INTRODUÇÃO: A esquistossomose se constitui em grande problema de saúde pública, sendo que estimativas apontam para 200 milhões de pessoas infectadas no mundo. No Brasil atinge 19 unidades federadas, apresentando áreas de alta e média endemicidade e, em uma grande extensão, áreas de baixa endemicidade. O município de Barra Mansa, Rio de Janeiro, Brasil, apresenta uma prevalência estimada de 1%. As áreas de baixa endemicidade (ABE) representam um novo desafio para o controle dessa helmintose, pois cerca de 75% dos indivíduos infectados são assintomáticos e cursam com infecções de baixa carga parasitária (<100 ovos por grama de fezes), ocorrendo uma diminuição da sensibilidade das técnicas parasitológicas de fezes, que são referência para o diagnóstico laboratorial dessa helmintose. OBJETIVO: Comparar o desempenho das técnicas de Kato-Katz (KK) e Hoffman, Pons e Janer (HH), do ensaio de ELISA-IgG e ELISA-IgM, da técnica de Imunofluorescência Indireta (RIFI), da técnica de qPCR TaqMan® em amostras de fezes e de soro (qPCR-fezes e qPCR-soro), tendo como referência a Reação Periovular (RPO), por meio de inquérito epidemiológico para obtenção de amostras de fezes e soro de indivíduos randomizados residentes nos bairros de Cantagalo, Nova Esperança, Santa Clara, São Luiz e Siderlândia, Barra Mansa/RJ. MÉTODOS: Estudo de corte transversal, no período de abril a dezembro de 2011, de amostragem probabilística, sendo coletadas 610 amostras de fezes e 612 amostras de soro. As técnicas de investigação diagnóstica laboratorial foram: KK e HH, ELISA-IgG e ELISA-IgM, RIFI-IgM, RPO, qPCR-fezes e qPCR-soro. RESULTADOS: Observaram-se os seguintes resultados, obtidos das diferentes técnicas diagnósticas: KK e HH, 0,8% (n=5); ELISA-IgG, 11,6% (n=71); ELISA-IgM, 21,4% (n=131); RIFI-IgM, 15,8 (n=97); RPO, 5,4% (n=33); qPCR-fezes, 9,8% (n=60); qPCR-soro, 1,5% (n=9). A maior positividade foi obtida no ensaio ELISA-IgM (21,4%), enquanto as técnicas de KK e HH foram as que menos denunciaram a presença da infecção (0,8%). Na comparação com a RPO, exceto a qPCR-soro, todas as outras técnicas apresentaram diferença estatisticamente significante na positividade (p < 0,05) e boa acurácia (82% a 95,5%), porém baixa concordância, sendo a melhor com ELISA-IgG (Kappa=0,377) e RIFI (Kappa=0,347). Na associação entre as variáveis sociodemográficas e as técnicas diagnósticas utilizadas, observou-se diferença estatisticamente significante (p =< 0,05) entre a variável residir no bairro Santa Clara, exceto com a técnica de qPCR-soro. CONCLUSÕES: As taxas de positividade das técnicas parasitológicas ficaram muito aquém daquelas apresentadas pelas outras técnicas. Vigilância inadequada nas áreas de baixa endemicidade de esquistossomose pode resultar na transformação das mesmas em áreas de média e alta endemicidade. Este estudo apresenta uma perspectiva de controle que aponta para a possibilidade de utilização de ferramentas laboratoriais combinadas para a identificação de casos nas áreas de baixa endemicidade / INTRODUCTION: Schistosomiasis constitutes a major public health problem, and estimates suggest that 200 million people are infected worldwide. In Brazil, it is reported in 19 federal units, showing areas of high and medium endemicity and a wide range of areas of low endemicity. Barra Mansa, Rio de Janeiro, Brazil, has an estimated prevalence of 1%. Areas of low endemicity (ALE) represent a new challenge for the helminth control because about 75% of infected individuals are asymptomatic and infections occur with low parasite load (< 100 eggs per gram of feces), causing a decrease in sensitivity of stool parasitological techniques, which are a reference for the laboratory diagnosis of this helminth. OBJECTIVE: To compare the performance of the techniques of Kato-Katz (KK), Hoffman, Pons and Janer (HH), ELISA-IgG and ELISA-IgM, the Indirect Immunofluorescence Technique (IFT) and the qPCR technique in samples of serum and stool (qPCR in feces and serum) using the Circumoval Precipitin Test (COPT) as reference, and epidemiological survey to obtain stool samples and sera from randomized residents in the neighborhoods of Cantagalo, Nova Esperança, Santa Clara, São Luiz and Siderlândia, Barra Mansa/RJ. METHODS: A cross-sectional study conducted from April to