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.

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.

Estudo do sistema inato de defesa de Biomphalaria tenagophila (d’ Orbigny, 1835) frente ao Schistosoma mansoni Sambon, 1907 / Study of the innate defense system of Biomphalaria tenagophila (d'Orbigny, 1835) against the Schistosoma mansoni Sambon, 1907

A fenoloxidase (PO) é uma enzima envolvida no processo de melanização. A melanização é um mecanismo de defesa contra patógenos, muito importante para diversos invertebrados, inclusive, alguns artigos já descreveram a existência dessa enzima em Biomphalaria glabrata. Utilizando B. tenagophila (linhagem resistente Taim e linhagem susceptível Cabo Frio), tentou-se esclarecer, por meio de experimentos “in vivo” e “in vitro”, a real importância desse sistema enzimático no processo de resistência da Biomphalaria ao S. mansoni. Verificou-se que, apesar do sistema fenoloxidase apresentar, em alguns experimentos, uma atividade significativamente maior, principalmente na linhagem Taim, na presença do S. mansoni, não foi possível afirmar que esse sistema tem importância no mecanismo de resistência da B. tenagophila Taim contra S. mansoni, pois a ativação enzimática ocorreu em todas as linhagens testadas. Quando os testes da atividade enzimática foram realizados, utilizando Echistosoma paraensei (trematódeo, digenea), que infecta a linhagem Taim, os resultados foram semelhantes. Outro aspecto estudado neste trabalho foi a resposta do sistema interno de defesa (SID - hemócitos, fração solúvel e hemolinfa total) da B. glabrata e B. tenagophila (Taim e Cabo Frio), frente aos esporocistos secundários, obtidos de caramujos B. glabrata infectadas. Já foi descrito na literatura o processo de mascaramento/mimetismo molecular como mecanismo de evasão do parasito.

Assim, utilizando os esporocistos secundários, foi possível demonstrar que os esporocistos primários são mais afetados pelo SID da Biomphalaria (principalmente Taim) do que os esporocistos secundários. Os esporocistos primários apresentaram maior mortalidade e danos no tegumento do que os esporocistos secundários. No entanto, a inoculação dos esporocistos secundários, provenientes de B. glabrata, obteve sucesso apenas na espécie análoga, resultando na eliminação de cercárias, trinta dias após a inoculação. O mesmo não ocorreu nas linhagen de B. tenagophila Taim e Cabo Frio. Dessa forma, o mimetismo/mascaramento molecular é um mecanismo possível, porém experimentos utilizando esporocistos secundários provenientes da B. tenagophila são necessários para verificar a real importância desse processo como mecanismo de escape do parasito em relação a B. tenagophila Taim

Estudo do sistema inato de defesa de Biomphalaria tenagophila (d’ Orbigny, 1835) frente ao Schistosoma mansoni Sambon, 1907

A fenoloxidase (PO) é uma enzima envolvida no processo de melanização. A melanização é um mecanismo de defesa contra patógenos, muito importante para diversos invertebrados, inclusive, alguns artigos já descreveram a existência dessa enzima em Biomphalaria glabrata. Utilizando B. tenagophila (linhagem resistente Taim e linhagem susceptível Cabo Frio), tentou-se esclarecer, por meio de experimentos “in vivo” e “in vitro”, a real importância desse sistema enzimático no processo de resistência da Biomphalaria ao S. mansoni. Verificou-se que, apesar do sistema fenoloxidase apresentar, em alguns experimentos, uma atividade significativamente maior, principalmente na linhagem Taim, na presença do S. mansoni, não foi possível afirmar que esse sistema tem importância no mecanismo de resistência da B. tenagophila Taim contra S. mansoni, pois a ativação enzimática ocorreu em todas as linhagens testadas. Quando os testes da atividade enzimática foram realizados, utilizando Echistosoma paraensei (trematódeo, digenea), que infecta a linhagem Taim, os resultados foram semelhantes. Outro aspecto estudado neste trabalho foi a resposta do sistema interno de defesa (SID - hemócitos, fração solúvel e hemolinfa total) da B. glabrata e B. tenagophila (Taim e Cabo Frio), frente aos esporocistos secundários, obtidos de caramujos B. glabrata infectadas. Já foi descrito na literatura o processo de mascaramento/mimetismo molecular como mecanismo de evasão do parasito.

