Comparative sequence analysis reveals regulation of genes in developing schistosomula of Schistosoma mansoni exposed to host portal serum.

Once inside a vertebrate host after infection, individual schistosomula of the parasite Schistosoma mansoni find a new and complex environment, which requires quick adjustments for survival, such as those that allow it to avoid the innate immune response of the host. Thus, it is very important for the parasite to remain within the skin after entering the host for a period of about 3 days, at which time it can then reach the venous system, migrate to the lungs and, by the end of eighth day post-infection, it reach the portal venous system, while undergoing minimal changes in morphology. However, after just a few days in the portal blood system, the parasite experiences an extraordinary increase in biomass and significant morphological alterations. Therefore, determining the constituents of the portal venous system that may trigger these changes that causes the parasite to consolidate its development inside the vertebrate host, thus causing the disease schistosomiasis, is essential. The present work simulated the conditions found in the portal venous system of the vertebrate host by exposing schistosomula of S. mansoni to in vitro culture in the presence of portal serum of the hamster, Mesocricetus auratus. Two different incubation periods were evaluated, one of 3 hours and one of 12 hours. These time periods were used to mimic the early contact of the parasite with portal serum during the course of natural infection. As a control, parasites were incubated in presence of hamster peripheral serum, in order to compare gene expression signatures between the two conditions. The mRNA obtained from parasites cultured under both conditions were submitted to a whole transcriptome library preparation and sequenced with a next generation platform. On average, nearly 15 million reads were produced per sample and, for the purpose of gene expression quantification, only reads mapped to one location of the transcriptome were considered. After statistical analysis, we found 103 genes differentially expressed by schistosomula cultured for 3 hours and 12 hours in the presence of hamster portal serum. After the subtraction of a second list of genes, also differentially expressed between schistosomula cultured for 3 hours and 12 hours in presence of peripheral serum, a set of 58 genes was finally established. This pattern was further validated for a subset of 17 genes, by measuring gene expression through quantitative real time polymerase chain reaction (qPCR). Processes that were activated by the portal serum stimulus include response to stress, membrane transport, protein synthesis and folding/degradation, signaling, cytoskeleton arrangement, cell adhesion and nucleotide synthesis. Additionally, a smaller number of genes down-regulated under the same condition act on cholinergic signaling, inorganic cation and organic anion membrane transport, cell adhesion and cytoskeleton arrangement. Considering the role of these genes in triggering processes that allow the parasite to quickly adapt, escape the immune response of the host and start maturation into an adult worm after contact with the portal serum, this work may point to unexplored molecular targets for drug discovery and vaccine development against schistosomiasis.

The skin migratory stage of the schistosomulum of Schistosoma mansoni has a surface showing greater permeability and activity in membrane internalisation than other forms of skin or mechanical schistosomula.

Skin schistosomula can be prepared by collecting them after isolated mouse skin have been penetrated by cercariae in vitro. The schistosomula can also migrate out of isolated mouse skin penetrated by cercariae in vitro and from mouse skin penetrated by cercariae in vivo. Schistosomula can also be produced from cercariae applied through a syringe or in a vortex. When certain surface properties of the different forms of schistosomula were compared, those migrating from mouse skin penetrated by cercariae in vivo or in vitro had greatly increased permeability to membrane impermeant molecules such as Lucifer yellow and high molecular weight dextrans. These migrating forms also possessed surfaces which showed greatly enhanced uptake into internal membrane vesicles of the dye FM 143, a marker for endocytosis. This greatly enhanced activity and permeability of the surfaces of tissue migrating schistosomula is likely to be of great importance in the adaptation to the new host.

Schistogram changes after administration of antischistosomal drugs in mice at the early phase of Schistosoma mansoni infection.

Mice infected with Schistosoma mansoni were treated with oxamniquine, praziquantel, artesunate at the pre-patent phase, aiming at observing schistogram alterations. Half of the animals were perfused five days post-treatment for counting and classification of immature worms, based on pre-established morphological criteria (schistogram); the remaining animals were evaluated 42 or 100 days after infection and perfusion of the portal-system was performed for collection and counting of adult worms and oogram. It was observed that oxamniquine and artesunate treatment administered at the pre-postural phase causes significant reduction in the number of immature and adult worms. However, there was little reduction with praziquantel when used at the dose of 400 mg/kg for treatments administered 14, 15, 21 or 23 days post-infection. Artesunate was responsible for significant alterations in development of young worms, as well as for a higher number of worms presenting intestinal damages. Immature adult worms were detected in mice treated with artesunate or oxamniquine at the pre-patent phase of infection and recovered by perfusion 100 days after infection. Schistogram proved to be a very useful tool for experimental evaluation of the activity of antischistosomal drugs and a good model to identify the most sensitive stages to drugs.

Schistogram changes after administration of antischistosomal drugs in mice at the early phase of Schistosoma mansoni infection

Mice infected with Schistosoma mansoni were treated with oxamniquine, praziquantel, artesunate at the pre-patent phase, aiming at observing schistogram alterations. Half of the animals were perfused five days post-treatment for counting and classification of immature worms, based on pre-established morphological criteria (schistogram); the remaining animals were evaluated 42 or 100 days after infection and perfusion of the portal-system was performed for collection and counting of adult worms and oogram. It was observed that oxamniquine and artesunate treatment administered at the pre-postural phase causes significant reduction in the number of immature and adult worms. However, there was little reduction with praziquantel when used at the dose of 400 mg/kg for treatments administered 14, 15, 21 or 23 days post-infection. Artesunate was responsible for significant alterations in development of young worms, as well as for a higher number of worms presenting intestinal damages. Immature adult worms were detected in mice treated with artesunate or oxamniquine at the pre-patent phase of infection and recovered by perfusion 100 days after infection. Schistogram proved to be a very useful tool for experimental evaluation of the activity of antischistosomal drugs and a good model to identify the most sensitive stages to drugs.