December 2011, using a probabilistic sampling that collected 610 fecal samples and 612 serum samples. The laboratory diagnostic techniques used were: KK and HH, ELISA-IgG and ELISA-IgM, IFA-IgM, COPT, qPCR-stool and qPCR-serum. RESULTS: We obtained the following results from different diagnostic techniques: KK and HH, 0.8% (n=5); ELISA-IgG, 11.6% (n=71); ELISA-IgM, 21.4% (n=131); IFA-IgM 15.8 (n=97); RPO 5.4% (n=33); qPCR-stools, 9.8% (n=60) and qPCR-serum, 1 5% (n=9). ELISA-IgM (21.4%) presented the highest positivity while the techniques of HH and KK were the least sensitive to indicate the presence of infection (0.8%). In comparison with COPT, except for qPCR-serum, all other techniques showed a statistically significant difference in positivity (p < 0.05) and high accuracy (from 82% to 95.5%), but poor agreement, and the best one was with ELISA-IgG (Kappa=0.377) and IFA (Kappa=0.347). Concerning the association between sociodemographic variables and diagnostic techniques used, we observed a statistically significant difference (p =< 0.05) between the variable living in the Santa Clara neighborhood with all techniques except qPCR-serum. CONCLUSIONS: The positivity rate of parasitological techniques was far from that presented by other techniques. The lack of adequate surveillance in areas of low endemicity of schistosomiasis may turn them into areas of medium and high endemicity. This study presents a control perspective, pointing to the possibility of using these combined laboratory tools in the diagnosis of schistosomiasis in low endemicity areas

O objetivo desse estudo foi comparar a pressão das varizes (PV), a tensão da parede (TP) e parâmetros endoscópicos, ultra-sonográficos e de Doppler em esquistossomóticos com (grupo I, n=7) e sem (grupo II, n=12) antecedente de sangramento. A PV and a TP no grupo I foi 14,7±3,0 mm Hg e 526,1±234,4, em contraste com 11,0±2,0 and 340,8±103,8 no grupo II (p=0,0053). PV acima de 12 mmHg foi observada em 71% dos casos com sangramento prévio e em 8% daqueles que nunca sangraram (0,0095). A PV correlacionou-se significantemente com a TP (p=0,0022). Nenhum parâmetro endoscópico, ultra-sonográfico ou de Doppler foi capaz de diferenciar os grupos I e II / This study was undertaken to compare variceal pressures (VP), wall tensions (WT), and endoscopic, ultrasound and Doppler parameters in schistosomiasis patients with (group I, n=7) and without (group II, n=12) previous bleeding. The VP and the WT in group I were 14.7±3.0 mm Hg and 526.1±234.4 respectively, in contrast to 11.0±2.0 and 340.8±103.8 in the group II (p=0,0053). VP over 12 mmHg was found in 71% of the patients with previous hemorrhage, compared with 8% of patients who had never bled (0,0095). VP significantly correlated with WT (p=0,0022). No endoscopic, ultrasound or Doppler parameters were shown to be different in patients with and without previous bleeding

Schistosomiasis mansoni is a serious parasitic disease, waterborne and chronic disease, which is the etiological agent of Schistosoma mansoni. This is one of the most prevalent parasitic disease in the world. In Sergipe, the disease has expanded from rural to peri-urban areas and the causal factors of this process of expansion and urbanization from this disease have not yet elucidated, thus characterizing a public health problem. This study aimed to determine the association between the risk factors identified in the distribution of infection by S. mansoni in the community of Santa Maria district, municipality of Aracaju-SE. It is a cross-sectional epidemiological study. The survey was conducted in 04 (four) times: a) survey malacological b) parasitological survey census c) a questionnaire to survey the risk factors socioeconomic, behavioral, and environmental contact with the water associated with the occurrence and transmission of disease d) georeferenced analysis of transmission focus disease and human cases of schistosomiasis. In the analysis of descriptive data were used programs BioEstat (version 5.0) and Microsoft Excel 2007. Spatial analysis of the distribution of the infection in the neighborhood led through programs and GPS TrackMaker and terraView 4.1.0 using kernel intensity estimator. Statistical analysis was performed using the chi-square test, G