Assim, utilizando os esporocistos secundários, foi possível demonstrar que os esporocistos primários são mais afetados pelo SID da Biomphalaria (principalmente Taim) do que os esporocistos secundários. Os esporocistos primários apresentaram maior mortalidade e danos no tegumento do que os esporocistos secundários. No entanto, a inoculação dos esporocistos secundários, provenientes de B. glabrata, obteve sucesso apenas na espécie análoga, resultando na eliminação de cercárias, trinta dias após a inoculação. O mesmo não ocorreu nas linhagen de B. tenagophila Taim e Cabo Frio. Dessa forma, o mimetismo/mascaramento molecular é um mecanismo possível, porém experimentos utilizando esporocistos secundários provenientes da B. tenagophila são necessários para verificar a real importância desse processo como mecanismo de escape do parasito em relação a B. tenagophila Taim

Association of oxamniquine praziquantel and clonazepam in experimental Schistosomiasis mansoni

The antischistosomal activity of clonazepam, when administered alone or in association with oxamniquine and praziquantel, was experimentally evaluated in mice infected with Schistosoma mansoni. The animals were treated 45 days post-infection with a single dose, by oral route, according to three treatment schedules: clonazepam 25 mg/kg and sacrificed 15 min, 1h or 4 h after treatment; clonazepam 1.0, 2.5 or 10.0 mg/kg and sacrificed 15 days post-treatment or with the dose of 10 mg/kg in association with oxamniquine 50 mg/kg or praziquantel 200 mg/kg, single dose, orally, every schedule with a control group. The efficacy of the drugs in vivo was assessed by means of worm counts and their distribution in mesentery and liver, mortality and oogram changes. In the chemotherapeutic schedules used, clonazepam did not present antischistosomal activity and the result of the association of this drug with oxamniquine or praziquantel was not significantly different from the one obtained when these two last drugs were administered alone. In the in vitro experiments, the worms exposed to 0.6 mg/mL clonazepam remained motionless throughout the 8-day-period of observation, without egg-laying, whereas the worms of the control group showed normal movements, egg-laying and hatching of miracidia on the last day of observation. The results obtained in the present study confirm the action of clonazepam on S. mansoni adult worm, in vitro, causing total paralysis of males and females. However, no additive or synergistic effects were observed when clonazepam were used in association with oxamniquine or praziquantel.

Oxamniquine, praziquantel and lovastatin association in the experimental Schistosomiasis mansoni.

The activity of lovastatin associated with oxamniquine or praziquantel against schistosomiasis mansoni was evaluated in mice infected with Schistosoma mansoni. Forty days after infection, mice were treated with lovastatin, 400 mg/kg for five consecutive days by oral route, and on the last day of this sequence with 50 mg/kg oxamniquine or with 200 mg/kg praziquantel, both by oral route, single dose. Fifteen days later, the animals were perfused in parallel with an untreated control group. Studies were carried out in vitro, using lovastatin in culture medium containing S. mansoni worms proceeding from experimentally infected mice. In the in vivo trials, the association of lovastatin with oxamniquine or praziquantel did not show any additive action, but there were oogram changes when lovastatin was associated with oxamniquine. In vitro lovastatin was able to interrupt the maturation of S. mansoni eggs, which remained at the 1st or 2nd stages, depending on the dose used. The total number of morphologically dead eggs found in culture of worms exposed to 2 microg/ml or 4 microg/ml concentrations of lovastatin was significantly higher than the number of viable eggs. Using the probe Hoescht 33258 it was observed that 70% of the eggs considered morphologically viable in the treated groups (against 16% in the control group) were labeled, indicating that the majority of the viable eggs had membrane permeability increased due to lovastatin action.

