, Bahia, Brazil was 16.7% [51]. The authors also showed that

50 8 Journal of Parasitology Research coinfected individuals tended to have small lesion size, however in the univariated model, the presence of helminth coinfection was associated with delayed lesion healing. When they evaluated infection with S. mansoni and Strongyloides stercoralis there was no difference in lesion healing compared to patients infected with geohelminths such as Ascaris lumbricoides, Ancylostoma duodenale, and Trichuristrichiura. Studies conducted by the same group in the endemic area of leishmaniasis in Brazil demonstrated that the use of immunomodulatory agents such as granulocyte/macrophage colony stimulating factor (GM-CSF) and pentoxifylline, an inhibitor of TNF production, can lead to faster healing time and higher cure rate in cutaneous leishmaniasis [52 55]. It has also been reported that intralesional injections of GM- CSF reduce healing time of CL ulcers by 5%. Moreover, Santos et al. [53] showed that GM-CSF applied locally in low doses as an adjuvant to pentavalent antimonial therapy, significantly decreases the healing time of CL ulcers, reducing the dose of antimony administered and/or the duration of antimonial therapy. These studies indicate that downmodulation of the inflammatory response in CL patients is associated with lesion cure and give support to the idea that inhibition of the strong inflammatory response early after Leishmania infection could avert tissue damage. In this context, the data presented herein suggest that S. mansoni antigens are capable of downregulating monocyte and T lymphocyte response to the Leishmania antigen in vitro, possibly through mechanisms that involve negative signaling by CTLA-4 and the action of regulatory T cells. This knowledge could contribute to the development of future strategies to prevent and treat cutaneous and mucosal leishmaniasis. Conflict of Interests The authors have no conflict of interests concerning the work reported in this paper. Acknowledgments The authors would like to thank the people of the leishmaniasis-endemic area in Corte de Pedra-Bahia for their participation in our study; Dr. Luiz Henrique Guimarães, Dr. Paulo Machado, and Ednaldo Lago who assisted in data collection and Hana Khidir and Whitney Oriana for the review of the paper. This work was supported by the Brazilian National Research Council (CNPq) and by the NIH Grant no. NIH AI M. I. Araujo, A. M. Góes, S. O. Costa, and E. M. Carvalho are investigators supported by CNPq. References [1] P. Desjeux, Leishmaniasis: public health aspects and control, Clinics in Dermatology, vol. 14, no. 5, pp , [2] A.Schriefer,A.L.F.Schriefer,A.Góes-Neto et al., Multiclonal Leishmania braziliensis population structure and its clinical implication in a region of endemicity for American tegumentary leishmaniasis, Infection and Immunity,vol.72,no.1,pp , 24. [3] O. Bacellar, H. Lessa, A. Schriefer et al., Up-regulation of Th1- type responses in mucosal leishmaniasis patients, Infection and Immunity, vol. 7, no. 12, pp , 22. [4] A. Ribeiro-de-Jesus, R. P. Almeida, H. Lessa, O. Bacellar, and E. M. Carvalho, Cytokine profile and pathology in human leishmaniasis, Brazilian Journal of Medical and Biological Research, vol. 31, no. 1, pp , [5] D. Sewell, Z. Qing, E. Reinke et al., Immunomodulation of experimental autoimmune encephalomyelitis by helminth ova immunization, International Immunology, vol. 15, no. 1, pp , 23. [6] A. Cooke, P. Tonks, F. M. Jones et al., Infection with Schistosoma mansoni prevents insulin dependent diabetes mellitus in non-obese diabetic mice, Parasite Immunology, vol. 21, no. 4, pp , [7] O. Atochina and D. Harn, Prevention of psoriasis-like lesions development in fsn/fsn mice by helminth glycans, Experimental Dermatology, vol. 15, no. 6, pp , 26. [8] A.M.Bafica,L.S.Cardoso,S.C.Oliveiraetal., Schistosoma mansoniantigens alter the cytokine response in vitro during cutaneous leishmaniasis, Memórias do Instituto Oswaldo Cruz, vol. 16, pp , 211. [9] A.J.G.Simpson,P.Hagan,F.Hackett,P.OmerAli,andS.R. Smithers, Epitopes expressed on very low M(r) Schistosoma mansoni adult tegumental antigens conform to a general pattern of life-cycle cross-reactivity, Parasitology, vol. 1, part 1, pp , 199. [1] F. C. Cardoso, J. M. R. Pinho, V. Azevedo, and S. C. Oliveira, Identification of a new Schistosoma mansoni membranebound protein through bioinformatic analysis, Genetics and Molecular Research, vol. 5, no. 4, pp , 26. [11] M. H. Tran, M. S. Pearson, J. M. Bethony et al., Tetraspanins on the surface of Schistosoma mansoni are protective antigens against schistosomiasis, Nature Medicine, vol.12,no.7,pp , 26. [12] C. Hirsch and A. M. Goes, Characterization of fractionated Schistosoma mansoni soluble adult worm antigens that elicit human cell proliferation and granuloma formation in vitro, Parasitology, vol. 112, no. 6, pp , [13] C.Hirsch,C.S.Zouain,J.B.Alves,andA.M.Goes, Induction of protective immunity and modulation of granulomatous hypersensitivity in mice using PIII, an anionic fraction of Schistosoma mansoni adult worm, Parasitology, vol. 115, part 1, pp , [14] L. S. Cardoso, S. C. Oliveira, R. P. Souza et al., Schistosoma mansoni antigens modulate allergic response in vitro in cells of asthmatic individuals, Drug Development Research, vol. 72, pp , 211. [15] L. Ziegler-Heitbrock, P. Ancuta, S. Crowe et al., Nomenclature of monocytes and dendritic cells in blood, Blood, vol. 116, no. 16, pp. e74 e8, 21. [16] K.L.Wong,J.J.Tai,W.C.Wongetal., Geneexpressionprofiling reveals the defining features of the classical, intermediate, and non-classical human monocyte subsets, Blood, vol. 118, pp , 211. [17] J. Cros, N. Cagnard, K. Woollard et al., Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors, Immunity, vol. 33, no. 3, pp , 21. [18] J. Y. Zhang, Z. S. Zou, A. Huang et al., Hyper-activated proinflammatory CD16 + monocytes correlate with the severity of liver injury and fibrosis in patients with chronic hepatitis B, PLoS ONE, vol. 6, no. 3, Article ID e17484, 211.

51 Journal of Parasitology Research 9 [19] Y. Rodriguez-Munoz, S. Martin-Vilchez, R. Lopez-Rodriguez et al., Peripheral blood monocyte subsets predict antiviral response in chronic hepatitis C, Alimentary Pharmacology & Therapeutics, vol. 34, pp , 211. [2] J. Han, B. Wang, N. Han et al., CD14 high CD16 + rather than CD14 low CD16 + monocytes correlate with disease progression in chronic HIV-infected patients, Journal of Acquired Immune Deficiency Syndromes, vol. 52, no. 5, pp , 29. [21] E. L. Azeredo, P. C. Neves-Souza, A. R. Alvarenga et al., Differential regulation of toll-like receptor-2, toll-like receptor-4, CD16 and human leucocyte antigen-dr on peripheral blood monocytes during mild and severe dengue fever, Immunology, vol. 13, no. 2, pp , 21. [22] G. Soares, A. Barral, J. M. Costa, M. Barral-Netto, and J. Van Weyenbergh, CD16 + monocytes in human cutaneous leishmaniasis: increased ex vivo levels and correlation with clinical data, Journal of Leukocyte Biology, vol. 79, no. 1, pp , 26. [23] K. S. Hathcock, G. Laszlo, C. Pucillo, P. Linsley, and R. J. Hodes, Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function, Journal of Experimental Medicine, vol. 18, no. 2, pp , [24] R. R. Oliveira, K. J. Gollob, J. P. Figueiredo et al., Schistosoma mansoni infection alters co-stimulatory molecule expression and cell activation in asthma, Microbes and Infection, vol. 11, no. 2, pp , 29. [25] T. L. Walunas, D. J. Lenschow, C. Y. Bakker et al., CTLA-4 can function as a negative regulator of T cell activation, Immunity, vol. 1, no. 5, pp , [26] J. F. Brunet, F. Denizot, and M. F. Luciani, A new member of the immunoglobulin superfamily CTLA-4, Nature, vol. 328, no. 6127, pp , [27] C. Favali, D. Costa, L. Afonso et al., Role of costimulatory molecules in immune response of patients with cutaneous leishmaniasis, Microbes and Infection, vol. 7, no. 1, pp , 25. [28] C.I.Brodskyn,G.K.DeKrey,andR.G.Titus, Influenceof costimulatory molecules on immune response to Leishmania major by human cells in vitro, Infection and Immunity, vol. 69, no. 2, pp , 21. [29] H. Yagi, T. Nomura, K. Nakamura et al., Crucial role of FOXP3 in the development and function of human CD25 + CD4 + regulatory T cells, International Immunology, vol. 16, no. 11, pp , 24. [3] C. Baecher-Allan, J. A. Brown, G. J. Freeman, and D. A. Hafler, CD4 + CD25 high regulatory cells in human peripheral blood, Journal of Immunology, vol. 167, no. 3, pp , 21. [31] C. Romagnani, M. Della Chiesa, S. Kohler et al., Activation of human NK cells by plasmacytoid dendritic cells and its modulation by CD4 + T helper cells and CD4 + CD25hi T regulatory cells, European Journal of Immunology, vol. 35, no. 8, pp , 25. [32] Y. Belkaid and K. Tarbell, Regulatory T cells in the control of host-microorganism interactions, Annual Review of Immunology, vol. 27, pp , 29. [33] Y. Belkaid, C. A. Piccirillo, S. Mendez, E. M. Shevach, and D. L. Sacks, CD4 + CD25 + regulatory T cells control Leishmania major persistence and immunity, Nature, vol. 42, no. 6915, pp , 22. [34] S. G. Reed, E. M. Carvalho, C. H. Sherbert et al., In vitro responses to Leishmania antigens by lymphocytes from patients with leishmaniasis or Chagas disease, Journal of Clinical Investigation, vol. 85, no. 3, pp , 199. [35] L. S. Cardoso, M. I. Araujo, A. M. Góes, L. G. Pacífico, R. R. Oliveira, and S. C. Oliveira, Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis, Microbial Cell Factories, vol. 6, article 1, 27. [36] M. S. Pearson, D. A. Pickering, H. J. McSorley et al., Enhanced protective efficacy of a chimeric form of the schistosomiasis vaccine antigen Sm-TSP-2, PLOS Neglected Tropical Diseases, vol. 6, article e1564, 211. [37] I. Follador, C. Araújo, O. Bacellar et al., Epidemiologic and immunologic findings for the subclinical form of Leishmania braziliensis infection, Clinical Infectious Diseases, vol. 34, no. 11, pp. E54 E58, 22. [38] L. R. V. Antonelli, W. O. Dutra, R. R. Oliveira et al., Disparate immunoregulatory potentials for double-negative (CD4 CD8 ) αβ and γδ T cells from human patients with cutaneous leishmaniasis, Infection and Immunity, vol. 74, no. 11, pp , 26. [39] S. T. Gaze, W. O. Dutra, M. Lessa et al., Mucosal leishmaniasis patients display an activated inflammatory T-cell phenotype associated with a nonbalanced monocyte population, Scandinavian Journal of Immunology, vol. 63, no. 1, pp. 7 78, 26. [4] P. N. Rocha, R. P. Almeida, O. Bacellar et al., Downregulation of Th1 type of response in early human American cutaneous leishmaniasis, Journal of Infectious Diseases, vol. 18, no. 5, pp , [41] M. I. A. S. Araujo, B. Hoppe, M. Medeiros et al., Impaired T helper 2 response to aeroallergen in helminth-infected patiente with asthma, Journal of Infectious Diseases, vol. 19, no. 1, pp , 24. [42] M. Hesse, C. A. Piccirillo, Y. Belkaid et al., The pathogenesis of schistosomiasis is controlled by cooperating IL-1- producing innate effector and regulatory T cells, Journal of Immunology, vol. 172, no. 5, pp , 24. [43] M. I. Araújo, B. S. Hoppe, M. Medeiros Jr., and E. M. Carvalho, Schistosoma mansoni infection modulates the immune response against allergic and auto-immune diseases, Memorias do Instituto Oswaldo Cruz, vol. 99, no. 5, pp , 24. [44] M. Rossol, S. Kraus, M. Pierer, C. Baerwald, and U. Wagner, The CD14 bright CD16 + monocyte subset is expanded in rheumatoid arthritis and promotes expansion of the Th17 cell population, Arthritis & Rheumatism, vol. 64, pp , 211. [45] W. K. Kim, Y. Sun, H. Do et al., Monocyte heterogeneity underlying phenotypic changes in monocytes according to SIV disease stage, Journal of Leukocyte Biology, vol. 87, no. 4, pp , 21. [46] L. Ziegler-Heitbrock, The CD14 + CD16 + blood monocytes: their role in infection and inflammation, Journal of Leukocyte Biology, vol. 81, no. 3, pp , 27. [47] K.L.Wong,W.H.Yeap,J.J.Tai,S.M.Ong,T.M.Dang,andS. C. Wong, The three human monocyte subsets: implications for health and disease, Immunology Research, vol. 53, pp , 212. [48] B. Passlick, D. Flieger, and H. W. Loms Ziegler-Heitbrock, Identification and characterization of a novel monocyte subpopulation in human peripheral blood, Blood, vol. 74, no. 7, pp , [49] L. S. Cardoso, S. C. Oliveira, and M. I. Araujo, Schistosoma mansoniantigens as modulators of the allergic inflammatory response in asthma, Endocrine, Metabolic & Immune Disorders, vol. 12, pp , 212.