Interaction between primary and secondary sporocysts of Schistosoma mansoni and the internal defence system of Biomphalaria resistant and susceptible to the parasite.

The outcome of the interaction between Biomphalaria and Schistosoma mansoni depends on the response of the host internal defence system (IDS) and the escape mechanisms of the parasite. The aim of this study was to evaluate the responsiveness of the IDS (haemocytes and soluble haemolymph factors) of resistant and susceptible Biomphalaria tenagophila lineages and Biomphalaria glabrata lineages in the presence of in vitro-transformed primary sporocysts and secondary sporocysts obtained from infected B. glabrata. To do this, we assayed the cellular adhesion index (CAI), analysed viability/mortality, used fluorescent markers to evaluate the tegumental damage and transplanted secondary sporocysts. B. tenagophila Taim was more effective against primary and secondary sporocystes than the susceptible lineage and B. glabrata. Compared with secondary sporocysts exposed to B. tenagophila, primary sporocysts showed a higher CAI, a greater percentage of dead sporocysts and were labelled by lectin from Glycine max and Alexa-Fluor 488 fluorescent probes at a higher rate than the secondary sporocysts. However, the two B. tenagophila lineages showed no cercarial shedding after inoculation with secondary sporocysts. Our hypothesis that secondary sporocysts can escape the B. tenagophila IDS cannot be confirmed by the transplantation experiments. These data suggest that there are additional mechanisms involved in the lower susceptibilty of B. tenagophila to S. mansoni infection.

Interaction between primary and secondary sporocysts of Schistosoma mansoni and the internal defence system of Biomphalaria resistant and susceptible to the parasite

The outcome of the interaction between Biomphalaria and Schistosoma mansoni depends on the response of the host internal defence system (IDS) and the escape mechanisms of the parasite. The aim of this study was to evaluate the responsiveness of the IDS (haemocytes and soluble haemolymph factors) of resistant and susceptible Biomphalaria tenagophila lineages and Biomphalaria glabrata lineages in the presence of in vitro-transformed primary sporocysts and secondary sporocysts obtained from infected B. glabrata. To do this, we assayed the cellular adhesion index (CAI), analysed viability/mortality, used fluorescent markers to evaluate the tegumental damage and transplanted secondary sporocysts. B. tenagophila Taim was more effective against primary and secondary sporocystes than the susceptible lineage and B. glabrata. Compared with secondary sporocysts exposed to B. tenagophila, primary sporocysts showed a higher CAI, a greater percentage of dead sporocysts and were labelled by lectin from Glycine max and Alexa-Fluor 488 fluorescent probes at a higher rate than the secondary sporocysts. However, the two B. tenagophila lineages showed no cercarial shedding after inoculation with secondary sporocysts. Our hypothesis that secondary sporocysts can escape the B. tenagophila IDS cannot be confirmed by the transplantation experiments. These data suggest that there are additional mechanisms involved in the lower susceptibilty of B. tenagophila to S. mansoni infection.

Antischistosomal activity of aminoalkanethiols, aminoalkanethiosulfuric acids and the corresponding disulfides.

This paper discusses the development of a series of sulfur-containing compounds that show an interesting in vivo activity against infection by Schistosoma mansoni. These substances include the aminoalkanethiols, aminoalkanethiosulfuric acids and aminoalkyl disulfides, among others. Although the aminoethanethiols and their disulfide derivatives have presented a relatively high toxicity for the host animal, the aminoalkanethiosulfuric acids have a low toxicity and a high specificity for the adult female S. mansoni worms. In vitro studies with schistosomula, lung-phase schistosomula and adult worms have demonstrated effects on the tegument and the metabolism on these different stages of S. mansoni worms. The encapsulation of these drugs in a nanoemulsion has resulted in an increase in the in vitro activity.

Synergistic action of praziquantel and host specific immune response against Schistosoma mansoni at different phases of infection.

The interaction between specific immune response to Schistosoma mansoni and praziquantel (PZQ) was studied in mice. In mice harboring concomitant immunity, 6-day-old parasites treated with PZQ were more effectively removed than 24 h treated parasites despite both had a significant worm burden reduction when compared with respective treated controls. These results show that PZQ can be effective at the skin and lung stages of parasite's development mainly acting with a established specific immune response, and particularly at the lung phase.

Synergistic action of praziquantel and host specific immune response against Schistosoma mansoni at different phases of infection

A interação entre a resposta imune específica ao Schistosoma mansoni e praziquantel (PZQ) foi avaliada em modelo murino. Em camundongos portadores de imunidade concomitante, parasitos com 6 dias de idade e tratados com PZQ, foram eliminados mais eficazmente do que parasitos de apenas 24 h, apesar de ambos mostrarem uma redução significativa da carga parasitária quando comparados com os respectivos controles tratados. Estes resultados mostram que o PZQ pode ser eficaz nos estágios de pele e pulmão durante o desenvolvimento do parasita, agindo principalmente com uma resposta imune específica estabelecida e, particularmente, na fase pulmonar.