Oxamniquine, praziquantel and lovastatin association in the experimental Schistosomiasis mansoni

The activity of lovastatin associated with oxamniquine or praziquantel against schistosomiasis mansoni was evaluated in mice infected with Schistosoma mansoni. Forty days after infection, mice were treated with lovastatin, 400 mg/kg for five consecutive days by oral route, and on the last day of this sequence with 50 mg/kg oxamniquine or with 200 mg/kg praziquantel, both by oral route, single dose. Fifteen days later, the animals were perfused in parallel with an untreated control group. Studies were carried out in vitro, using lovastatin in culture medium containing S. mansoni worms proceeding from experimentally infected mice. In the in vivo trials, the association of lovastatin with oxamniquine or praziquantel did not show any additive action, but there were oogram changes when lovastatin was associated with oxamniquine. In vitro lovastatin was able to interrupt the maturation of S. mansoni eggs, which remained at the 1st or 2nd stages, depending on the dose used. The total number of morphologically dead eggs found in culture of worms exposed to 2 µg/ml or 4 µg/ml concentrations of lovastatin was significantly higher than the number of viable eggs. Using the probe Hoescht 33258 it was observed that 70 percent of the eggs considered morphologically viable in the treated groups (against 16 percent in the control group) were labeled, indicating that the majority of the viable eggs had membrane permeability increased due to lovastatin action.

Association of oxamniquine praziquantel and clonazepam in experimental Schistosomiasis mansoni.

The antischistosomal activity of clonazepam, when administered alone or in association with oxamniquine and praziquantel, was experimentally evaluated in mice infected with Schistosoma mansoni. The animals were treated 45 days post-infection with a single dose, by oral route, according to three treatment schedules: clonazepam 25 mg/kg and sacrificed 15 min, 1h or 4 h after treatment; clonazepam 1.0, 2.5 or 10.0 mg/kg and sacrificed 15 days post-treatment or with the dose of 10 mg/kg in association with oxamniquine 50 mg/kg or praziquantel 200 mg/kg, single dose, orally, every schedule with a control group. The efficacy of the drugs in vivo was assessed by means of worm counts and their distribution in mesentery and liver, mortality and oogram changes. In the chemotherapeutic schedules used, clonazepam did not present antischistosomal activity and the result of the association of this drug with oxamniquine or praziquantel was not significantly different from the one obtained when these two last drugs were administered alone. In the in vitro experiments, the worms exposed to 0.6 mg/mL clonazepam remained motionless throughout the 8-day-period of observation, without egg-laying, whereas the worms of the control group showed normal movements, egg-laying and hatching of miracidia on the last day of observation. The results obtained in the present study confirm the action of clonazepam on S. mansoni adult worm, in vitro, causing total paralysis of males and females. However, no additive or synergistic effects were observed when clonazepam were used in association with oxamniquine or praziquantel.

Avaliação do efeito de drogas esquistossomicidas sobre a fase intramolusco do Schistosoma mansoni / Evaluation of the effect of schistosomicidal drugs on the phase intramolluscan of the Schistosoma mansoni