test and multiple logistic regression. It was found that the prevalence of infection was was 5.4% in 2011; prevailed mild infection with 72.7% according to parasite load, in relation to sex of subjects infected, infection with S. mansoni prevalent in males 63.7%. We identified 444 cases of schistosomiasis in the year under study. The eliminations largest egg schistosomiasis were most prevalent adolescents and young adults in the age group 10-39 years. Individuals most at risk for getting sick of schistosomiasis are residing near the springs with their homes accumulating water in the backyard in the winter, unpaved streets, where the individual and the family head have low education, male and age productive (10-59 years), who do not carry water treatment at home and have constant contact with water. In Malacological survey were collected 147 snails of the species Biomphalaria glabrata, being 19.17% rate of infection by S. mansoni in 22 transmitters of disease outbreaks. Spatial analysis of foci of transmission of schistosomiasis indicates the existence of three major clusters in the neighborhood and viewing areas of greatest concentration of cases exposed to different degrees of risk. The survey results allow offering, the municipal health services, a tool that facilitates the understanding of the occurrence and spatial distribution of schistosomiasis. / A esquistossomose mansônica é uma doença parasitária grave, de veiculação hídrica e evolução crônica, cujo agente etiológico é o Schistosoma mansoni. Trata-se de uma das doenças parasitárias mais prevalentes no mundo. Em Sergipe, a doença vem se expandindo da zona rural para áreas periurbanas, sendo os fatores causais desse processo de expansão e urbanização dessa endemia ainda não elucidados, caracterizando assim um problema de saúde pública. Este estudo objetivou determinar a associação entre os fatores de riscos identificados com a distribuição da infecção pelo S. mansoni, na comunidade do bairro Santa Maria, município de Aracaju-SE. Trata-se de um estudo epidemiológico e transversal. A pesquisa foi realizada em quatro momentos: a) inquérito malacológico; b) inquérito coproscópico censitário; c) aplicação de questionário para levantamento dos fatores de risco socioeconômicos, comportamentais, ambientais e de contato com as águas associados à ocorrência e transmissão da doença; d) análise georeferenciada dos focos de tranmissão da doença e dos casos humanos de Esquistossomose. Na análise dos dados descritivos foram utilizados os programas Microsoft Excel 2007 e BioEstat (versão 5.0). A análise espacial da distribuição da infecção no bairro foi realizada através dos programas GPS TrackMaker e TerraView 4.1.0 utilizando o estimador de intensidade Kernel. A análise estatística foi realizada através dos Teste Qui-Quadrado, Teste G e Regressão Logística Múltipla. Constatou-se que a prevalência da infecção foi foi de 5,4%, em 2011; prevaleceu a infecção leve com 72,7% segundo carga parasitária; em relação ao sexo dos sujeitos infectados, a infecção pelo S. mansoni prevaleceu no sexo masculino 63,7%. Foram identificados 444 casos de esquistossomose mansônica no ano em estudo. As eliminações maiores de ovos de esquistossomose acometeram mais os adolescentes e adultos jovens da faixa etária de 10 a 39 anos. Os indivíduos sob o maior risco para adoecer de esquistossomose são os que residem próximo aos mananciais, com suas residências acumulando água no quintal no inverno, em ruas não asfaltadas, onde o indivíduo e o chefe da família possuem baixa escolaridade, do sexo masculino e em idade produtiva (10 59 anos), que não realizam tratamento da água no domicílio e que tem constante contato com águas. No inquérito malacológico, foram levantados 147 caramujos da espécie Biomphalaria glabrata, sendo 19,17% a taxa de infecção pelo S. mansoni em 22 focos transmissores da doença. A análise espacial dos focos de transmissão da esquistossomose mansônica aponta a existência de três grandes aglomerados no bairro e a visualização de áreas de maior concentração de casos expostos a diferentes graus de risco. Os resultados da pesquisa possibilitam oferecer, aos serviços municipais de saúde, um instrumento que facilite a compreensão da ocorrência e distribuição espacial da Esquistossomose.