52 1 Journal of Parasitology Research [5] L. G. G. Pacífico, F. A. V. Marinho, C. T. Fonseca et al., Schistosoma mansoni antigens modulate experimental allergic asthma in a murine model: a major role for CD4 + CD25 + Foxp3 + T cells independent of interleukin-1, Infection and Immunity, vol. 77, no. 1, pp , 29. [51] S. E. O Neal, L. H. Guimarães, P. R. Machado et al., Influence of helminth infections on the clinical course of and immune response to Leishmania braziliensis cutaneous leishmaniasis, Journal of Infectious Diseases, vol. 195, no. 1, pp , 27. [52] H. A. Lessa, P. Machado, F. Lima et al., Successful treatment of refractory mucosal leishmaniasis with pentoxifylline plus antimony, American Journal of Tropical Medicine and Hygiene, vol. 65, no. 2, pp , 21. [53] J. B. Santos, A. R. De Jesus, P. R. Machado et al., Antimony plus recombinant human granulocyte-macrophage colonystimulating factor applied topically in low doses enhances healing of cutaneous leishmaniasis ulcers: a randomized, double-blind, placebo-controlled study, Journal of Infectious Diseases, vol. 19, no. 1, pp , 24. [54] R. P. Almeida, J. Brito, P. L. Machado et al., Successful treatment of refractory cutaneous leishmaniasis with GM-CSF and antimonials, American Journal of Tropical Medicine and Hygiene, vol. 73, no. 1, pp , 25. [55] R. Almeida, A. D Oliveira Jr., P. Machado et al., Randomized, double-blind study of stibogluconate plus human granulocyte macrophage colony-stimulating factor versus stibogluconate alone in the treatment of cutaneous leishmaniasis, Journal of Infectious Diseases, vol. 18, no. 5, pp , 1999.

53 Hindawi Publishing Corporation Journal of Parasitology Research Volume 212, Article ID 48824, 7 pages doi:1.1155/212/48824 Research Article Transcriptional Profile and Structural Conservation of SUMO-Specific Proteases in Schistosoma mansoni Roberta Verciano Pereira, 1 Fernanda Janku Cabral, 2 MatheusdeSouzaGomes, 3 Liana Konovaloff Jannotti-Passos, 4 William Castro-Borges, 1 and Renata Guerra-Sá 1 1 Departamento de Ciências Biológicas/Núcleo de Pesquisas em Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro, 354- Ouro Preto, MG, Brazil 2 Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Lineu Prestes, 1374-Butantan, São Paulo, SP, Brazil 3 Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Av. Getúlio Vargas, Palácio dos Cristais-Centro, Patos de Minas, MG, Brazil 4 Fundação Oswaldo Cruz, Centro de Pesquisas René Rachou, Laboratório de Malacologia, Av. Augusto de Lima, 1715-Barro Preto, Belo Horizonte, MG, Brazil Correspondence should be addressed to Renata Guerra-Sá, rguerra@iceb.ufop.br Received 24 April 212; Revised 9 August 212; Accepted 23 August 212 Academic Editor: Cristina Toscano Fonseca Copyright 212 Roberta Verciano Pereira et al. 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. 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 by Sentrin/SUMO-specific proteases (SENPs). Here we report the characterization of these proteases in Schistosoma mansoni. Using in silico analysis, we identified two SENPs sequences, orthologs of mammalian SENP1 and SENP7, confirming their identities and conservation through phylogenetic analysis. In addition, the transcript levels of Smsenp1/7 in cercariae, adult worms, and in vitro cultivated schistosomula were measured by qrt-pcr. Our data revealed upregulation of the Smsenp1/7 transcripts in cercariae and early schistosomula, followed by a marked differential gene expression in the other analyzed stages. However, no significant difference in expression profile between the paralogs was observed for the analyzed stages. Furthermore, in order to detect desumoylating capabilities in crude parasite extracts, SmSENP1 enzymatic activity was evaluated using SUMO-1- AMCsubstrate. Theendopeptidase activity related tosumo-1precursorprocessing didnotdiffer significantly between cercariae and adult worms. Taken together, these results support the developmentally regulated expression of SUMO-specific proteases in S. mansoni. 1. Introduction Reversible posttranslational modification by ubiquitin and ubiquitin-like proteins (Ubls) plays a crucial role in dynamic regulation of protein function, fate, binding partners, activity, and localization. Small ubiquitin-related modifier (SUMO) is a member of Ubls family that is covalently attached to lysine residues of their protein targets in cells, altering function and subcellular localization [1]. SUMOylation has been implicated in the regulation of numerous biological processes including transcription [2] and cell cycle control[3]. SUMO conjugation mechanism is a highly dynamic and reversible process closely related to ubiquitylation. The sequential actions of E1, E2, and E3 enzymes catalyze the attachment of SUMO to target proteins, while deconjugation is promoted by SUMO-specific proteases. In addition, like other Ubls, SUMO polypeptide is synthesized as an inactive precursor, which requires a C- terminal cleavage to expose the glycine residue for substrate conjugation. This process is also catalyzed by SUMO-specific protease through its hydrolase activity. The catalytic activity is maintained within a highly conserved 2 amino acid region at the C-terminus of the proteases [4 6]. Recently, the importance of reversible SUMOylation for normal cell physiology has been demonstrated. Excessive SUMO conjugation using knockouts for either SENP1 or

54 2 Journal of Parasitology Research SENP2 induced an embryonic lethal phenotype [7, 8] and deregulation of either SUMO conjugation or deconjugation can contribute to cancer progression as reviewed by [9]. In humans there are six SENP enzymes (SENP1, -2, -3, -5, -6, and -7) that deconjugate mono-sumoylated proteins or disassemble polymeric SUMO side chains, while in Saccharomyces cerevisiae there are only two desumoylating enzymes, Ulp1 and Ulp2. Based on these activities and affinities for SUMO isoforms, SENPs can be categorized into three independent subfamilies: SENP1 and SENP2; SENP3 and SENP5; SENP6 and SENP7 [9]. The initial molecular characterization of SUMOylation pathway in S. mansoni showed the presence of two SUMO paralogs called SMT3C and SMT3B [1], suggesting a variety of targeted substrates for these modifications. Recently, our group reported the conservation of SUMO conjugating enzyme SmUBC9 by phylogeny, primary and modeled tertiary structures, as well as mrna transcription and protein levels throughout the S. mansoni life cycle. Our data showed that Smubc9 transcription levels are upregulated in early schistosomula, suggesting the utilization of this posttranslational modification mechanism S. mansoni development, particularly at the initial phase of invasion of the vertebrate host [11]. To extend the investigation of the SUMOylation pathway in S. mansoni, we firstly retrieved through homology-based searches putative SmSENPs using the publically available S. mansoni databases. Furthermore, the levels of Smsenp1/7 transcripts were evaluated by qrt-pcr using mrna from cercariae, adult worms and mechanically-transformed schistosomula (MTS) in vitro-cultivated during 3.5h, 1, 2, 3, 5, and 7 days. We also measured the endopeptidase activities of SmSENPs in cercariae and adult worm crude extracts. The present study reveals differences in the gene expression profile of Smsenp1/7 during schistosomula development. In addition, similar SmSENP1 activity was observed in cercariae and adult worms, suggesting the importance of this proteolytic activity during the parasite s life cycle. 2. Materials and Methods 2.1. Ethics Statement. All experiments involving animals were authorized by the Ethical Committee for Animal Care of Federal University of Ouro Preto, and were in accordance with the national and international regulation accepted for laboratory animal use and care Parasites. S. mansoni. LE strain was maintained by routine passage through Biomphalaria glabrata snails and BALB/c mice. The infected snails were induced to shed cercariae under light exposure for 2 h and the cercariae were recovered by sedimentation on ice. Adult worm parasites were obtained by liver perfusion of mice after 5 days of infection. Mechanically transformed schistosomula (MTS) were prepared as described by Harrop and Wilson [12]. Briefly, cercariae were recovered, and washed in RPMI 164 medium (Invitrogen), before vortexing at maximum speed for 9 s and immediately cultured during 3.5 h at 37 C, 5% CO 2. Then the recovered schistosomula were washed with RPMI 164 until no tails were detected. For subsequent incubations, the parasites were maintained in M169 medium supplemented with 1% FBS, penicillin, and streptomycin at 1 μg/ml and 5% Schneider s medium [13] at37 Con5% CO 2 during3.5h,1,2,3,5,and7days Identification and Computational Analysis of SmSENP1/7. SmSENP1/7 sequences were retrieved from the S. mansoni genome database version 5. available at through BLAST searches. Amino acid sequences from Drosophila melanogaster, Caenorhabditis elegans and Homo sapiens SENP orthologs were used as queries. The BLASTp algorithm, underpinned by the Pfam (v26.), allowed detection of conserved protein domains or motifs from S. mansoni sequences. Multiple alignments of SmSENP1/7 were performed by ClustalX 2. and phylogenetic analysis was conducted in MEGA 5 [14]. A phylogenetic tree of these sequences was inferred using the Neighbor-Joining method [15]. The bootstrap consensus tree inferred from 1 replicates was used to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 5% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1 replicates) is shown next to the branches. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. All positions containing gaps and missing data were eliminated from the dataset RNA Preparation and Expression Analysis of Smsenp1/7 by qrt-pcr. Total RNA from adult worms, cercariae, and schistosomula was obtained using a combination of the Trizol reagent (GIBCO-BRL) and chloroform for extraction, and then on column purified using the SV total RNA Isolation System (Promega, Belo Horizonte, Brazil). The preparation was treated with RNase-free DNase I in 3 different rounds by decreasing enzyme concentration (RQ1 DNase; Promega). RNA was quantified using a spectrophotometer and an aliquot containing 1 μg of total RNA reverse transcribed using an oligodt primer from the Thermoscript RT-PCR System (Invitrogen São Paulo, Brazil) as described by the manufacturer. The efficiency of DNAse I treatment was evaluated by PCR amplification of the cdna reaction mix without the addition of the Thermoscript enzyme. S. mansoni specific SENP1/7 primers were designed using the program GeneRunner for SmSENP1 (GeneDB access number Smp ) (forward 5 -AGGAAACGGAGGCGG- GATTC-3, reverse 5 -ACACTGGAGACACGGGATGAGC- 3 )andforsmsenp7 (GeneDB access number Smp 15912) (forward 5 -TCAGTTACACGGCCCTTTATC-3, reverse 5 - CCTGAGAAGTGGATGCGATC-3 ). Reverse-transcribed cdna samples were used as templates for PCR amplification using SYBR Green Master Mix UDG-ROX (Invitrogen) and 73 Real Time PCR System (Applied Biosystems, Rio de Janeiro, Brazil). Specific primers for S. mansoni

