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Infection with the helminth parasite Schistosoma mansoni leads to chronic inflammation and Th2 mediated fibrosis, which result in severe pathology characterised by hepatosplenomegaly. Dendritic cells (DCs) are adept initiators of CD4+ T cell responses, but their fundamental importance in this regard in Th2 settings remains to be demonstrated. Indeed, the role of DCs at different stages of infection with S. mansoni is also yet to be determined. In addition, the importance of the interaction of DCs with tissue factors in the tissue microenvironment on the development of Th2 response to S. mansoni antigens is an area of active research and debate. This thesis is comprises of four studies. The first study tackles the involvement and importance of DCs in the induction and development of Th2 responses against S. mansoni using CD11c–diphtheria toxin receptor mice to deplete CD11c+ cells during the priming stage of the CD4+ Th2 response against S. mansoni. Diphtheria toxin treatment significantly depleted CD11c+ DCs from all tissues tested, with 70-80% efficacy. Even this incomplete depletion resulted in dramatically impaired CD4+ T cell production of Th2 cytokines, altering the balance of the immune response and causing a shift towards IFN-γ production. In contrast, basophil depletion using Mar-1 antibody had no measurable effect on Th2 induction in this system. These data underline the vital role that CD11c+ antigen presenting cells can play in orchestrating Th2 development against helminth infection in vivo, a response that is ordinarily balanced so as to prevent the potentially damaging production of inflammatory cytokines. The second study addresses whether the exposure of DCs to the cercarial stage of the parasite is critical for either parasite survival or the subsequent development of the Th2 immune response against later stages of infection. It was found that CD11c depletion prior to infection resulted in increased parasite survival, but did not impair the development of CD4+ T cell Th2 response later in infection. The third study asked whether DCs continue to be necessary for the maintenance of the chronic immune response during infection with S. mansoni. In contrast, depletion of CD11c+ cells during the initiation (4 to 6 weeks) or maintenance (6 to 8 weeks or 12 to 14 weeks) of Th2 response to eggs, resulted in severely impaired Th2 cytokine production. Interestingly, depletion during the later stages of infection led to dramatic weight loss and mortality, coincident with impaired CD4+ T cell responses. These data suggest that CD11c+ antigen presenting cells, in addition to being important in the early priming phase, also play a vital role in the maintenance and homeostasis of chronic CD4+ T cell responses in a Th2 infection setting, the disruption of which can have lethal consequences. The final study in this thesis aimed to establish whether the tissue factor thymic stromal lymphopoietin (TSLP) is able to enhance or modulate the Th2 responses initiated by DCs stimulated with SEA. Contrary to previous studies, it was found that BMDCs do not become phenotypically activated by TSLP, in particular, they do not up-regulate the costimulatory molecule OX40L, nor does TSLP suppress the production of IL-12p40 or IL-12p70 in response to LPS or CpG. Further, exposure to TSLP had no impact on DC cytokine production or survival. Irrespective of this unaltered profile in vitro, TSLP exposed DCs transferred in vivo induced the production of significantly more Th1 and Th2 cytokines from polyclonally restimulated splenocytes than DCs exposed to medium alone. In addition to this, TSLP altered the kinetic of the immune response induced by DCs stimulated with the soluble egg antigen (SEA) of S. mansoni. This was characterised by the antigen specific production of T cell cytokines starting more rapidly than with non-TSLP treated control DCs. The alteration in the kinetics of the immune response was not restricted to Th2 antigens and was also seen to some extent in Propionibacterium acnes stimulated DCs. This suggests a possible role for TSLP in either inducing faster DC migration or greater production of T cell chemoattractants and thus, enhancing the rate of DC interaction with T cells.