O objetivo desse estudo foi comparar a pressão das varizes (PV), a tensão da parede (TP) e parâmetros endoscópicos, ultra-sonográficos e de Doppler em esquistossomóticos com (grupo I, n=7) e sem (grupo II, n=12) antecedente de sangramento. A PV and a TP no grupo I foi 14,7±3,0 mm Hg e 526,1±234,4, em contraste com 11,0±2,0 and 340,8±103,8 no grupo II (p=0,0053). PV acima de 12 mmHg foi observada em 71% dos casos com sangramento prévio e em 8% daqueles que nunca sangraram (0,0095). A PV correlacionou-se significantemente com a TP (p=0,0022). Nenhum parâmetro endoscópico, ultra-sonográfico ou de Doppler foi capaz de diferenciar os grupos I e II / This study was undertaken to compare variceal pressures (VP), wall tensions (WT), and endoscopic, ultrasound and Doppler parameters in schistosomiasis patients with (group I, n=7) and without (group II, n=12) previous bleeding. The VP and the WT in group I were 14.7±3.0 mm Hg and 526.1±234.4 respectively, in contrast to 11.0±2.0 and 340.8±103.8 in the group II (p=0,0053). VP over 12 mmHg was found in 71% of the patients with previous hemorrhage, compared with 8% of patients who had never bled (0,0095). VP significantly correlated with WT (p=0,0022). No endoscopic, ultrasound or Doppler parameters were shown to be different in patients with and without previous bleeding

A esquistossomose é incidente em países tropicais e subtropicais e o uso de moluscicidas é adequado para prevenir a infecção de pessoas. O objetivo deste estudo foi buscar compostos em Piper ativos em Biomphalaria glabrata e avaliar a atividade esquistossomicida e toxicidade do composto mais ativo. O fracionamento biomonitorado de Piper diospyrifolium resultou no isolamento da flavocavaína A e ácido 4-hidroxi-3-[3,7,11-trimetildodeca-2,6,10-trienil]benzoico. A busca de compostos também foi realizada em amidas e chalconas e dos oito compostos avaliados, quatro foram ativos, sendo a piplartina mais ativa. A piplartina não foi letal a miracídios e cercárias de Schistosoma mansoni e foi tóxica em Daphnia similis e Danio rerio, contudo ainda foi menos tóxica que a niclosamida. Neste estudo, os compostos moluscicidas foram obtidos pelo fracionamento biomonitorado de P. diospyrifolium e pela avaliação da atividade de amidas e chalconas. Ambas as metodologias foram adequadas e a análise de componente principal mostrou ser uma ferramenta viável para a busca de compostos. / Schistosomiasis is a parasitic disease and the use of molluscicides has been considered an appropriate method to prevent human infection. The aim of this study was to search for compounds in Piper active in B. glabrata and evaluate schistosomicidal activity and toxicity of the most active compound. The P. diospyrifolium bioguided fractionation resulted in flavokavain A and 4-hydroxy-3-[3,7,trimetildodeca-11-2,6,10-trienil]benzoic acid isolation. The search for active compounds was carried out in amides and chalcones. Eight compounds were evaluated, four were active and piplartine was the most active. There was no mortality of miracidia and cercariae exposed to piplartine. It was classified as toxic to D. similis and D. rerio; nevertheless was less toxic than niclosamide. In this study, molluscicidal compounds were obtained from bioguided fractionation of P.diospyrifolium and by evaluating the activity of amides and chalcones. Both methods were suitable to obtain active compounds and principal component analysis also proved to be a viable tool for obtain compounds.