55 Journal of Parasitology Research 3 α-tubulin were used as an endogenous control (GenBank access number M8214) (forward 5 -CGTATTCGCAAG- TTGGCTGACCA-3, reverse 5 -CCATCGAAGCGCAGT- GATGCA-3 )[16]. The efficiency of each pair of primers was evaluated according to the protocol developed by the Applied Biosystems application (cdna dilutions were 1 : 1, 1 : 1 and 1 : 1). For all investigated transcripts three biological replicates were performed and their gene expression normalized against the α-tubulin transcript according to the 2 ΔCt method [17] using the Applied Biosystems 73 software Endopeptidase Activity. To determine the enzymatic activity of SENP proteases present in adult worms and cercariae crude extracts we used the fluorogenic substrate SUMO-1-AMC that is specific for SENP1 (Enzo Life Sciences, São Paulo, Brazil). In these assays 1 μg of total protein were used and,45 μm of the fluorogenic substrate in 5 mm Tris-HCl ph 8, 1 mm MgCl 2 ±,45 μm SUMO-1- aldehyde (Enzo Life Sciences) to control for specific enzyme inhibition. Each enzymatic assay was conducted in a final volume of 1 μl for both extracts, followed by 6 minutes incubation at 37 C. The reaction was stopped by addition of 2 ml of 99.5% ethanol. The fluorometric readings were taken at wavelengths of 38 nm (excitation) and 44 nm (emission) in a spectrofluorimeter (Turner QuantechTM Fluorometer), and the results expressed in fluorescence units per μg of total protein Statistical Analysis. Statistical analysis was performed using GraphPad Prism version 5. software package (Irvine, CA, USA). Normality of the data was established using one way analysis of variance (ANOVA). Tukey post tests were used to investigate significant differential expression of SUMO proteases throughout the investigated stages. In all cases, the differences were considered significant when P values were < Results 3.1. SUMO Specific-Proteases: The Conservation of SmSENP1/7. In silico analysis of SmSENPs revealed the conserved putative domains of SUMO proteases in S. mansoni. The parasite database has two sequences encoding putative SENPs: Smp (putative SENP1) and Smp (putative SENP7). SENP1 has an alternative spliced form annotated as Smp We evaluated the conservation of these proteins throughout evolution using a phylogenetic approach. Our results showed that each SENP was grouped in 2 distinct clades, reinforcing their structural conservation among the thirteen analyzed orthologs (Figure 1). To confirm that these predicted proteases are cysteinefamily members, analyses using the Pfam protein domain database were conducted. For both SmSENPs entries a conserved Peptidase C48 domain was revealed. Our in silico analysis demonstrated SmSENP1 more closely related to HsSENP1 (GenBank access number NP ), whereas SmSENP7 is related to HsSENP7 (GenBank access number NP ). Alignment of their catalytic domains with the human SENPs showed the presence of the three essential catalytic residues (Cys-His-Asp), highlighted in Figure Smsenp1/7 Are Differentially Expressed in S. mansoni. The gene expression profile of Smsenp1 and Smsenp7 was determined using qrt-pcr. We designed specific primers to amplify Smsenp1 and Smsenp7 transcripts in the cercariae-schistosomula transition and adult worm (Figure 3). We observed that Smsenp1 and Smsenp7 transcripts are expressed in basal levels from MTS-2d to 7 days and adult worms. The levels of the SENPs transcripts were significantly higher in MTS-3.5 h being approximately 1- fold higher when compared to levels in adult worms and during 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 transcription levels were not significantly different (P <.5) within a given stage, except for MTS-3.5 h where Smsenp7 levels are 3 fold-higher than Smsenp SmSENP1 Enzymatic Activity in Cercariae and Adult Worms. In vitro endopeptidase assays were performed using crude extract from cercariae and adult worms (Figure 4). The SmSENP1 enzymatic activity was slightly higher in cercariae compared to adult worms, although the levels were not significantly different (P <.5). Enzyme activities were also recorded in the presence of a commercially available HsSENP1 inhibitor but significant differences were not observed in the analyzed stages. 4. Discussion Previous reports from our group showed the presence of two SUMO paralogs (SMT3C and SMT3B) in S. mansoni, exhibiting differential expression between larvae and adult worms. The electrophoretic pattern of SUMO-conjugated molecules also differed comparing the analyzed stages [1]. Furthermore, our group demonstrated a differential expression profile for UBC9 throughout the parasite life cycle [11]. The structural SUMO conservation in S. mansoni reinforces SUMO as a candidate for a transcription regulator and a degradation signal by facilitating ubiquitylation of certain target proteins during parasite development. In order to understand the role of SUMOylation in S. mansoni, we evaluated the conservation of SUMO proteases and their gene expression profile in cercariae, schistosomula, and adult worm. Firstly, we retrieved, through homologybased searches, two sequences containing conserved domains of SUMO proteases. A phylogenetic approach then revealed two putative proteases closely associated to human SENP1 and SENP7. Alignment of parasite sequences with their human counterparts also demonstrated conservation of the catalytic triad for both proteases [18]. The active site residues within the core domain of Ulp1 and Ulp2 have been proven to be necessary for catalytic activity and in vivo function in yeast [19, 2]. Furthermore, Ulp1 lacks obvious sequence similarity to any known deubiquitinating enzyme so it is

56 4 Journal of Parasitology Research NP (Homo sapiens) NP (Mus musculus) XP (Danio rerio) XP (Branchiostoma floridae) XP (Strongylocentrotus purpuratus) XP (Apis mellifera) NP (Drosophila melanogaster) SENP1 or ULP1-like 94 Smp AAX (Schistosoma japonicum) XP (Caenorhabditis briggsae) 85 NP (Caenorhabditis elegans) NP (Arabidopsis thaliana) NP (Saccharomyces cerevisiae S288c) NP (Arabidopsis thaliana) NP (Saccharomyces cerevisiae S288c) XP (Strongylocentrotus purpuratus) 83 XP (Caenorhabditis briggsae) NP (Caenorhabditis elegans) XP (Branchiostoma floridae) XP (Apis mellifera) NP (Drosophila melanogaster) SENP7 or ULP2-like 86 Smp CAX (Schistosoma japonicum) XP (Danio rerio) 55 NP (Homo sapiens) 78 NP (Mus musculus) Figure 1: Consensus phylogenetic tree based on amino acid sequences of SmSENPs. The tree construction and analysis of bootstrap were performed using ClustalX 2. and MEGA 4.. For the consensus tree and reliability of the branches formed test was used phylogenetic bootstrap using 1 replicates for each sequence, and 5% 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 (Drosophila melanogaster),cb (Caenorhabditis briggsae) and Ce (Caenorhabditis elegans).

57 Journal of Parasitology Research 5 : : : : : :.: Hs SENP7 VPIHLG-VHWCLAVVD-----ITYYDSMG IPQQMNGSDCGMFACKY Sm SENP7 IPIHDRGMHWCLSCID-----ITYYDSMG VPQQYNGSDCGVFLCTF : :.: :.:: : ::.. : : Hs SENP1 VPVNESSHWYLAVIC RPCILILDSLKA VPKQDNSSDCGVYLLQY Sm SENP1 IPINECAHWFLGLVC MPCVLLFDSLPC VPVQSNLVDCGIYLLHY Figure 2: S. mansoni has two predicted SUMO-Specific Proteases. ClustalX 2. alignment of SENP1 and SENP7 catalytic residues from human and S. mansoni. The domains position of proteins in S. mansoni were based on Pfam database as well as the e-value score. The grey boxes 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. Relative expression of SmSENPs Adult worm Cercariae MTS-3, 5 h MTS-1 d MTS-2 d MTS-3 d MTS-5 d MTS-7 d SENP1 SENP7 Figure 3: SmSENPs are differentially expressed throughout the S. mansoni life cycle. The mrna expression levels were measured based on 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 an endogenous control (one-way variance analysis followed by Tukey pairwise comparison P <.5). Different from adult worm, different from cercariae, and different from MTS-3.5 h. Fluorescence (µg/min) Cercariae SUMO1-AMC SUMO1 aldehyde Adult worms Figure 4: SmSENP1 enzymatic activity in S. mansoni. The fluorescence levels were measured based on three replicates, in cercariae and adult worm stages. Statistical analysis was performed using t tests followed by unpaired test P <.5. Different from cercariae. unlikely that it is able to process ubiquitin-linked substrates [21]. Considering that the SUMOylation pathway dictates the function of a variety of substrates [22], and the existence of two SUMO paralogs in S. mansoni [1], it is plausible that the worm utilizes distinct SUMO proteases for SUMO maturation and desumoylation of target substrates. Discrimination between SUMO-1 and SUMO-2/3 paralogs may also account for distinct functions of SENP proteases in S. mansoni. In mammalian cells, proteomic studies have identified proteins that are selectively modified by either SUMO-1 or SUMO-2/3 [23]. It is well established that SENP1 processes SUMO-1 in preference to SUMO-2, and has a very low catalytic constant for SUMO-3 [24]. In contrast, the recent characterization of the catalytic domain of SENP7 in humans demonstrated a greater deconjugating activity for SUMO-2/3 chains [25]. As discussed by Shen [26] SENP7 acts as a SUMO-2/3 specific protease that is likely to regulate the metabolism of polysumo-2/3 rather than SUMO-1 conjugation in vivo.

58 6 Journal of Parasitology Research In the present investigation, the transcriptional profile of SUMO proteases was also evaluated by qpcr, using total RNA from distinct stages of the S. mansoni life cycle. The gene expression data revealed similar expression profile for Smsenp1 and Smsenp7 at any given stage, but with a remarkable differential expression for both proteases comparing the larvae and adult stages. The differential gene expression profile observed for Smsenp1/7 throughout the S. mansoni life cycle attests for the distinct patterns of SUMO conjugates observed during parasite development [1]. Poly- SUMOylation has been reported to increase during heatshock treatment and stress conditions, with SENP7 being responsible for dismantling SUMO polymers [25, 27].Given that the levels of Smsenp1/7 transcripts were significantly higher in cercariae and MTS-3.5 h, we suggest a stressinduced expression of the SUMOylation machinery during the parasite s body adaptation to the new metabolic and morphological alterations it undergoes [28]. Our findings are also in agreement with Pereira [11] which demonstrated a similar pattern of gene expression profile for Smubc9 in the aforementioned parasite stages. We have not observed a positive correlation between differential transcriptional levels of Smsenp1 and enzymatic activity evaluated using synthetic SUMO-1-AMC substrate. The levels of enzymatic activity for SmSENP1 were slightly higher in extracts of cercariae compared to adult worms. In the presence of a specific inhibitor, no significant change in enzymatic activity was detected. This may account for the limited amino acid sequence identity (28%) shared between SmSENP1 and its human ortholog, from which the synthesis of SUMO-1 aldehyde is based. Additional studies aimed at elucidating substrate selection of SmSENP1/7 in S. mansoni and a detailed kinetic analysis of their enzymatic activities, particularly during the cercarie to schistosomula transition may provide further insights into the biological activities of these enzymes and the role of SUMO conjugation in S. mansoni. Acknowledgments The authors thank the transcriptome initiatives: São Paulo Transcriptome Consortium; Minas Gerais GenomeNetwork; Wellcome Trust Genome Iniatiative (UK). This work was supported by the following Brazilian research agencies: FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais, CBB 558/9), Núcleo de Bioinformática da Universidade Federal de Ouro Preto (NuBio UFOP) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). References [1] J. R. Gareau and C. D. Lima, The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition, Nature Reviews Molecular Cell Biology, vol. 11, no. 12, pp , 21. [2] J. M. P. Desterro, M. S. Rodriguez, and R. T. Hay, SUMO- 1 modification of IκBα inhibits NF-κB activation, Molecular Cell, vol. 2, no. 2, pp , [3] H. D. Ulrich, Regulating post-translational modifications of theeukaryoticreplicationclamppcna, DNA Repair, vol. 8, no. 4, pp , 29. [4] D. Mukhopadhyay and M. Dasso, Modification in reverse: the SUMO proteases, Trends in Biochemical Sciences, vol. 32, no. 6, pp , 27. [5] J. H. Kim and S. H. Baek, Emerging roles of desumoylating enzymes, Biochimica et Biophysica Acta, vol. 1792, no. 3, pp , 29. [6] Z. Xu, H. Y. Chan, W. L. Lam et al., SUMO proteases: redox regulation and biological consequences, Antioxidants and Redox Signaling, vol. 11, no. 6, pp , 29. [7]J.Cheng,X.Kang,S.Zhang,andE.T.H.Yeh, SUMOspecific protease 1 is essential for stabilization of HIF1α during hypoxia, Cell, vol. 131, no. 3, pp , 27. [8] S. Y. Chiu, N. Asai, F. Costantini, and W. Hsu, SUMOspecific protease 2 is essential for modulating p53-mdm2 in development of trophoblast stem cell niches and lineages, PLOS Biology, vol. 6, no. 12, p. e31, 28. [9] T. Bawa-Khalfe and E. T. H. Yeh, SUMO losing balance: SUMO proteases disrupt SUMO homeostasis to facilitate cancer development and progression, Genes and Cancer, vol. 1, no. 7, pp , 21. [1] F. J. Cabral, O. S. Pereira Jr., C. S. Silva, R. Guerra-Sá, and V. Rodrigues, Schistosoma mansoni encodes SMT3B and SMT3C molecules responsible for post-translational modification of cellular proteins, Parasitology International, vol. 57, no. 2, pp , 28. [11] R. V. Pereira, F. J. Cabral, M. S. Gomes et al., Molecular characterization of SUMO E2 conjugation enzyme: differential expression profile in Schistosoma mansoni, Parasitology Research, vol. 19, no. 6, pp , 211. [12] R. Harrop and R. A. Wilson, Protein synthesis and release by cultured schistosomula of Schistosoma mansoni, Parasitology, vol. 17, no. 3, pp , [13] P. F. Basch and J. J. DiConza, In vitro development of Schistosoma mansoni cercariae, Journal of Parasitology, vol. 63, no. 2, pp , [14] S. Kumar, K. Tamura, and M. Nei, MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment, Briefings in bioinformatics, vol. 5, no. 2, pp , 24. [15] N. Saitou and M. Nei, The neighbor-joining method: a new method for reconstructing phylogenetic trees, Molecular Biology and Evolution, vol. 4, no. 4, pp , [16] P. J. Webster, K. A. Seta, S. C. Chung, and T. E. Mansour, A cdna encoding an α-tubulin from Schistosoma mansoni, Molecular and Biochemical Parasitology, vol. 51, no. 1, pp , [17] K. J. Livak and T. D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2- ΔΔCT method, Methods, vol. 25, no. 4, pp , 21. [18] S. J. Li and M. Hochstrasser, The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization, and substrate specificity, Journal of Cell Biology, vol. 16, no. 7, pp , 23. [19] S. J. Li and M. Hochstrasser, A new protease required for cellcycle progression in yeast, Nature, vol. 398, no. 6724, pp , [2] A. V. Strunnikov, L. Aravind, and E. V. Koonin, Saccharomyces cerevisiae SMT4 encodes an evolutionarily conserved protease with a role in chromosome condensation regulation, Genetics, vol. 158, no. 1, pp , 21.