Schistosoma mansoni is a member of a genus of platyhelminths whose members cause the disease schistosomiasis. Particularly prevalent in sub-Saharan Africa, it is thought to be directly responsible for approximately 5500 deaths per year, as well as contributing significantly to morbidity, being responsible for 3.3 million lost disability-adjusted life years. Schistosomes are dioecious and male and female worms find one another and pair in the blood vessels of the host's liver. This sets in motion a unique feature of schistosome biology, the pairing-dependent sexual maturation of the female worms. Over the course of the next three weeks, the females fully develop their reproductive organs, especially ovaries and vitellarian tissue, to allow for the production of large quantities of eggs, which not only play a crucial role in the transmission of the parasites but are also responsible for much of the pathology associated with schistosomiasis. This thesis aims to explore the changes in gene expression which take place following pairing and result in the sexual maturation of females. To do so, RNA-Seq data was produced from male and female worms from mixed sex as well as single sex infections at 18, 21, 28, 35, 38 and 49 days post infection and analysed to understand when and how gene expression changes in paired worms. Then gene expression was examined in worms that had been removed from their partner to examine the process of regression, where female worms lose much of their reproductive tissue. The last experiments describe examine gene expression in the testes and ovaries of schistosomes, to reveal differences between the gonads of worms from mixed and single sex infections and understand in more detail how these worms may regulate the growth of their reproductive organs, contributing to our knowledge of schistosome biology.

Thesis (M. S. in Chemistry)--Vanderbilt University, Dec. 2009. / Title from title screen. Includes bibliographical references.

A G protein-coupled receptor with structural characteristics of a biogenic amine GPCR was cloned from Schistosoma mansoni (SmGPCR). SmGPCR was codon-optimized and double-tagged with FLAG and His epitopes at the N- and C-terminal ends, respectively. Immunofluorescence experiments targeting these epitopes revealed that the expression of codon-optimized SmGPCR was highly increased compared to wild-type in mammalian cells. These studies also demonstrated that SmGPCR has a typical GPCR topology, the N-terminus being extracellular and C-terminus intracellular. Functional assays revealed that codon-optimized SmGPCR was responsive only to histamine, which caused a dose-dependent increase in intracellular Ca2+ (EC50 = 0.54 +/- 0.05 muM), but not cAMP, consistent with a Gq pathway of signal transduction. In vitro behavioral studies showed that treatment of S. mansoni cercaria with exogenous histamine caused a dose-dependent increase in the motility of the parasite.

Tübingen, Univ., Diss., 2003.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-17T15:55:52Z No. of bitstreams: 1 faro_final.pdf: 19982397 bytes, checksum: 9dce53f5afd040c6e74278cf677ce010 (MD5) / Made available in DSpace on 2013-09-17T15:55:52Z (GMT). No. of bitstreams: 1 faro_final.pdf: 19982397 bytes, checksum: 9dce53f5afd040c6e74278cf677ce010 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A castração parasitária pode ser entendida como qualquer alteração na função reprodutiva do molusco que seja decorrente da interferência das larvas dos trematódeos em desenvolvimento em seu interior, podendo levar a interrupção completa da atividade ovipositória ou apenas causar a sua redução. Neste trabalho, foi feito um estudo para observar a castração parasitária de Biomphalaria glabrata infectada com Schistosoma mansoni nos períodos pré-patente e patente. Foi estudado o efeito da infecção nos parâmetros relativos à fecundidade e fertilidade, taxa de crescimento e sobrevivência em um período de observação de 62 dias. Além disso, foram realizados estudos bioquímicos e