O presente trabalho avaliou a ação da oxaminiquina (OXA), do praziquantel (PZQ) e da associação OXA/PZQ sobre a fase intramolusco do Schistosoma mansoni. Nos estudos "in vivo", caramujos Biomphalaria glabrata infectados com S. mansoni foram tratados com 500mg/Kg de OXA ou 1000mg/Kg de PZQ ou 250mg/Kg de OXA+500mg/Kg de PZQ, na fase pré-patente e patente da infecção. Nos experimentos "in vitro", esporocistos primários transformados "in vitro" foram expostos a 5 micrograma/mL e 10 micrograma/mL de PZQ. [...] Já o tratamento com a associação OXA/PZQ, neste mesmo período, retardou ainda mais o desenvolvimento do parasito, aumentando em até 56 dias o período pré-patente. O tratamento, no período patente, com OXA ou PZQ, isoladamente, interrompeu a eliminação de cercárias. A eliminação de cercárias foi restabelecida aproximadamente 15 dias após o tratamento, porém em quantidades bem menores às anteriores ao tratamento, quando os caramujos foram tratados com 500mg/Kg de OXA. A associação OXA/PZQ, no período patente, não só interrompeu a eliminação de cercárias, como levou à "cura" dos caramujos. Esses resultados sugerem um efeito sinérgico dessas drogas quando administradas associadas. Em relação aos experimentos "in vitro", foi possível verificar que o PZQ causa uma nítida contração na musculatura do esporocisto, alterando o formato do parasito. O PZQ gera, ainda, dano no tegumento do S. mansoni, evidenciado pela marcação com as sondas Hoechst 33258 e Lectina de Glicine max. Além disso, verificou-se um aumento da área marcada pela sonda LysoTracker Red, após o contato dos esporocistos com a droga, sugerindo maior atividade das vesículas ácidas presentes. A partir dos resultados obtidos, nos quais caramujos infectados e tratados com 1000mg/Kg de PZQ param de eliminar cercárias e retornam a eliminá-las, em números bem menores aos encontrados antes do tratamento, 15 dias após o término do tratamento, avaliamos se cercárias provenientes destes caramujos tratados poder.

Avaliação do efeito de drogas esquistossomicidas sobre a fase intramolusco do Schistosoma mansoni / Evaluation of the effect of drugs on the esquistossomicidas phase intramolusco of Schistosoma mansoni

O presente trabalho avaliou a ação da oxamniquina (OXA), do praziquantel (PZQ) e da associação OXA/ PZQ sobre a fase intramolusco do Schistosoma mansoni. Nos estudos in vivo, caramujos Biomphalaria glabrata infectados com S. mansoni foram tratados com 500mg/Kg de OXA ou 1000mg/Kg de PZQ ou 250mg/Kg de OXA+ 500mg/Kg de PZQ, na fase pré-patente e patente da infecção. Nos experimentos in vitro, esporocistos primários transformados in vitro foram expostos a 5μg/mL e 10μg/mL de PZQ. Avaliou-se, através de marcadores fluorescentes específicos, a ação do PZQ sobre o tegumento e sobre as vesículas ácidas, descritas pela primeira vez em esporocistos primários em nosso trabalho. Nossos resultados mostraram que tanto o tratamento com OXA quanto com PZQ, isoladamente, no período pré-patente, retardam o desenvolvimento do parasito, aumentando em aproximadamente dez dias o período pré-patente. Já o tratamento com a associação OXA!PZQ, neste mesmo período, retardou ainda mais o desenvolvimento do parasito, aumentando em até 56 dias o período pré-patente. O tratamento, no período patente, com OXA ou PZQ, isoladamente, interrompeu a eliminação de cercárias. A eliminação de cercárias foi restabelecida aproximadamente 15 dias após o tratamento, porém em quantidades bem menores às anteriores ao tratamento, quando os caramujos foram tratados com 1000mg/Kg de PZQ. O mesmo resultado não foi observado quando os caramujos foram tratados com 500mg/Kg de OXA. A associação OXA/PZQ, no período patente, não só interrompeu a eliminação de cercárias, como levou à cura dos caramujos. Esses resultados sugerem um efeito sinérgico dessas drogas quando administradas associadas. Em relação aos experimentos in vitro, foi possível verificar que o PZQ causa uma nítida contração na musculatura do esporocisto, alterando o formato do parasito. O PZQ gera, ainda, dano no tegumento do S mansoni, evidenciado pela marcação com as sondas Hoechst 33258 e Lectina de Glicine max...