59 Journal of Parasitology Research 7 [21] S. J. Li and M. Hochstrasser, The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein, Molecular and Cellular Biology, vol. 2, no. 7, pp , 2. [22] G. Rosas-Acosta, W. K. Russell, A. Deyrieux, D. H. Russell, and V. G. Wilson, A universal stategy for proteomic studies of SUMO and other ubiquitin-like modifiers, Molecular and Cellular Proteomics, vol. 4, no. 1, pp , 25. [23] A. C. O. Vertegaal, J. S. Andersen, S. C. Ogg, R. T. Hay, M. Mann, and A. I. Lamond, Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics, Molecular and Cellular Proteomics, vol. 5, no. 12, pp , 26. [24] L. N. Shen, C. Dong, H. Liu, J. H. Naismith, and R. T. Hay, The structure of SENP1-SUMO-2 complex suggests a structural basis for discrimination between SUMO paralogues during processing, Biochemical Journal, vol. 397, no. 2, pp , 26. [25] C. D. Lima and D. Reverter, Structure of the human SENP7 catalytic domain and poly-sumo deconjugation activities for SENP6 and SENP7, Journal of Biological Chemistry, vol. 283, no. 46, pp , 28. [26] L. N. Shen, M. C. Geoffroy, E. G. Jaffray,andR.T.Hay, Characterization of SENP7, a SUMO-2/3-specific isopeptidase, Biochemical Journal, vol. 421, no. 2, pp , 29. [27] F. Golebiowski, I. Matic, M. H. Tatham et al., System-wide changes to sumo modifications in response to heat shock, Science Signaling, vol. 2, no. 72, p. ra24, 29. [28] J. R. Lawson and R. A. Wilson, Metabolic changes associated with the migration of the schistosomulum of Schistosoma mansoni in the mammal host, Parasitology, vol. 81, no. 2, pp , 198.

60 Hindawi Publishing Corporation Journal of Parasitology Research Volume 212, Article ID , 8 pages doi:1.1155/212/ Review Article Schistosoma Tegument Proteins in Vaccine and Diagnosis Development: An Update Cristina Toscano Fonseca, 1, 2 Gardênia Braz Figueiredo Carvalho, 1 Clarice Carvalho Alves, 1 and Tatiane Teixeira de Melo 1 1 Laboratório de Esquistossomose, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Avenida Augusto de Lima 1715, Belo Horizonte,MG 319-2, Brazil 2 Instituto Nacional de Ciências e Tecnologia em Doenças Tropicais (INCT-DT), Avenida Augusto de Lima 1715, Belo Horizonte, MG 319-2, Brazil Correspondence should be addressed to Cristina Toscano Fonseca, ctoscano@cpqrr.fiocruz.br Received 27 July 212; Accepted 24 September 212 Academic Editor: Andrea Teixeira-Carvalho Copyright 212 Cristina Toscano Fonseca et al. 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. The development of a vaccine against schistosomiasis and also the availability of a more sensitive diagnosis test are important tools 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 targets and 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 tegument represents the interface between host and parasite and its molecules are responsible for essential functions to parasite survival. In this paper we will present the advances in the development of vaccines and diagnosis tests achieved with the use of the information from schistosome genome focused on parasite tegument as a source for antigens. 1. Introduction Schistosomiasis is still a significant public health problem in tropical countries despite the existence of effective drugs against the parasite [1]. Chemotherapy as a strategy for disease control has proved ineffective in controlling transmission [1] therefore, the development of a vaccine against the disease and also a more sensitive diagnosis test is necessary to assist chemotherapy in control programs [1, 2]. In this context, the recent availability of schistosome genomes information represents an important toll to be used in the discovery of new targets for vaccine and diagnosis. Schistosoma mansoni genome, published in 29 [3] described genes while Schistosoma japonicum genome [4] has been described to be composed of genes. Their assemblies were generated by conventional capillary sequencing resulting in scaffolds (S. mansoni) and scaffolds (S. japonicum). More recently an improved version of the S. mansoni genome was published [5], utilizing a combination of traditional Sanger capillary sequencing and deep-coverage Illumina sequencing that refined gene prediction resulting in a reduction in the number of predicted genes from to Illumina-based technology was also used in Schistosoma haematobium genome sequencing, which described genes [6]. Simultaneously to genome publication, an important tool to access and analyze parasite genome has been developed, the SchistoDB ( database [7]. The SchistoDB enables access to information on the parasite genome even to those researchers not specialized in computer language. The current 3. database version provides access to the latest draft of S. mansoni genome sequence and annotation and also to S. japonicum and S. haematobium genome annotation. The bioinformatics tools, together with the availability to access parasite genome, should have helped the knowledge of parasite biology and the search for new vaccines, therapeutic targets, and antigens to be used in the disease diagnosis. In

61 2 Journal of Parasitology Research this paper we will present the advances in the development of vaccines and diagnostics tests achieved with the use of the information from schistosome genome, focus will be given to the parasite tegument as a source for antigens. 2. Host-Parasite Relationship: Role for the Parasite Tegument Highly adapted to parasitic life, schistosomes can live for years or decades even in a hostile environment as the circulatory system from vertebrate host where the parasite has an intimate contact with circulating elements of the immune system [8]. In this successful host-parasite relationship, the host immune system plays an important role in both parasite development and elimination. CD4+ cells, hormones, and cytokines 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, andtnf-α has been described to be involved in parasite elimination in the irradiated cercariae vaccine model [16 18]. Moreover, the highly adapted relationship between schistosomes and the mammalian definitive host also involves the effective mechanisms for evading the immune response that they provoke. In this context, the parasite tegument plays an important role [19, 2]. After penetration, the parasite surface undergoes a profound change that allows parasite adaptation into the host internal microenvironment where the parasite switches from its immune-sensitive to an immune-refractory state [21]. In cercariae, the surface is characterized by a single bilayer membrane covered by a dense glicocalyx. During penetration, the glicocalyx is lost and the membrane transforms into a double bilayer membrane [22]. Evading mechanisms as antigenic mimicry, membrane turnover, production of immunomodulatory molecules and modulation of surface antigens expression also takes place in the parasite surface and contributes to schistosome survival [23, 24]. Trying to eliminate the parasite, host immune system targets the antigens in parasite surface. Studies in mice have shown that the developmental stage most susceptible to the host immune system attack is the schistosomula stage. Very early after infection, schistosomula are susceptible to cellular and humoral immunity, however, in the course of parasite development the susceptibility is rapidly lost [25, 26]. The resistance to host immune response acquired by parasites can be in part explained by surface changes independently of host antigens adsorption [27 29]. In addition, El Ridi and colleagues [3], demonstrated that lung-stage schistosomulum protect themselves from the host immune system by confining antigenic molecules in lipid-rich sites of surface membrane. In contrast, McLaren, in 1989 [31], demonstrated that both skin and lung schistosomula phases are targets of the immune system in the radiation-attenuated vaccine model which trigger an inflammatory reaction around the larvae inhibiting their migration. Since schistosomula is the major target of the host immune system attack and its tegument represents the interface between parasite and host, also performing vital functions that ensure parasite survival [32], the study of its structure and how it interacts with the host immune system can provide important information about disease control, especially to those related to the search for new drugs and vaccine development. We have recently demonstrated that the schistosomula tegument from S. mansoni (Smteg) is recognized by TLR4 in dendritic cells (DC) leading to DC activation and production of proinflammatory cytokines as IL- 12 and TNF-α [33]. In contrast to this inflammatory profile, Smteg also induce IL-1 production by DC in a TLR (Toll like receptors) 2, 3, 4, and 9 independent manner (unpublished data) once again demonstrating that schistosomula tegument can both activate or modulate host immune system. 3. The Tegument as Antigen Source for Vaccine Development Most of the studies that aimed to identify membrane proteins in parasite tegument were performed in adult worms [34 36]. Although schistosomula is the major target for host immunity, its tegument proteins have still not been characterized, mainly due to the difficulty in obtaining sufficient quantities of material for such protein studies [37]. Indeed protective antigens are found in S. mansoni schistosomula tegument (Smteg) since mice immunization with Smteg formulated with Freunds adjuvant [38] or Alum + CPG- ODN (unpublished data) is able to reduce significantly worm burden and egg elimination with the feces. The characterization of these protective antigens is being performed using immune-proteomics analysis and genome databases to identify candidates to be used in a vaccine formulation against schistosomiasis. Other omics technologies are also being used to identify schistosoma proteins, mainly those expressed in schistosomula. In this context, two studies, using cdna microarrays technologies assessed the most relevant transcriptional changes in the schistosomula development phase. These studies demonstrated that tetraspanin, Sm22.6, Sm29, Sm2 and phosphadiesterase are membrane proteins are highly expressed during schistosomula phase [39, 4]. Furthermore, the studies that used gene silencing through RNAi technique could clarify the importance of some proteins, such as cathepsins [41, 42] and tetraspanins [43] forparasite development and survival. The same membrane protein was identified in adult worm tegument preparations using Mass spectrometry (MS-)-basedproteomics [33, 34] together with genome, transcriptome and genetic maps information [3, 44 46]. Recently a proteomic analysis demonstrated that Sm29 and Sm2 are linked to parasite surface membrane through a GPI-anchor [47] while the most abundant protein in adult worm tegument, among the investigated molecules, are aquaporin, dysferlin, TSP-2, and ATP diphosphohydrolase [48]. Among this expressive catalogue of protein expressed in the schistosome tegument, some of them have been evaluated as vaccine antigen in immunization protocols in mice. The Table 1 summarizes the results observed in these preclinical trials using tegument proteins. Sm29 was identified by Cardoso and coworkers using in silicoanalysis to identify in S. mansoni transcriptome putative expressed proteins localized in the parasite tegument [49].