A esquistossomose é incidente em países tropicais e subtropicais e o uso de moluscicidas é adequado para prevenir a infecção de pessoas. O objetivo deste estudo foi buscar compostos em Piper ativos em Biomphalaria glabrata e avaliar a atividade esquistossomicida e toxicidade do composto mais ativo. O fracionamento biomonitorado de Piper diospyrifolium resultou no isolamento da flavocavaína A e ácido 4-hidroxi-3-[3,7,11-trimetildodeca-2,6,10-trienil]benzoico. A busca de compostos também foi realizada em amidas e chalconas e dos oito compostos avaliados, quatro foram ativos, sendo a piplartina mais ativa. A piplartina não foi letal a miracídios e cercárias de Schistosoma mansoni e foi tóxica em Daphnia similis e Danio rerio, contudo ainda foi menos tóxica que a niclosamida. Neste estudo, os compostos moluscicidas foram obtidos pelo fracionamento biomonitorado de P. diospyrifolium e pela avaliação da atividade de amidas e chalconas. Ambas as metodologias foram adequadas e a análise de componente principal mostrou ser uma ferramenta viável para a busca de compostos. / Schistosomiasis is a parasitic disease and the use of molluscicides has been considered an appropriate method to prevent human infection. The aim of this study was to search for compounds in Piper active in B. glabrata and evaluate schistosomicidal activity and toxicity of the most active compound. The P. diospyrifolium bioguided fractionation resulted in flavokavain A and 4-hydroxy-3-[3,7,trimetildodeca-11-2,6,10-trienil]benzoic acid isolation. The search for active compounds was carried out in amides and chalcones. Eight compounds were evaluated, four were active and piplartine was the most active. There was no mortality of miracidia and cercariae exposed to piplartine. It was classified as toxic to D. similis and D. rerio; nevertheless was less toxic than niclosamide. In this study, molluscicidal compounds were obtained from bioguided fractionation of P.diospyrifolium and by evaluating the activity of amides and chalcones. Both methods were suitable to obtain active compounds and principal component analysis also proved to be a viable tool for obtain compounds.

MPH / Department of Public Health

La bilharziose reste un problème de santé majeur. L'équipe du Pr Dessein a montré que les infections élevées étaient déterminées par un locus majeur en 5q31 et que des polymorphismes dans un gène à ce locus,IL13, aggravent l'infection. Notre premier objectif était d'évaluer si des variants d'autres gènes de la voie de l'IL13 intervenaient dans le contrôle de l'infection. Nous avons observé une association entre le SNP rs324013, dans le promoteur de STAT6,et les niveaux d'infection à S. haematobium. Ce polymorphisme a un effet additif avec le polymorphisme IL13rs1800925. Ce SN modifie la fixation de facteurs nucléaires au niveau du promoteur de STAT6. L'équipe du Pr Dessein avait également montré que les fibres hépatiques avancées et sévères étaient déterminées par un autre locus majeur localisé en 6q23. Notre deuxième objectif fut d'évaluer dans le laboratoire du Pr Dessein et en étroite collaboration avec le laboratoire du Pr Li(Yueyang Institute of Parasitic disease)deux gènes candidats(IFNGR1 et CTGF) situés dans cette région chromosomique. Nous avons observé une association entre les deux polyporphismes(rs17066192 er rs673156)localisés dans le promoteur du gène. Nous avons observé une association entre les deux polymorphismes(rs17066192 et rs673156)localisés dans le promoteur du gène IFNGR1 et la fibrose hépatique: le génotype rs673156A/A et rs17066192C/C sont associés à un risque 7.3 fois et 1.5 fois plus élevé, respectivement, de fibrose avancée. Nous avons également montré que les variants rs9402373 et rs12526196 du gène CTGF sont indépendamment associés à la fibrose chez les fermiers et pêcheurs chinois infectés par S.japonicum. Sur la population chinoise d'étude, les risques relatifs associés aux polymorphismes rs9402373 et rs12526196 sont de 2.8 et 3 / Schistosomiasis remains one of the world’s most prevalent diseases. It comprises a group of chronic diseases caused by helminths of the Schistosoma genus. Schistosoma haematobium causes obstructive nephropathy that can be aggravated by urinary bacterial infections. S.japonicum and S.mansoni cause hepatic fibrosis associated with portal blood hypertension, which can be lethal. In previous studies, our laboratory had shown that worm burden in