histológicos neste mesmo período. A população de B. glabrata infectada com a linhagem BH do S. mansoni apresentou 50% de positividade e 100% de sobrevivência nos dois grupos analisados. O período pré-patente foi de 39 dias após a exposição aos miracídios. Foi observada pouca diferença no crescimento dos três grupos estudados (exposto que não eliminaram cercárias, controle e positivos que eliminaram cercárias). Moluscos expostos que não eliminaram cercárias e controle não apresentaram diferenças nos parâmetros estudados, porém os moluscos positivos (eliminando cercárias) apresentaram um decréscimo de fecundidade e fertilidade resultando em cessação da oviposição a partir de 55 dias. Quanto as alterações fisiológicas, foi observada uma oscilação dos níveis de glicose na hemolinfa com uma diminuição nos períodos onde ocorre maior mobilização energética por parte das larvas, assim como uma depleção de glicogênio na massa cefalopodal e glândula digestiva. A histopatologia mostrou que, aos 55 dias, o ovoteste apresenta uma atrofia considerável com o desaparecimento quase que total das células germinativas e o estroma de sustentação formando uma rede quase vazia. Quanto à glândula digestiva infectada aos 45 dias, evidenciou-se um foco de multiplicação parasitária com espocistos e cercárias, com o tecido da glândula bem preservada. O conteúdo dos grânulos de secreção da glândula de albúmen apresentou coloração pelo Alcian Blue (AB) pH 1,0 e 2,5 nos animais infectados, indicando a presença de carboidratos sulfatados e carboxilados. Desta forma, pode-se concluir a existência de castração parasitária em Biomphalaria glabrata infectada por S.mansoni no período patente da infecção, de forma direta e indireta. Quando a infecção foi realizada na fase adulta (maturidade sexual) observou-se a diminuição da fecundidade e fertilidade. / The parasitic castration can be understood as any change in the reproductive function of the snail which is due to interference of the larvae developing inside, leading to complete disruption of the egg laying activity or just its reduction. In this work, there was made a study to observe the parasite castration of Biomphalaria glabrata infected with Schistosoma mansoni in the pre-patent and patent phases. The effect of infection on parameters related to fecundity and fertility, growth rate and survival was studied in an observation period of 62 days. In addition, biochemical and histological features were studied in the same period. The population of B. glabrata infected with the BH strain of S. mansoni showed 50% positive and 100% survival in both groups. The prepatent period was 39 days after exposure to miracidia. We observed little difference in the growth of the three groups. Exposed snails without cercarial shedding and control group showed no differences in the parameters studied, the positive snails shedding showed a decrease in fecundity and fertility resulting in cessation of egg-laying from 55 days. The physiological changes, there was a fluctuation of glucose levels in the hemolymph with a decrease in periods where there is greater mobilization of energy by the larvae, as well as a depletion of glycogen in the cephalopodal mass and digestive gland. The histopathology showed that, at 55 days the ovotestis presented a considerable atrophy with almost complete disappearance of germ cells and supporting stroma forming a nearly empty net. The infected digestive gland at 45 days showed a focus of parasite multiplication and sporocysts with cercariae, with the gland tissue well preserved. The contents of the secretory granules of the albumen gland were stained with Alcian Blue (AB) pH 1.0 and 2.5 in infected animals, indicating the presence of sulfated and carboxylated carbohydrates. Thus, we can conclude the existence of parasitic castration in Biomphalaria glabrata infected S.mansoni the patent period of infection, directly and indirectly. When the infection was performed in adult phase (sexual maturity) a decrease in fecundity and fertility was observed.