62 Journal of Parasitology Research 3 Table 1: Schistosome tegument protein evaluated as vaccine candidates in preclinical studies. Protein Vaccine type Protection level Egg reduction Bioinformatictoolusedin antigen selection References Sm 21.7 Recombinant protein 41% 7% ND ND [63] Sm 21.7 DNA vaccine 41.5% 62% (liver) 67% (intestine) ND [64] Cu/Zn superoxide dismutase DNA vaccine 44% 6% ND ND [65] Sm TSP2 Recombinant protein 57% 64% (liver) 65% (feces) BLAST [57, 83] Sm29 Recombinant protein 51% 6% (intestine) InterProScan, SignalIP 3., Signal IP Neural, NetNGlyc 1., BLAST, WolfpSORT, [49, 5] SOSUI, Compute pi/mw tool, ECL (2 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 associated with a reduction of 51% in worm burden when used in vaccine formulation [5]. The tegumental protein, Sm22.6 and its homologue in S. japonicum (Sj22.6), are involved in resistance to reinfection in endemic areas [51, 52]. Immunization of mice with recombinant 22.6 formulated with Freund adjuvant resulted in 34.5% reduction on worm burden [53] while Sm22.6 formulated with alum failed to induce protection against schistosomiasis but induced a regulatory response able to modulate allergic asthma in mice [54, 55]. Tetraspanins (TSP) 1 and 2 were identified in a cdna library from S. mansoni based on their membrane-targeting signal [56]. Immunization of mice with TSP1 recombinant protein resulted in a reduction of 57% in worm burden and reduction in the number of eggs in liver (64%) and intestine (65%), TSP2 recombinant protein was less effective in reducing worm burden (34%) but had similar effects in reducing the number of eggs trapped in the liver (52%) and intestine (69%) [57]. The TSP-2 homologue in S. japonicum has also been evaluated in murine immunization however no protection was observed [58]. ECL or Sm2 is a GPI-anchored protein in the S. mansoni tegument that has also been associated with praziquantel efficacy, since antibodies against this protein can restore drug efficacy in B cells depleted mice [59, 6]. Murine DNA vaccination with the gene encoding Sm2 elicited 38.1% protection while immunization of mice with enzymatically cleaved GPI-anchored proteins from the S. mansoni tegument, in which Sm2 represent the most abundant protein result 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 recombinant Sm21.7 resulted in a decrease of 41% 7% in worm burden while DNA vaccination resulted in of 41.5% worm burden reduction [63, 64]. The schistosome antioxidant enzymes (Cu/Zn superoxide dismutase-sod, glutathione-s-peroxidase-gpx) are developmentally regulated. The lowest level of gene expression and enzyme-specific activity was found in the larval stages while the highest level of gene expression was observed in adult worms [65 68]. This suggests that antioxidant enzymes are important in immune evasion by adult schistosome parasites [67]. Also RNAi assays demonstrated that knocking down the antioxidants enzymes GPX and GST result in dramatic decreases in sporocysts survival indicating that these enzymes are capable of enhancing parasite survival in an oxidative environment [69]. Mice immunized with the antioxidant enzyme Cu-Zn superoxide dismutase in a DNA vaccine strategy resulted in 44 6% reduction in worm burden [65]. 4. Antigens to Be Used in Schistosomiasis Diagnostic Test Currently, all available techniques for the diagnosis of schistosomiasis are characterized by having some limitations, especially when it becomes necessary to detect infection in a large number of patients with low parasite load [7]. One of the initial difficulties in the development of a test for the diagnosis of schistosomiasis is the choice of an appropriate antigen. There are several factors that influence this choice: easily of production, high stability in sample storage, immunogenicity, specificity, and ability to be incorporated to low costs test platforms [71]. In this context, the availability of the complete genome sequences in combination with other technologies such as bioinformatics and proteomics, provides important tolls to seek for an ideal candidate to compose an efficient immunodiagnostic test. With this in mind, our group have recently designed anin silico strategy based in the principles of reverse vaccinology, and using a rational criteria to mine candidates in parasite genome to be used in the immunodiagnosis of schistosomiasis [72]. Six antigens were selected based on the evidence of gene expression at different phases of the parasite

63 4 Journal of Parasitology Research Protein Table 2: Schistosoma mansoni protein selected by genome mining to be used in serological diagnosis for schistosomiasis. SchistoDB number Sm2 Smp 1773 Annotation 2-kDa GPI-anchored surface glycoprotein Number of amino acid Base pairs Predicted molecular weight Predicted isoelectric point ,5 kda 4.97 Predicted location Tegument surface membranes Sm12.8 Smp Expressed protein ,8 kda 6.88 Extracellular Sm43.5 Smp 4291 Expressed protein ,5 kda 8.43 Extracellular Sm127.9 Smp 1713 Hypothetical protein ,9 kda 6.63 Extracellular Sm18.9 Smp Cytochrome oxidase subunit, putative ,9 kda 9.3 Extracellular Sm16.5 Smp Cytochrome oxidase subunit, putative ,5 kda 9.14 Extracellular Adapted of Carvalho et al., 211 [72]. life cycle in the definitive host, accessibility to host immune system (exposed proteins), low similarity with human and other helminthic proteins, and presence of predicted B cells epitopes (Table 2)[72]. Although our in silico analysis led to identification of six candidates, this strategy has not been yet experimentally validated. Other groups have also used bioinformatics analysis to select target sequence from S. japonicum genome to be used for the detection of parasite DNA in blood samples. A 23- bp sequence from the highly repetitive retrotransposon SjR2 was identified and it was demonstrated that PCR test to detect SjR2 is highly sensitive and specific for detection S. japonicum infection in the sera of infected rabbits and patients [73]. More recently the same group performed a comparative study to determine the best target to be used in a molecular diagnosis test for schistosomiasis japonicum in 29 retrotransposons identified by bioinformatics analysis. A 33-bp sequence had the highest sensitivity and specificity for the detection of S. japonicum DNA in serum samples [74]. Proteomics analysis has also been used in the identification of candidates to the immunodiagnosis of schistosomiasis. Western Blot with sera from S. japonicum infected rabbit in a two-dimensional gel loaded with adult worm preparation identified 1 spots that were demonstrated by LC/MS- MS to correspond to four different proteins: SjLAP (Leucine aminopeptidases), SjFBPA (fructose-1,6-bisphosphate aldolase), SjGST (Glutathione-S-transferase) and SJ22.6 [75]. Recombinant SjLAP and SjFBPA were tested in ELISA assay and presented high efficacy for the diagnosis of S. japonicum infection, with 96.7% specificity for both proteins and 98.1% or 87.8% sensitivity to detect acute and chronically infected individuals, respectively, when SjLAP was used as antigen or a sensitivity of 1% (acute) and 84.7% (chronic infection) whensjfbpawasusedasantigen[75]. 5. Other Membrane Proteins Candidates to Be Used in Vaccine Formulation and Diagnosis Tests Aquaporins are small integral membrane proteins involved in the selective transportation of water and other solutes through plasma membranes of mammals, plants and lower organisms [76]. This protein was described to be abundant in schistosome tegument and due to its physiological function and abundance represent an interesting target to vaccines and diagnosis tests [48]. Characterization of the S. japonicum aquaporin-3 using bioinformatics tools demonstrated that this 32.9 kda transmembrane protein has predicted B cells epitopes with the most likely epitopes present in the N- terminal portion of the protein, located outside the membrane [77]. Other abundant protein in schistosoma tegument is dysferlin, based on analogy with homologues from other organisms, this protein seems to be involved in membrane repair and/or vesicle fusion in tegument surface [34]. ATP-diphosphohydrolases are enzymes involved in ADP and ATP hydrolysis that has been related to host immune system evasion, since this enzyme could hydrolyze the ATP produced in response to parasite induced stress in the endothelio thus modulating the DAMP (danger associated molecular pattern)-mediated inflammatory signaling [78, 79]. In schistosomes two different proteins have been described SmATPDase 1 and SmATPDase2 with approximately 63 and 55 kda [8, 81]. SmATPDase 1 is located in the border of the tegument while SmATPDase2 is located in internal structure of the tegument syncytium and can be secreted [81]. The immunogenicity of the synthetic peptide (r175 19) from SmATPDase2 has been demonstrated in Balb-c mice, however the protection induced by this epitope has not been evaluated [82]. Although most tegument protein listed in this paper has been identified in adult worm tegument, an in silico analysis performed in SchistoDB ( demonstrates that some of them are also expressed in the schistosomula stage as demonstrated in Figure 1 reinforcing their potential to be used in a vaccine formulation or in the early diagnosis of schistosome infection. 6. Conclusion So far the genome, transcriptome, and proteome information provided many targets to be tested in schistosomiasis vaccine and diagnosis and also new knowledge about schistosome biology. However approximately 4% of the schistosome genome is composed of hypothetical proteins with unknown function that represents interesting targets to be

64 Journal of Parasitology Research 5 Numbers of EST Sm2 Sm29 TSP-2 TSP-1 Dysferlin Sm22.6 Sm21.7 Cercariae Schistosomula Adult worm Egg Figure 1: Predicted expression of schistosome tegument proteins in the different parasite life stage in the definitive host. schistosome tegument protein identified by proteomics analysis of the adult worm tegument was analyzed in SchistoDB database ( Bars represent the numbers of EST in each parasite life stage whose annotation correspond to Sm2, Sm29, TSP-2, TSP-1, Dysferlin, Sm22.6, or Sm21.7. tested and characterized. An increase in the knowledge about parasite biology, pathogenesis, and host-parasite relationship can 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 and C. C. Alves both received fellowship from Fapemig. C. T. Fonseca received a fellowship from PQ/CNPq. References [1] N. R. Bergquist, L. R. Leonardo, and G. F. Mitchell, Vaccinelinked chemotherapy: can schistosomiasis control benefit from an integrated approach? Trends in Parasitology, vol. 21, no. 3, pp , 25. [2] N. Berhe, G. Medhin, B. Erko et al., Variations in helminth faecal egg counts in Kato-Katz thick smears and their implications in assessing infection status with Schistosoma mansoni, Acta Tropica, vol. 92, no. 3, pp , 24. [3] M. Berriman, B. J. Haas, P. T. Loverde et al., The genome of the blood fluke Schistosoma mansoni, Nature, vol. 46, no. 7253, pp , 29. [4] Y. Zhou, H. Zheng, Y. Chen et al., The Schistosoma japonicum genome reveals features of host-parasite interplay, Nature, vol. 46, no. 7253, pp , 29. [5] A. V. Protasio, I. J. Tsai, A. Babbage et al., A systematically improved high quality genome and transcriptome of the human blood fluke Schistosoma mansoni, PLoS Neglected Tropical Diseases, vol. 6, no. 1, Article ID e1455, 212. [6] N. D. Young, A. R. Jex, B. Li et al., Whole-genome sequence of Schistosoma haematobium, Nature Genetics, vol. 44, no. 2, pp , 212. [7] A. Zerlotini, M. Heiges, H. Wang et al., SchistoDB: a Schistosoma mansoni genome resource, Nucleic Acids Research, vol. 37, no. 1, pp. D579 D582, 29. [8] A.R.C.Harris,R.J.Russell,andA.D.Charters, Areviewof schistosomiasis in immigrants in Western Australia, demonstrating the unusual longevity of Schistosoma mansoni, Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 78, no. 3, pp , [9] S.J.Davies,J.L.Grogan,R.B.Blank,K.C.Lim,R.M.Locksley, and J. H. McKerrow, Modulation of blood fluke development in the liver by hepatic CD4 + lymphocytes, Science, vol. 294, no. 5545, pp , 21. [1] R. L. De Mendonça, H. Escrivá, D. Bouton, V. Laudet, and R. J. Pierce, Hormones and nuclear receptors in schistosome development, Parasitology Today, vol. 16, no. 6, pp , 2. [11] P. Saule, E. Adriaenssens, M. Delacre et al., Early variations of host thyroxine and interleukin-7 favor Schistosoma mansoni development, Journal of Parasitology, vol. 88, no. 5, pp , 22. [12] P. Amiri, R. M. Locksley, T. G. Parslow et al., Tumour necrosis factor α restores granulomas and induces parasite egg-laying in schistosome-infected SCID mice, Nature, vol. 356, no. 637, pp , [13] P. T. LoVerde, A. Osman, and A. Hinck, Schistosoma mansoni: TGF-β signaling pathways, Experimental Parasitology, vol. 117, no. 3, pp , 27. [14] I. Wolowczuk, S. Nutten, O. Roye et al., Infection of mice lacking interleukin-7 (IL-7) reveals an unexpected role for IL- 7 in the development of the parasite Schistosoma mansoni, Infection and Immunity, vol. 67, no. 8, pp , [15] R. B. Blank, E. W. Lamb, A. S. Tocheva et al., The common γ chain cytokines interleukin (IL)-2 and IL-7 indirectly modulate blood fluke development via effects on CD4 + Tcells, Journal of Infectious Diseases, vol. 194, no. 11, pp , 26. [16] D. A. A. Vignali, P. Crocker, Q. D. Bickle, S. Cobbold, H. Waldmann, and M. G. Taylor, A role for CD4 + but not CD8 + T cells in immunity to Schistosoma mansoni induced by 2 kradirradiated and Ro terminated infections, Immunology, vol. 67, no. 4, pp , [17] D. Jankovic, T. A. Wynn, M. C. Kullberg et al., Optimal vaccination against Schistosoma mansoni requires the induction of both B cell- and IFN-γ-dependent effector mechanisms, Journal of Immunology, vol. 162, no. 1, pp , [18] M. Street, P. S. Coulson, C. Sadler et al., TNF is essential for the cell-mediated protective immunity induced by the radiation-attenuated schistosome vaccine, Journal of Immunology, vol. 163, no. 8, pp , [19] F. G. C. Abath and R. C. Werkhauser, The tegument of Schistosoma mansoni: functional and immunological features, Parasite Immunology, vol. 18, no. 1, pp. 15 2, [2] Z. G. Han, P. J. Brindley, S. Y. Wang, and C. Zhu, Schistosoma genomics: new perspectives on schistosome biology and hostparasite interaction, Annual Review of Genomics and Human Genetics, vol. 1, pp , 29. [21] M. K. Jones, G. N. Gobert, L. Zhang, P. Sunderland, and D. P. McManus, The cytoskeleton and motor proteins of human schistosomes and their roles in surface maintenance and hostparasite interactions, BioEssays, vol. 26, no. 7, pp , 24. [22] D. J. Hockley and D. J. McLaren, Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm, International Journal for Parasitology, vol. 3, no. 1, pp. 13 2, [23] M. Salzet, A. Capron, and G. B. Stefano, Molecular crosstalk in host-parasite relationships: Schistosome- and leech-host