S.haematobium infections were aggravated by IL13 variants and that severe hepatic fibrosis (HF) was controlled by gene(s) located on 6q23. The present study is to further evaluate other IL-13 pathway genes (STAT6) in the control of infection in Malian farmers and to test candidate genes in the 6q23 region in hepatic fibrosis (HF) in S.japonicum infected Chinese fishermen and farmers. First we have developped an improved FTA® technology technique to perform SNP genotyping. This technique allows us to use saliva samples for genotyping SNPs. Subsequently, this improved FTA® technology was used in our study on HF.Our work on a Malian sample infected with S. haematobium indicated that a polymorphism (rs324013) in the promoter of STAT6 gene was associated with the control of S. haematobium infection levels and has an additive effect with IL13rs1800925, a polymorphism previously associated with infection in this same population. Both SNPs modify the binding of nuclear factors to the promoter regions of their respective genes. Thus, both SNPs may play a crucial role in controlling S. haematobium infection levels. In order to study HF in S.japonicum infections, we have participated actively in the study that recruited of a large sample of Chinese fishermen and farmers who had been exposed to the infection for most of their life. HF was evaluated by ultrasound and covariates that could affect HF were evaluated by interviews. Then, we tested two genes (IFNGR1, CTGF) of the 6q23 region that were good candidates for the control of HF on these samples. Both genes encode molecules that were shown in animal and human studies to have strong effect on extracellular matrix proteins deposition and turnover. We found that two polymorphisms (rs17066192 and rs673156) in IFNGR1 promoter were associated with HF: the rs673156A/A genotype was associated with a 7.3-fold increased risk of advanced HF; and rs17066192C/C genotype with a 1.5-fold increased risk of HF. These results must now be confirmed in another population sample. We also found that variants of CTGF rs9402373 and rs12526196 were independently associated with HF in Chinese fishermen and farmers, in Sudanese, and in Brazilians infected with either S. japonicum or S. mansoni. Our results provide additional evidence for a protective role of IL-13 in schistosome infections, and they also demonstrate that TGFβ / CTGF pathway plays a key role in HF and should be targeted by chemotherapy. Ongoing studies evaluate whether CTGF variants could be used in the prognosis of the HF caused by schistosomes and also by other infectious agents.

Les bilharzioses hépatospléniques sont des maladies parasitaires provoquées essentiellement par Schistosoma japonicum et Schistosoma mansoni. Ces infections provoquent une fibrose hépatique sévère chez 5 à 20% des sujets infectées vivant en zone endémique. Plusieurs études ont démontré que le développement de cette fibrose était régulé par des cytokines et chemokines et que la susceptibilité génétique à cette maladie dépendait chez l'homme d'un locus majeur sur le chromosome humain 6 en position q23. L'IL-22 est une cytokine produite essentiellement par les lymphocytes T auxiliaires et les cellules NK et qui a été impliqué dans la protection du foie et de l'intestin chez la souris. Le gène IL22RA2 codant pour le récepteur inhibiteur soluble de l'IL-22 (IL-22BP) est localisé dans la région de susceptibilité à la FH (6q23). Nous avons donc étudié l'implication de l'IL-22 dans la pathologie bilharzienne par une approche immunogénétique. Notre étude démontre que la production d'IL-22 est augmentée en réponse aux œufs de S. japonicum et aux antigènes d'œufs de S. mansoni dans des cultures de cellules mononuclées du sang périphérique d'individus chinois et brésiliens vivant dans des zones endémiques. Le traitement par le Praziquantel, qui favorise l'élimination des parasites et la réversion de la fibrose, est associé à une augmentation des taux d'IL-22 en culture. Nous avons observé que dans le sang des patients chinois, l'IL-22 est produit majoritairement par les lymphocytes T CD4+ et des cellules CD3-CD4- ne produisant pas d'IL-17A. Les taux d'IL-22 en culture et la proportion des cellules CD3-CD4-IL22+ sont inversement corrélés à la FH. / Hepatosplenic schistosomiasis is a parasitic disease caused primarily by Schistosoma japonicum and Schistosoma mansoni. These infections cause severe hepatic fibrosis (HF) in 5-20% of infected subjects living in endemic areas. Several studies have shown that the development of this fibrosis was regulated by cytokines and chemokines. Our laboratory has shown that genetic susceptibility to HF map to a major locus on human chromosome 6 at position q23. IL-22 is a cytokine produced primarily by T cells and NK cells and has been involved in protecting the liver and intestine in mice. The gene IL22RA2, encoding the soluble inhibitor receptor of IL-22 (IL-22BP), is located in the region of susceptibility to HF (6q23). We therefore examined the involvement of IL-22 in schistosomiasis pathology by an immunogenetic approach. Our study shows that the production of IL-22 is increased in response to eggs of S. japonicum and egg antigens of S. mansoni in cultures of peripheral blood mononuclear cells from Chinese and Brazilian subjects living in endemic areas. Treatment with praziquantel, which helps eliminate parasites and reversion of HF, is associated with increased levels of IL-22 in culture. We observed that in the blood of Chinese patients, IL-22 is produced mainly by CD4+ T cells and CD3-CD4-cells that do not produce IL-17A. The levels of IL-22 in culture and the proportion of CD3+CD4-IL22+ are inversely correlated with HF. These observations suggest that IL-22 may play a protective role in HF. To confirm this implication, we performed association studies between SNPs located in IL22 and IL22RA2 genes and HF.

La schistosomiase représente actuellement la seconde endémie parasitaire mondiale après le paludisme. Annuellement, cette pathologie est responsable de 280 000 décès et 700 millions d’individus y sont exposés dans 74 pays à travers le monde. Actuellement, le traitement de la schistosomiase repose sur l’utilisation d’un seul médicament, le Praziquantel®. Ainsi, le développement de nouveaux médicaments est devenu une priorité absolue pour l’OMS. Dans cette étude, notre objectif a été d’identifier de nouvelles cibles thérapeutiques afin de développer de nouveaux précurseurs de médicaments. Au cours de ce projet, nous avons focalisé nos recherches sur les enzymes impliquées dans la modification des histones et plus particulièrement sur les sirtuines, qui sont des lysines désacétylases NAD+ dépendantes.Dans une première partie, nous avons caractérisé 5 orthologues de sirtuines de mammifères chez Schistosoma mansoni (SmSirt1, 2, 5, 6 et 7). De plus, nous avons étudié le potentiel des sirtuines comme cibles thérapeutiques pour le traitement de la schistosomiase en évaluant la toxicité d’inhibiteurs génériques de sirtuines humaines sur des parasites maintenus en culture. Ainsi, nous avons montré que les inhibiteurs de sirtuines humaines affectent in vitro la viabilité des schistosomules ainsi que la stabilité de l’accouplement et la production d’oeufs des vers adultes. De plus, ces inhibiteurs induisent des changements morphologiques de l’appareil génital du ver femelle.Dans une seconde partie, nous avons entrepris d’étudier plus spécifiquement le rôle de SmSirt2 en tant que cible thérapeutique. Ainsi, l’expression de la protéine recombinante en bactérie E. coli (collaboration: C. Romier, IGBMC, Illkirch) ainsi que l’optimisation d’un dosage fluorimétrique nous ont permis de montrer que SmSirt2 présente une activité lysine désacétylase in vitro (collaboration: M. Jung, Université Albert-Ludwigs, Freibourg). De plus, l’utilisation de ce dosage nous a permis de mettre en place le criblage à haut débit d’une chimiothèque de plus de 80 000 composés afin d’identifier de nouvelles molécules inhibitrices de l’enzyme SmSirt2 (collaboration: J. Schultz, Kancera AB, Stockholm). Les composés les plus prometteurs, ont été testés in vitro sur des parasites en culture. Les résultats obtenus démontrent que les inhibiteurs de SmSirt2 affectent également la viabilité des schistosomules ainsi que la stabilité de l’accouplement et la production d’oeufs des vers adultes.Dans une dernière partie, nous avons mis en place un criblage d’une banque d’ADNc de vers adultes par la technique du double hybride en levure dans le but d’identifier les partenaires protéiques de Sirt1 chez S. mansoni. L’analyse partielle des résultats nous a permis de mettre en évidence que SmSirt1 interagit avec plusieurs protéines impliquées dans la régulation des gènes chez le schistosome. Au cours de ce projet, nous avons également développé et optimisé un protocole permettant