Submitted by Ana Paula Macedo (ensino@ioc.fiocruz.br) on 2013-10-03T12:30:30Z No. of bitstreams: 1 Flávia Rachel Moreira Lamarão.pdf: 5085186 bytes, checksum: 2eec081eb56cb6c2a5e03f4e14d27b2f (MD5) / Made available in DSpace on 2013-10-03T12:30:30Z (GMT). No. of bitstreams: 1 Flávia Rachel Moreira Lamarão.pdf: 5085186 bytes, checksum: 2eec081eb56cb6c2a5e03f4e14d27b2f (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A esquistossomose é uma doença complexa no qual o parasita passa por um hospedeiro intermediário até finalmente chegar ao definitivo, em uma complexa interação. Algumas consequências são consideradas como o dano tecidual, fibrose variável, câncer e morte. Tais respostas dependem de características intrínsecas do parasita, do hospedeiro, e também da interação parasita-hospedeiro. O metabolismo do hospedeiro pode ser determinante no desfecho da doença. Neste estudo o papel do ferro à luz das interações moleculares, morfológicas e parasita-hospedeiro dessa doença complexa foi observado. Foi observado o papel do ferro utilizando camundongos infectados por S. mansoni tratados com suplemento de ferro (Iron Sulfate-IS) e quelante de ferro (Deferoxamina-DFO) observando-se os seguintes parâmetros: a sobrevivência dos animais, a saturação de transferrina, a análise de aglomeração gênica, análise individual de genes do metabolismo do ferro, fibrose, apoptose, ciclo celular, estresse oxidativo, reparo do DNA, a senescência celular e sobrevivência. Alterações morfológicas, volume de colágeno do granuloma e viabilidade do miracídio também foram acessados. Os camundongos infectados tratados com as drogas mostraram aumento da sobrevida quando comparados com os animais infectados. Já os genes reguladores de ferro e seu status foram acessados por PCR em tempo real e imuno-ensaio. Para uma análise mais detalhada, um agrupamento Bayesiano da expressão do gene foi realizado demonstrando o desequilíbrio de ferro em grupos tratados e infectados formado um cluster interessante no fígado, que não foi observado no intestino. De acordo com a análise de expressão gênica, o grupo tratado com DFO apresentou desorganização na formação do granuloma. Já os genes de estresse NDRG-1 e MMT, e de ciclo celular p53, p16 e p21 mostraram expressão gênica aumentada enquanto expressão TERT foi diminuída. O volume de colágeno no granuloma e viabilidade miracídio foi diminuído neste grupo. Nossos resultados sugerem que a esquistossomose é um modelo interessante para analisar a instabilidade genética, fibrose e interação parasita-hospedeiro dentro do qual o metabolismo do ferro desempenha um papel importante. O metabolismo desequilibrado nos animais tratados com DFO interferiu na sobrevivência animal e na modulação de proteção no hospedeiro. Análises complementares são necessárias para um profundo entendimento do papel do metabolismo do ferro, o que pode modificar as perspectivas terapêuticas desta doença. / Schistosomiasis is a complex disease where the parasite passes through an intermediate host to finally reach a definitive mammalian, in a complex interaction. Some consequences take into account tissue damage, variable fibrosis, cancer and death. Such responses depend on intrinsic characteristics of the parasite, host and also host-parasite interactions. Host metabolism can be determinant on disease outcome. The role of iron in the light of molecular, morphological and host-parasite interactions of this complex disease are observed. We analyzed the role of iron using S. mansoni infected mice treated with iron supplement (Iron Sulfate-IS) and iron chelator (Deferoxamine-DFO) and observed the following parameters: animal survival, transferrin saturation, gene cluster analysis, gene-by-gene analysis of iron metabolism, fibrosis, apoptosis, cell cycle, oxidative stress, DNA repair, cell senescence and survival. Morphological changes, granuloma collagen volume and miracidium viability were also accessed. Treated infected mice showed increased survival when compared to infected animals. Iron regulatory genes and status were accessed by real-time PCR and immunoassay. For detailed analysis, a Bayesian clustering of gene expression was performed demonstrating that iron imbalance in treated and infected groups formed an interesting cluster in the liver, which was not observed in intestine. According to gene expression analysis, the DFO treated group presented disorganization of granuloma formation. Stress genes (NDRG-1 and MMT), p53, p16 and p21 showed increased gene expression while TERT expression was decreased. The granuloma collagen volume and miracidium viability was decreased in this group. Our findings suggest schistosomiasis as an interesting model to analyze fibrosis, genetic instability and host-pathogen interaction in which iron metabolism plays an important role within. DFO imbalanced metabolism interfered in: animal survival, protective modulation for host in host-pathogen interaction. Major analyses are required to understand the role of iron metabolism, which holds therapeutic prospects, in this disease.