65 6 Journal of Parasitology Research interactions, Parasitology Today, vol. 16, no. 12, pp , 2. [24] R. T. Damian, Molecular mimicry revisited, Parasitology Today, vol. 3, no. 9, pp , [25] S. R. Smithers, D. J. McLaren, and F. J. Rahalho-Pinto, Immunity to schistosomes: the target, American Journal of Tropical Medicine and Hygiene, vol. 26, no. 6, pp , [26] F. Santoro, P. J. Lachmann, A. Capron, and M. Capron, Activation of complement by Schistosoma mansoni schistosomula: killing of parasites by the alternative pathway and requirement of IgG for classical pathway activation, Journal of Immunology, vol. 123, no. 4, pp , [27] D. A. Dean, Decreased binding of cytotoxic antibody by developing Schistosoma mansoni. Evidence for a surface change independent of host antigen adsorption and membrane turnover, Journal of Parasitology, vol. 63, no. 3, pp , [28] A.Dessein,J.C.Samuelson,andA.E.Butterworth, Immune evasion by Schistosoma mansoni: loss of susceptibility to antibody or complement-dependent eosinophil attack by schistosomula cultured in medium free of macromolecules, Parasitology, vol. 82, no. 3, pp , [29] E. L. Racoosin, S. J. Davies, and E. J. Pearce, Caveolae-like structures in the surface membrane of Schistosoma mansoni, Molecular and Biochemical Parasitology, vol. 14, no. 2, pp , [3] R. El Ridi, S. H. Mohamed, and H. Tallima, Incubation of Schistosoma mansoni lung-stage schistosomula in corn oil exposes their surface membrane antigenic specificities, Journal of Parasitology, vol. 89, no. 5, pp , 23. [31] D. J. McLaren, Will the real target of immunity to schistosomiasis please stand up, Parasitology Today, vol. 5, no. 9, pp , [32] G. N. Gobert, M. Chai, and D. P. McManus, Biology of the schistosome lung-stage schistosomulum, Parasitology, vol. 134, no. 4, pp , 27. [33] F. V. Durães, N. B. Carvalho, T. T. Melo, S. C. Oliveira, and C. T. Fonseca, IL-12 and TNF-α production by dendritic cells stimulated with Schistosoma mansoni schistosomula tegument is TLR4- and MyD88-dependent, Immunology Letters, vol. 125, no. 1, pp , 29. [34] B. W. M. Van Balkom, R. A. Van Gestel, J. F. H. M. Brouwers et al., Mass spectrometric analysis of the Schistosoma mansoni tegumental sub-proteome, Journal of Proteome Research, vol. 4, no. 3, pp , 25. [35] S. Braschi, W. C. Borges, and R. A. Wilson, Proteomic analysis of the shistosome tegument and its surface membranes, Memorias do Instituto Oswaldo Cruz, vol. 11, supplement 1, pp , 26. [36] S. Braschi and R. A. Wilson, Proteins exposed at the adult schistosome surface revealed by biotinylation, Molecular and Cellular Proteomics, vol. 5, no. 2, pp , 26. [37] A. Loukas, S. Gaze, J. P. Mulvenna et al., Vaccinomics for the major blood feeding helminths of humans, OMICS A Journal of Integrative Biology, vol. 15, no. 9, pp , 211. [38] T. Teixeira De Melo, J. Michel De Araujo, F. Do Valle Durães et al., Immunization with newly transformed Schistosoma mansoni schistosomula tegument elicits tegument damage, reduction in egg and parasite burden, Parasite Immunology, vol. 32, no , pp , 21. [39] G. P. Dillon, T. Feltwell, J. P. Skelton et al., Microarray analysis identifies genes preferentially expressed in the lung schistosomulum of Schistosoma mansoni, International Journal for Parasitology, vol. 36, no. 1, pp. 1 8, 26. [4] G. N. Gobert, M. H. Tran, L. Moertel et al., Transcriptional changes in Schistosoma mansoni during early schistosomula development and in the presence of erythrocytes, PLoS Neglected Tropical Diseases, vol. 4, no. 2, article e6, 21. [41] J.M.Correnti,P.J.Brindley,andE.J.Pearce, Long-termsuppression of cathepsin B levels by RNA interference retards schistosome growth, Molecular and Biochemical Parasitology, vol. 143, no. 2, pp , 25. [42] M. E. Morales, G. Rinaldi, G. N. Gobert, K. J. Kines, J. F. Tort, and P. J. Brindley, RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade, Molecular and Biochemical Parasitology, vol. 157, no. 2, pp , 28. [43]M.H.Tran,T.C.Freitas,L.Cooperetal., Suppressionof mrnas encoding tegument tetraspanins from Schistosoma mansoni results in impaired tegument turnover, PLoS pathogens, vol. 6, no. 4, Article ID e184, 21. [44] F. Liu, S. J. Cui, W. Hu, Z. Feng, Z. Q. Wang, and Z. G. Han, Excretory/secretory proteome of the adult developmental stage of human blood fluke, Schistosoma japonicum, Molecular and Cellular Proteomics, vol. 8, no. 6, pp , 29. [45] S. Verjovski-Almeida, R. DeMarco, E. A. L. Martins et al., Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni, Nature Genetics, vol. 35, no. 2, pp , 23. [46] C. D. Criscione, C. L. L. Valentim, H. Hirai, P. T. LoVerde, and T. J. C. Anderson, Genomic linkage map of the human blood fluke Schistosoma mansoni, Genome Biology, vol. 1, no. 6, article R71, 29. [47] W. Castro-Borges, A. Dowle, R. S. Curwen, J. Thomas-Oates, and R. A. Wilson, Enzymatic shaving of the tegument surface of live schistosomes for proteomic analysis: a rational approach to select vaccine candidates, PLoS Neglected Tropical Diseases, vol. 5, no. 3, article e993, 211. [48] W. Castro-Borges, D. M. Simpson, A. Dowle et al., Abundance of tegument surface proteins in the human blood fluke Schistosoma mansoni determined by QconCAT proteomics, Journal of Proteomics, vol. 74, pp , 211. [49] F. C. Cardoso, J. M. R. Pinho, V. Azevedo, and S. C. Oliveira, Identification of a new Schistosoma mansoni membranebound protein through bioinformatic analysis, Genetics and Molecular Research, vol. 5, no. 4, pp , 26. [5] F. C. Cardoso, G. C. Macedo, E. Gava et al., Schistosoma mansoni tegument protein Sm29 is able to induce a Th1-type of immune response and protection against parasite infection, PLoS Neglected Tropical Diseases, vol. 2, no. 1, article e38, 28. [51] D. W. Dunne, M. Webster, P. Smith et al., The isolation of a 22 kda band after SDS-PAGE of Schistosoma mansoni adult worms and its use to demonstrate the IgE responses against the antigen(s) it contains are associated with human resistance to reinfection, Parasite Immunology, vol. 19, no. 2, pp , [52] M. L. Santiago, J. C. R. Hafalla, J. D. Kurtis et al., Identification of the Schistosoma japonicum 22.6-kDa antigen as a major target of the human IgE response: similarity of IgE-binding epitopes to allergen peptides, International Archives of Allergy and Immunology, vol. 117, no. 2, pp , [53] L. G. G. Pacífico, C. T. Fonseca, L. Chiari, and S. C. Oliveira, Immunization with Schistosoma mansoni 22.6 kda antigen induces partial protection against experimental infection in a recombinant protein form but not as DNA vaccine, Immunobiology, vol. 211, no. 1-2, pp , 26.

66 Journal of Parasitology Research 7 [54] L. G. G. Pacífico, C. T. Fonseca, M. M. Barsante, L. S. Cardoso, M. I. Araújo, and S. C. Oliveira, Aluminum hydroxide associated to Schistosoma mansoni 22.6 kda protein abrogates partial protection against experimental infection but not alter interleukin-1 production, Memorias do Instituto Oswaldo Cruz, vol. 11, supplement 1, pp , 26. [55] L. S. Cardoso, S. C. Oliveira, A. M. Góes et al., Schistosoma mansoni antigens modulate the allergic response in a murine model of ovalbumin-induced airway inflammation, Clinical and Experimental Immunology, vol. 16, no. 2, pp , 21. [56] D. Smyth, D. P. McManus, M. J. Smout, T. Laha, W. Zhang, and A. Loukas, Isolation of cdnas encoding secreted and transmembrane proteins from Schistosoma mansoni by a signal sequence trap method, Infection and Immunity,vol.71,no.5, pp , 23. [57] M. H. Tran, M. S. Pearson, J. M. Bethony et al., Tetraspanins on the surface of Schistosoma mansoni are protective antigens against schistosomiasis, Nature Medicine, vol. 12, no. 7, pp , 26. [58] W. Zhang, J. Li, M. Duke et al., Inconsistent protective efficacy and marked polymorphism limits the value of Schistosoma japonicum tetraspanin-2 as a vaccine target, PLoS Neglected Tropical Diseases, vol. 5, no. 5, Article ID e1166, 211. [59] S. Y. Sauma and M. Strand, Identification and characterization of glycosylphosphatidylinositol-linked Schistosoma mansoni adult worm immunogens, Molecular and Biochemical Parasitology, vol. 38, no. 2, pp , 199. [6] P. J. Brindley, M. Strand, A. P. Norden, and A. Sher, Role of host antibody in the chemotherapeutic action of praziquantel against Schistosoma mansoni: identification of target antigens, Molecular and Biochemical Parasitology, vol. 34, no. 2, pp , [61] E. J. M. Nascimento, R. V. Amorim, A. Cavalcanti et al., Assessment of a DNA vaccine encoding an anchored- glycosylphosphatidylinositol tegumental antigen complexed to protamine sulphate on immunoprotection against murine schistosomiasis, Memorias do Instituto Oswaldo Cruz, vol. 12, no. 1, pp , 27. [62] V. P. Martins, C. S. Pinheiro, B. C. P. Figueiredo et al., Vaccination with enzymatically cleaved GPI-anchored proteins from schistosoma mansoni induces protection against challenge infection, Clinical and Developmental Immunology, vol. 212, Article ID , 11 pages, 212. [63] H. M. Ahmed and M. H Romeih, Protection against Schistosoma mansoni infection with recombinant schistosomula 21.7 kda protein, Arab Journal of Biotechnology, vol. 24, pp , 21. [64] H. M. Ahmed, M. H. Romeih, and T. S. Abou-Shousha, DNA immunization with the gene encoding Sm21.7 protects mice against S. mansoni infections, American Journal of Science, vol. 2, pp , 26. [65] K. A. Shalaby, L. Yin, A. Thakur, L. Christen, E. G. Niles, and P. T. LoVerde, Protection against Schistosoma mansoni utilizing DNA vaccination with genes encoding Cu/Zn cytosolic superoxide dismutase, signal peptide-containing superoxide dismutase and glutathione peroxidase enzymes, Vaccine, vol. 22, no. 1, pp , 23. [66]Z.Hong,D.J.Kosman,A.Thakur,D.Rekosh,andP.T. LoVerde, Identification and purification of a second form of Cu/Zn superoxide dismutase from Schistosoma mansoni, Infection and Immunity, vol. 6, no. 9, pp , [67] H. Mei and P. T. LoVerde, Schistosoma mansoni: the developmental regulation and immunolocalization of antioxidant enzymes, Experimental Parasitology, vol. 86, no. 1, pp , [68] H.Mei,A.Thakur,J.Schwartz,andP.T.LoVerde, Expression and characterization of glutathione peroxidase activity in the human blood fluke Schistosoma mansoni, Infection and Immunity, vol. 64, no. 1, pp , [69] M. D. M. Mourão, N. Dinguirard, G. R. Franco, and T. P. Yoshino, Role of the endogenous antioxidant system in the protection of Schistosoma mansoni primary sporocysts against exogenous oxidative stress, PLoS Neglected Tropical Diseases, vol. 3, no. 11, article e55, 29. [7] J. V. Hamilton, M. Klinkert, and M. J. Doenhoff, Diagnosis of schistosomiasis: antibody detection, with notes on parasitological and antigen detection methods, Parasitology, vol. 117, pp. S41 S57, [71] M. J. Doenhoff, P. L. Chiodini, and J. V. Hamilton, Specific and sensitive diagnosis of schistosome infection: can it be done with antibodies? Trends in Parasitology, vol. 2, no. 1, pp , 24. [72] G.B.F.Carvalho,R.A.daSilva-Pereira,L.G.G.Pacífico, and C. T. Fonseca, Identification of Schistosoma mansoni candidate antigens for diagnosis of schistosomiasis, Memorias do Instituto Oswaldo Cruz, vol. 16, no. 7, pp , 211. [73] C. M. Xia, R. Rong, Z. X. Lu et al., Schistosoma japonicum: a PCR assay for the early detection and evaluation of treatment in a rabbit model, Experimental Parasitology, vol. 121, no. 2, pp , 29. [74] J.-J. Guo, H.-J. Zheng, J. Xu, X.-Q. Zhu, S.-Y. Wang, and C.- M. Xia, Sensitive and specific target sequences selected from retrotransposons of Schistosoma japonicum for the diagnosis of schistosomiasis, PLoS Neglected Tropical Diseases, vol. 6, no. 3, Article ID e1579, 212. [75] Z. R. Zhong, H. B. Zhou, X. Y. Li et al., Serological proteome-oriented screening and application of antigens for the diagnosis of Schistosomiasis japonica, Acta Tropica, vol. 116, no. 1, pp. 1 8, 21. [76] A. S. Verkman, Physiological importance of aquaporin water channels, Annals of Medicine, vol. 34, no. 3, pp , 22. [77] J. Song and Q.-F. He, Bioinformatics analysis of the structure and linear B-cell epitopes of aquaporin-3 from Schistosoma japonicum, Asian Pacific Journal of Tropical Medicine, vol. 5, no. 2, pp , 212. [78] E. G. Vasconcelos, P. S. Nascimento, M. N. L. Meirelles, S. Verjovski-Almeida, and S. T. Ferreira, Characterization and localization of an ATP-diphosphohydrolase on the external surface of the tegument of Schistosoma mansoni, Molecular and Biochemical Parasitology, vol. 58, no. 2, pp , [79] R. Bhardwaj and P. J. Skelly, Purinergic signaling and immune modulation at the schistosome surface? Trends in Parasitology, vol. 25, no. 6, pp , 29. [8] R. DeMarco, A. T. Kowaltowski, R. A. Mortara, and S. Verjovski-Almeida, Molecular characterization and immunolocalization of Schistosoma mansoni ATP-diphosphohydrolase, Biochemical and Biophysical Research Communications, vol. 37, no. 4, pp , 23. [81] J. Levano-Garcia, R. A. Mortara, S. Verjovski-Almeida, and R. DeMarco, Characterization of Schistosoma mansoni ATP- Dase2 gene, a novel apyrase family member, Biochemical and Biophysical Research Communications, vol. 352, no. 2, pp , 27. [82]R.G.P.R.Mendes,M.A.N.Gusmão,A.C.R.G.Maia et al., Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydrolase isoform