d’étudier l’activité enzymatique de SmSirt1 par injection d’ARNm dans des ovocytes de Xénope. Ainsi, nous avons pu montrer que le sirtinol et la salermide, deux inhibiteurs de Sirt1 humaine, présentent également une activité inhibitrice sur l’enzyme du parasite (collaboration: K. Cailliau, Université des Sciences et Technologies, Lille).L’ensemble des résultats obtenus au cours de ce projet de thèse suggère que les sirtuines sont des cibles thérapeutiques potentielles dans le traitement de la schistosomiase. Parmi les 5 orthologues identifiés chez S. mansoni, SmSirt2 semble une cible prometteuse. De plus, le criblage à haut débit que nous avons réalisé sur l’enzyme recombinante a permis d’identifier des molécules qui, après bio-optimisation, pourront être des candidats médicaments. Pour finir, ces résultats participent à une meilleure compréhension du rôle biologique des sirtuines chez S. mansoni et plus particulièrement sur leur implication dans la survie et la reproduction du parasite. / Schistosomiasis is the second most important parasitic disease worldwide after malaria. It is responsible for about 280 000 deaths annually and 700 million people in 74 countries are exposed to infection. Treatment of schistosomiasis currently depends on the use of the only available drug, praziquantel, and for this reason the development of new drugs is a strategic priority of the W.H.O. In this study, our objective was to identify novel therapeutic targets in order to develop new lead molecules for drug development. During this project we have focused our research on enzymes involved in histone modification, and more particularly on sirtuines, which are NAD+-dependent lysine deacetylases.In the first part of the project, we have identified 5 homologues of mammalian sirtuins in Schistosoma mansoni (SmSirt1, 2, 5, 6 and 7). Moreover, we studied the potential of sirtuins as therapeutic targets for the treatment of schistosomiasis by evaluating the toxicity for parasites maintained in culture of generic inhibitors of human sirtuins. In this way we showed that these inhibitors affect the viability of schistosomula and the stability of pairing and egg production of adult worms. Moreover, these inhibitors caused major morphological changes, particularly to the female worm genital apparatus.A second part of our work was devoted to the more detailed study of SmSirt2 as a therapeutic target. Immunisation of mice with the recombinant protein allowed us to obtain specific antibodies and to show that SmSirt2 protein is expressed at all parasite developmental stages. Furthermore, the use of the recombinant SmSirt2 expressed in E. coli (collaboration: C. Romier, IGBMC, Illkirch) and the optimization of a fluorimetric assay allowed us to show that SmSirt2 possesses a lysine deacetylase activity (collaboration: M. Jung, University Albert-Ludwigs, Freibourg). Moreover, the use of this assay allowed the setting up of a high-throughput screen (collaboration: J. Schultz, Kancera AB, Stockholm) of more than 80 000 compounds in order to identify novel inhibitors. The most promising candidates were tested on parasites in culture and the results obtained showed that SmSirt2 inhibitors also affect the viability of schistosomula, as well as the stability of pairing and egg production of adult worms.In parallel, we have carried out screening of a yeast two-hybrid cDNA library in order to identify protein partners of Sirt1 in S. mansoni. The partial analysis of the results obtained shows that SmSirt1 interacts with several proteins involved in gene regulation. In the course of this project, using the enzyme expressed in Xenopus oocytes we were able to show that both sirtinol and salermide, inhibitors of human Sirt1, also inhibit the schistosome enzyme (Collaboration: K. Cailliau, University of Sciences and Technologies, Lille).Taken together, the results of this thesis project suggest that sirtuins are potential therapeutic targets for the treatment of schistosomiasis. Of the five orthologues of human sirtuins identified in S. mansoni, SmSirt2 seems to be a promising target. Moreover, high-throughput screening using the recombinant enzyme identified inhibitors that, after bio-guided optimization, could be drug candidates. Finally, these results contribute to a better understanding of the biological role of S. mansoni sirtuins and in particular their importance in parasite survival and reproduction.