The aim of the present study was to evaluate the activity of oxamniquine (OXA), praziquantel (PZQ), and OXA/PZQ association on the intramolluscan phase of Schistosoma mansoni. In vivo studies were carried out using Biomphalaria glabrata snails, infected with S. mansoni, and treated with 500 mg/Kg OXA or 1000 mg/Kg PZQ or 250 mg/Kg OXA +500 mg/Kg PZQ, at the pre-patent and patent phases of infection. In vitro studies were also performed with in vitro transformed primary sporocysts, exposed to 5_g/mL and 10_g/mL PZQ. By means of specific fluorescent labels, it was possible to evaluate the activity ofpraziquantel on the tegument and acidic vesicles in primary sporocysts, described for the first time in our study. Our results showed that treatments administered either with OXA or with PZQ, separately, retarded the parasite´s development at the pre-patent phase, and raised upthis period for 10 days as well. On the other hand, treatment performed with the association OXA/PZQ delayed even more the parasite´s development, increasing up to 56 days the prepatent period. Treatment carried out with OXA or PZQ, separately, interrupted cercarial shedding. Approximately 15 days after treatment, cercarial shedding was restablished, but insmaller quantities than those observed before treatment, when the snails were treated with 1000 mg/Kg PZQ. The same result could not be obtained when the snails were treated with 500 mg/Kg PZQ. OXA/PZQ association administered at the pre-patent period, not only interrupted cercarial shedding, but provided also a “cure” for the snails. These resultssuggested a synergistic effect of these drugs, when they are associately administered. In relation to the in vitro experiments, it was possible to observe that PZQ caused an evident contraction of the sporocyst musculature, changing the parasite´s structure. PZQ caused damage to the tegument of S. mansoni, as it could be seen by labelling using the probes Hoechst 33258 and...

Avaliação do efeito de drogas esquistossomicidas sobre a fase intramolusco do Schistosoma mansoni

O presente trabalho avaliou a ação da oxaminiquina (OXA), do praziquantel (PZQ) e da associação OXA/PZQ sobre a fase intramolusco do Schistosoma mansoni. Nos estudos "in vivo", caramujos Biomphalaria glabrata infectados com S. mansoni foram tratados com 500mg/Kg de OXA ou 1000mg/Kg de PZQ ou 250mg/Kg de OXA+500mg/Kg de PZQ, na fase pré-patente e patente da infecção. Nos experimentos "in vitro", esporocistos primários transformados "in vitro" foram expostos a 5 micrograma/mL e 10 micrograma/mL de PZQ. [...] Já o tratamento com a associação OXA/PZQ, neste mesmo período, retardou ainda mais o desenvolvimento do parasito, aumentando em até 56 dias o período pré-patente. O tratamento, no período patente, com OXA ou PZQ, isoladamente, interrompeu a eliminação de cercárias. A eliminação de cercárias foi restabelecida aproximadamente 15 dias após o tratamento, porém em quantidades bem menores às anteriores ao tratamento, quando os caramujos foram tratados com 500mg/Kg de OXA. A associação OXA/PZQ, no período patente, não só interrompeu a eliminação de cercárias, como levou à "cura" dos caramujos. Esses resultados sugerem um efeito sinérgico dessas drogas quando administradas associadas. Em relação aos experimentos "in vitro", foi possível verificar que o PZQ causa uma nítida contração na musculatura do esporocisto, alterando o formato do parasito. O PZQ gera, ainda, dano no tegumento do S. mansoni, evidenciado pela marcação com as sondas Hoechst 33258 e Lectina de Glicine max. Além disso, verificou-se um aumento da área marcada pela sonda LysoTracker Red, após o contato dos esporocistos com a droga, sugerindo maior atividade das vesículas ácidas presentes. A partir dos resultados obtidos, nos quais caramujos infectados e tratados com 1000mg/Kg de PZQ param de eliminar cercárias e retornam a eliminá-las, em números bem menores aos encontrados antes do tratamento, 15 dias após o término do tratamento, avaliamos se cercárias provenientes destes caramujos tratados poder