67 8 Journal of Parasitology Research (SmATPDase 2) and immunolocalisation of this protein in the schistosoma mansoni egg, Memorias do Instituto Oswaldo Cruz, vol. 16, no. 7, pp , 211. [83] M. E. Hemler, Specific tetraspanin functions, JournalofCell Biology, vol. 155, no. 7, pp , 21.

68 Hindawi Publishing Corporation Journal of Parasitology Research Volume 212, Article ID , 9 pages doi:1.1155/212/ Review Article Pathogenicity of Trichobilharzia spp. for Vertebrates Lichtenbergová Lucie and Horák Petr Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, Prague 2, Czech Republic Correspondence should be addressed to Lichtenbergová Lucie, lichten@centrum.cz Received 13 July 212; Accepted 13 September 212 Academic Editor: Rashika El Ridi Copyright 212 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 type I, immediate hypersensitivity response, followed by a late phase reaction. The immune response is Th2 polarized. Primary infection leads to an inflammatory reaction that is insufficient to eliminate the schistosomes and schistosomula may continue its migration through the body of avian as well as mammalian hosts. However, reinfections of experimental mice revealed an immune reaction leading to destruction of the majority of schistosomula in the skin. Infection with the nasal schistosome Trichobilharzia regenti probably 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 waterfowl where 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 the vicinity of the schistosomulum. Such infections are manifest by neuromotor disorders. 1. Introduction Despite their worldwide distribution, avian schistosomes were neglected by parasitologists who assumed that they have no or minor pathogenic impact on birds or mammals, including humans. Nowadays, many studies focus on these parasites since it has been recognized that they can be severe pathogens of birds. Moreover, their larval stages (cercariae) frequently infect humans and cause cercarial dermatitis. The most reported agents of swimmer s itch are cercariae of the genus Trichobilharzia [1]. Human infections by bird schistosomes are associated mostly with the development of cercarial dermatitis (swimmer s itch), an allergic skin response, which develops after repeated contact with cercariae penetrating into the skin. For a long time, it was assumed that the reaction eliminated the majority of the schistosomes that had penetrated into the skin. However, the studies on mice infected experimentally with bird schistosomes showed that soon after the penetration, the cercariae transform to schistosomula. Under certain circumstances, these schistosomula are able to resist host immune response, escape from the skin, and migrate further to target organs [2, 3]. In mammals, bird schistosomes can survive for several days or weeks, but they never mature [4, 5]. The exact reason why bird schistosomes die in mammalian hosts has not been known until the present. Studies on bird schistosomes disclosed a new species Trichobilharzia regenti [4] with unusual behavior in compatible as well, noncompatible hosts. In comparison to the majority of bird schistosome species living in the blood system of visceral organs, mature T. regenti occur in the nasals of their definitive host where they lay eggs. Migration of the worms from the skin to the nasals is via the spinal cord and brain [4]. Experimental infections of mice showed that T. regenti schistosomula can evade attack by immune cells in the skin of mammalian hosts allowing them to migrate further through the central nervous system (CNS) where immature worms die after several days [5, 6]. Migration of the parasites through CNS of both bird and mammalian hosts causes severe 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 conditions with regard to their development, physiology (including

69 2 Journal of Parasitology Research enzymes participating in host tissue degradation and digestion), immunomodulation of the host immune response, and pathogenicity towards natural and accidental hosts. In field studies, the emphasis is on the study of species spectrum, inter- and intraspecific variability, and prevalence of bird schistosomes (see, e.g., Brant and Loker [8], Jouet et al. [9], and Korsunenko et al. [1]; see also review by Horák and Kolářová [1]). With regard to T. szidati and T. regenti, their occurrence has been reported from several countries. In particular, T. regenti cercariae have been found in freshwater ponds for example, in Russia [11]and cercariae of T. szidati in Russia, Belorussia [11], Germany [12] and France [13]. Several findings of T. szidati infections in birds were reported, for example, from France [9], Poland, and Czech Republic [14]. Infections of birds with T. regenti were detected for example, in Iceland [15] and in France, where the prevalence on three studied localities reached 4% [9]. Based on findings of Rudolfová et al. [14, 16], prevalence of T. 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 distributed throughout Europe, there is a report of their occurrence in snails collected from Michigan and Montana in the United States [8]. The main aim of our review is to summarize the present knowledge of the pathogenesis of bird schistosomiasis and the immune reactions to bird schistosomes presence in avian and mammalian hosts, with a special emphasis on T. regenti. The neurotropic species T. regenti,due to its unusualmode of migration and potential pathogenic impact on avian as well as mammalian hosts, deserves more attention. Therefore, a major part of this review is dedicated to this species of schistosome. 2. Skin Infection After leaving the snail intermediate hosts, bird schistosome cercariae have a tendency to cling to the water surface and wait for their definitive host. They react to shadow stimuli and start to swim with a negative phototactic orientation from the water surface toward the definitive host [17]. Except for physical stimuli such as shadow, water turbulence, and warmth, cercariae respond to host chemical cues like duckfoot skin lipids cholesterol and ceramides [17, 18]. Once attached to the host skin, cercariae creep on the skin and search for a suitable penetration site [19]. In contrast to human schistosomes, such as Schistosoma mansoni,which penetrate smooth skin, bird schistosome cercariae prefer skin wrinkles and hair follicles for penetration [2]. Studies on cercarial behavior of S. mansoni and T. szidati revealed differences in the speed of migration through the host skin. For example, cercariae of T. szidati invade human skin more efficiently than S. mansoni such that they are able to locate entry sites and penetrate through the skin more rapidly than S. mansoni [2]. Skin penetration by cercariae is stimulated by fatty acids [19]. According to the study of Haas and Haeberlein [2], T. szidati cercariae respond to linolenic acid with higher sensitivity if compared to S. mansoni. This feature seems to represent an adaptation to invade duck skin that has a lower content of free fatty acids compared to human skin [2]. Therefore, human skin with higher amount of surface lipids is likely more attractive to bird schistosome cercariae than duck skin [19]. Penetration through the skin is facilitated by a number ofproteolyticenzymes,whicharereleasedfromcercarial circum- and postacetabular glands immediately after attaching to the host skin. In the case of bird schistosomes, glandular secretion is stimulated mainly by fatty acids, ceramides, and cholesterol [2]. Cercarial glands fill about one-third of the cercarial body [21] and contain many of the potentially antigenic proteins. The most important penetration enzyme of S. mansoni is probably a serine protease, elastase [22]. Nevertheless, Mikeš et al. [23] and Kašný et al. [24] did not find any elastase activity in the secretions of T. szidati and T. regenti cercariae, and it was not found in the congener S. japonicum [25]. However, cathepsin B-like activity was detected in the aforementioned species. This enzyme from cercarial penetration glands is considered to be the main component in the cercarial penetration process [23 25].Thesametypesofenzymes could be the reason for similar penetration speed of S. japonicum and Trichobilharzia cercariae [2]. In addition, six isoforms of cathepsin B1 (TrCB1.1 TrCB1.6) and cathepsin B2 (TrCB2) were identified in an extract of migrating T. regenti schistosomula [26, 27]. Two isoforms, TrCB1.1 and TrCB1.4, degrade myelin basic protein, but do not efficiently cleave hemoglobin [26]. The recombinant form of TrCB2 is able to cleave protein components of the skin (keratin, collagen, and elastin) as well as nervous tissue (myelin basic protein), but has negligible activity towards hemoglobin [27]. The enzyme could, therefore, serve as a tool for migration through the host skin and subsequently through the nervous tissue. Host fatty acids seem to stimulate not only the penetration of cercaria through the host skin, but also transformation of their tegument as a part of parasite immune evasion [19]. Penetration of the cercariae into the host skin is accompanied by cercaria/schistosomulum transformation with reconstruction of tegumental surface. Transformation starts with loss of tail, a process supported by a sphincter muscle in cercarial hindbody [19], then the cercariae shed the glycocalyx and start to form a surface double membrane. Creation of a new surface is accompanied by the disappearance of lectin and antibody targets on the surface of the schistosomula [28]. In the skin of the bird hosts, schistosomula move through the skin towards deeper layers and, therefore, require information for orientation. Studies on visceral schistosomes invading humans, S. mansoni, and birds, T. ocellata, showed that schistosomula use negative photo orientation to move away from light source [29]. The other stimulus involved in navigation of the visceral schistosomula is represented by the concentration gradient of chemicals, such as D-glucose and L-arginine [3]. Unfortunately, data about orientation of the nasal species T. regenti are not complete, but there is an indication that the stimuli differ from those used by visceral species (unpublished).

70 Journal of Parasitology Research 3 3. Cercarial Dermatitis In humans, the skin infection is a result of the development of an inflammatory reaction known as swimmer s itch or cercarial dermatitis. Cercarial dermatitis can occur after contact with water containing cercariae from snails infected by bird schistosomes. Chances of getting cercarial dermatitis increase with repeated exposures to the parasite. Higher incidence of the infection is connected with bathing in shallow water, which is the preferred habitat for water snails and, therefore, a place where cercariae accumulate [31]. Penetration of cercariae into the skin may result in an immediate prickling sensation that lasts for about 1 hour [32]. Severity and intensity of cercarial dermatitis depend on various factors including the number and duration of exposures to the cercariae, and host immune status, that is, history of cercarial dermatitis, and individual susceptibility to the infection [32]. After a primary infection, the skin reaction is unapparent or mild with small and transient macules or maculopapules, which develop after 5 14 days [32]. The most pronounced disease occurs after repeated exposures that result in a strong inflammatory reaction against the parasites [33]. The skin disease manifests by maculo-papulovesicular eruptions accompanied by intense itching and, occasionally, by erythema, fever, local lymph node swelling, oedema. Massive infections may also cause nausea and diarrhea (for a review see Horák et al. [34]). Skin lesions develop only on those parts of body where there was cercarial penetration [35]. Diagnosis of cercarial dermatitis is based on anamnesis and clinical findings [36]. Some work has been done using serological tests for confirmation of the diagnosis [37]. Nevertheless, immunological tests are not routinely available and laboratory confirmation of causative agents of the dermatitis remains difficult. 4. Skin Immune Response Clinical pattern of cercarial dermatitis is linked with histopathological reactions to the infection. In the case of the human schistosome, Schistosoma mansoni, infections in naive mice led to a mild skin response with contribution of neutrophils and mononuclear cells. In contrast, a more severe cellular reaction developed in the mouse skin after repeated infections with S. mansoni [38]. Similarly as for human schistosomes, infections of mice with the bird schistosomes T. szidati (T. ocellata)andt. regenti initiate the development of a skin immune response [2, 39]. Primary mouse infection with T. regenti initiates an acute inflammation with oedema, vasodilatation, and tissue infiltration by neutrophils, macrophages, mast cells, and MHC II antigen presenting cells (APCs), and a weak infiltration by CD4 + lymphocytes; repeated infections cause substantially elevated infiltration of all cells mentioned above [2, 6]. Trichobilharzia regenti primoinfection leads to the development of an inflammatory reaction in the murine skin within 1 6 h after exposure [2]. Detection of cytokine production by in vitro cultured biopsies of pinnae (later skin biopsies) obtained from 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 with higher Th1-associated IFN-γ production by cells from skindraining lymph nodes [2]. Similarly, the study of Hogg et al. [4] using S. mansoni illustrates rapid host immune response to parasite penetration with production of acute phase cytokines, such as IL-1β and IL-6 which were detected in the supernatants of skin biopsies from wild-type mice. Both IL- 1β and IL-6 promote Th17-cell differentiation [41], therefore implying that primary mouse infection with human as well as avian schistosomes induces a Th17 polarized response. Skin immune response to challenge infections leads to capture and elimination of the majority of schistosomula in the skin [2]. In the early phase after re-infection with T. regenti cercariae, infiltration of mouse skin with inflammatory cells (high density of granulocytes and neutrophils, abundant MHC II APCs, macrophages and CD4 + lymphocytes) was also accompanied by oedema caused by local vascular permeability that was initiated by histamine produced by activated mast cells and basophils [2]. Degranulation of mast cells and basophils with release of histamine and IL-4 is realized after binding of IgE-antigen complex via high affinity receptors FcεRI on the cell surface [42, 43]. Histamine has been described previously as a potent effector of Th1 and Th2 responses as well as immunoglobulin synthesis [44, 45]. Repeated infections with T. regenti evoke dominant production of Th2-type cytokines, and the first and most abundant cytokine detected in supernatants of skin biopsies from mice after repeated infections is IL-6 [2], which can initiate Th2-type polarization via induction of IL-4 [46]. Within 1 hour after the penetration by T. regenti cercariae a massive upregulation of IL-4 and IL-1 can be observed in the skin biopsies, and the level of these cytokines declines after 48 h. This upregulation during T. regenti infection is accompanied by release of histamine and proliferation of mast cells [2]. Production of histamine and IL-4 detected in skin biopsies immediately after the last infection of reinfected mice was realized via IgE-dependent mast cell degranulation [2]. IL-4 plays also a crucial role in the development of Th2-type immune responses to S. mansoni antigens and regulation of immunoglobulin isotype switch to IgE [47]. CD4 + cells, numbers of which significantly increase in the skin after challenge infections, are potential sources of IL-4 associated with Th2 response [2]. Like mast cells, basophils possess high-affinity IgE surface receptors (FcεRI) that, after antigen-specific crosslink, induce production and release of mediators such as histamine and IL-4 [48]. In vitro stimulation of basophils obtained from healthy (nonsensitized) humans by homogenate of cercariae and excretory/secretory (E/S) products of T. regenti cercariae revealed that these antigens induce basophil degranulation and release of IL-4 [49]. Antigens stimulated basophil release of IL-4 in a dose-dependent manner, and antigens from E/S products were more potent inducers of IL-4 release than cercarial homogenate [49]. Elevated levels of skin mast cells [2] and high titers of serum IgE in mice infected repeatedly with T. regenti [49] showed that the cells of mast cell/basophil lineage play an important

71 4 Journal of Parasitology Research role in development of Th2 responses during Trichobilharzia infections. 5. Antibody Response and Antigens of Bird Schistosomes Domination of Th-2 polarization of the immune response after repeated T. regenti infections was confirmed by measurement of antigen-specific antibody levels. Similarly as in the study of Kouřilová et al. [2], the increase of Th2-associated antigen-specific IgG1 and total serum IgE antibodies with concurrent decline of Th1-associated IgG2b antibody was demonstrated in sera of mice repeatedly infected with T. regenti [49]. During T. regenti primary infection, IgM response against glycan structures of the cercariae and their excretory/secretory (E/S) products was observed [49]. This indicates that early antibody response is directed against components of highly antigenic cercarial glycocalyx as well as against glycoproteins contained in E/S products of circumand postacetabular glands of cercariae [49]. The glycocalyx of T. regenti cercariae was described as the most antigenic structure and its remnants were still present on 1-day old schistosomula transformed in vitro [5], therefore IgM antibodies could recognize components of the glycocalyx during primary infection. After penetration into the host skin, cercariae transform to schistosomula by shedding their tails, releasing E/S products, and rebuilding their surface [51]. The dominant part of the cercarial surface is represented by glycocalyx, which is likely the main component responsible for complement activation [52]. Cercarial surface of human and bird schistosomes is recognized by antibodies from humans and mice infected with T. regenti, T. szidati, ands. mansoni [53]. In the skin, parasites need to avoid destruction by the host immune system, thus the transformation of cercaria to schistosomulum is accompanied by the decrease of surface saccharides and antigen epitopes which could be recognized by lectins and antibodies, respectively [28]. Although a substantial part of glycocalyx is removed by the cercariae during penetration [54], some of glycocalyx components remain associated with surface of the schistosomula for some time after transformation [54, 55]. Therefore, not only the cercarial surface but also the surface of the early in vitro transformed (5 h) schistosomula was strongly recognized by IgG and total Ig antibodies from mice repeatedly infected by T. szidati [28]. E/S products of human as well as bird schistosome cercariae, mainly the products released by transforming larvae, are rich in components of glycocalyx and secretions of circum- and post-acetabular glands [23, 54]. Immunohistochemical staining of ultrathin sections of particular developmental stages revealed variable distribution of antigens recognized by IgG antibodies from sera of mice re-infected with T. regenti [5]. Except for antibody binding to the surface of cercariae and 1-day-old schistosomula, a positive reaction with spherical bodies located in subtegumental cells of cercariae and early schistosomula was recorded [5]. In schistosomula, spherical bodies represented the most reactive structure. Further development of the parasite was accompanied with loss of immunoreactivity. In adult worms, the antibodies recognized the surface and subtegumental cells, but with lower intensity if compared to the larval stages [5]. Spherical bodies primary located within subtegumental cells were transferred via cytoplasmic bridges to the surface of schistosomula. As in human schistosomes [56], these bodies probably release their content on surface of the tegument where the antigenic molecules could be recognized by the host immune system [5]. However, composition of the spherical body content is not known till present. Nevertheless, based on Western blot analysis of cercarial homogenate probed with sera from re-infected mice, several antigens (14.7, 17, 28, 34, and 5 kda) were identified by IgG and IgE antibodies. Antigens of 34 and 5 kda were also recognized in cercarial gland secretions [49]. A precise identification of the 34 kda molecule which is regarded as a major immunogen is in progress. 6. CNS Infection The bird schistosome T. regenti exhibits an unusual mode of behavior. After successful escape from the host skin, schistosomula migrate further to the CNS of both specific avian and accidental mammalian hosts [5, 7]. CNS represents an obligatory part on the migration pathway of T. regenti to the nasal mucosa [4]. In specific bird hosts, schistosomula grow and mature during the migration, and their development is completed in the nasal area of the host [4]. In accidental mammalian hosts, the parasite is not able to complete its development and dies as an immature schistosomula in the spinal cord or brain [4]. Soon after penetration of cercariae into the skin of specific as well as accidental definitive hosts, schistosomula locate peripheral nerves, enter nerve fascicles (Figure 1) or epineurium, and reach the spinal cord via spinal roots [5, 57]. Schistosomula appear in the spinal cord from day 2 post infection (p.i.); intact parasites can be detected in the duck spinal cord even 23 days p.i. [7], and days p.i. in the spinal cord of mice [5]. Then, schistosomula migrate to the brain 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 days p.i. in the case of infected mice [5]. At the beginning of the brain infection, schistosomula are located mainly in medulla oblongata and further in subarachnoidal area of cerebellum [5, 57]. Other localization in the brain is mostly restricted to the subarachnoidal space and the area of the fourth ventricle [57, 58]. Migration through the host CNS requires parasites adaptations to this environment. Light-brown-pigmented granules in the intestine of T. regenti schistosomula [59], which exhibit immunoreactivity with antibodies against components of CNS [57], proved that schistosomula utilize host nervous tissue for nutrition during their migration via CNS. For this purpose the parasite should have proteases capable of cleaving components of the nervous tissue. Until the present only cathepsins B1 and B2 with the capability

72 Journal of Parasitology Research 5 (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 1 μm; (b) myelin sheets visualized by binding of anti-mbp (myelin basic protein) antibody and secondary anti-rabbit IgG-Cy3 (red), neurofilaments stained by anti-smi-32 (neurofilament H nonphosphorylated) and secondary anti-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 as candidate molecules. Except localization in CNS that is typical for this species, schistosomula were accidentally observed in the lungs of specific avian [4] aswellasnon-compatiblemammalian hosts [5, 57]. Moreover, schistosomula were also found in skin capillaries [53]. However, in vitro tests of blood vessel attractiveness did not show any positive results [5]. Therefore, it seems that schistosomulum presence in the lungs represents an ectopic localization of the parasite in heavily infected 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 were located mostly in meninges of thoracic and synsacral spinal cord and in the white and gray matter of synsacral spinal cord [7]. The surroundings of the worms were infiltrated with eosinophils, heterophils, and less frequently with plasma cells, histiocytes, lymphocytes, and macrophages [7]. Despite dense infiltration with the immune cells, the parasites were not destroyed by host immune response [7]. The parasites located in brain, predominantly in meninges, were surrounded by macrophages and endothelial cells [7]. At present, details about the immune response in the infected ducks are still missing. Morestudieshavebeendoneonmiceasamodelof non-compatible host. Early infections (3 days p.i.) of the mouse spinal cord did not initiate any inflammatory reaction or damage to the nervous tissue; only focal oedema was observed [6, 57]. Signs of the infection were observed 6 or 7 days p.i., when the vicinity of schistosomula was infiltrated with granulocytes, predominantly neutrophils, activated microglia, macrophages and, less frequently, CD3 + lymphocytes [6, 57]. It seems that microglia and macrophages, are responsible for destruction of schistosomula in the mouse CNS [57]. Similarly, microglia are the most important cells in prevention against Toxoplasma gondii tachyzoite proliferation in the brain [6]. The presence of T. regenti schistosomula in the nervous tissue triggered activation and proliferation of astrocytes, which were detected in the tracks after migrating schistosomula [57]. Formation of glial scars by astrocytic processes around inflammatory infiltrates was also observed in the brain of human patients infected with Taenia solium metacestodes [61]. The presence of the metacestodes was accompanied by astrocytic activation and increased expression of glial fibrillary acidic protein (GFAP) [61]. Similar activation of astrocytes was detected in the brain of Toxocara-infected mice [62]. Progress of T. regenti infection led to the formation of inflammatory lesions surrounding, destroyed schistosomula in the white matter of the spinal cord. Lesions were formed by microglia, macrophages, eosinophils, neutrophils and CD3 + lymphocytes, and damaged axons were detected in the tissue surrounding the lesions. On the other hand, presence of the parasites outside of parenchyma, in subarachnoidal space of the spinal cord and brain, and in the cavity of the 4th ventricle of the brain, did not initiate any heavy inflammation, nor pathological changes in the surrounding tissue. This implies that schistosomula are able to prolong their survival while they migrate outside of parenchyma [57]. Nevertheless, progression of the infection inevitably led to elimination of the parasite. Most schistosomula were destroyed on day 21 p.i. [57]. Challenge infections initiated development of a strong immune response leading to rapid destruction and elimination of schistosomula. No intact worms, only parasite remnants surrounded by inflammatory foci, were observed in 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 to development of mild immune reaction, which is not sufficient for elimination of the parasite [6, 57]. Even challenge infections did not induce proper immune reactions leading