Neusa Araújo

Estudo de uma cepa humana de Schistosoma mansoni resistente a agentes esquistossomicidas

There has been isolated a Schistosoma mansoni strain from two patients submitted to two courses of treatment with hycanthone (2,5mg/kg, i.m.), in January and April, 1970, and to one course with niridazole (25mg/kg/day x 5, per os), in April, 1971. Before treatment, the number of eggs in the faeces of those patients was, per gram, 2,675 and 1,025, respectively; after completion of treatment, such number had come down to around 100 eggs/gram. Miracidia hatched from the patients faeces could infect Biomphalaria glabrata snails, which elimmated cercariae (WW strain) that were used for experimental infection of albino mice. The infected animais were, afterwards, treated with hycanthone, niridazole and oxamniquine under various schedules. Comparative studies of WW and LE strains (the latter being routinely kept in our laboratories) revealed marked differences in their sensitivity to the schistosomicides employed. Actually, after treatment with hycanthone, at the dosage of 80 mg/kg, i.m., a 100% oogram changes were observed in the intestinal wall of mice inoculated with LE strain, whereas no alterations could be detected in the mice infected with WW strain. As regards oxamniquine and niridazole the changes were smaller although still quite sufficient to indicate greater resistance of WW strain to these schistosomicides. It is worth while remembering that, in the pertaining literature, it is the first time that resistance in S. mansoni strains from treated patients has been demonstrated.

Schistosoma mansoni: a method for inducing resistance to praziquantel using infected Biomphalaria glabrata snails

To elucidate the mechanisms of antischistosoma resistance, drug-resistant Schistosoma mansoni laboratory isolates are essential. We developed a new method for inducing resistance to praziquantel (PZQ) using successive drug treatments of Biomphalaria glabrata snails infected with S. mansoni. Infected B. glabrata were treated three times with 100 mg/kg PZQ for five consecutive days with a one-week interval between them. After the treatment, the cercariae (LE-PZQ) produced from these snails and the LE strains (susceptible) were used to infect mice. Forty-five days after infection, mice were treated with 200, 400 or 800 mg/kg PZQ. Thirty days post-treatment, we observed that the mean number of worms recovered by perfusion was significantly higher in the group of mice infected with the LE-PZQ isolate treated with 200 and 400 mg/kg in comparison to the LE strain with the same treatment. Moreover, there was a significant difference between the ED50 (effective dose required to kill 50% of the worms) of the LE-PZQ isolate (362 mg/kg) and the LE strain (68 mg/kg). In the in vitro assays, the worms of the LE-PZQ isolate were also less susceptible to PZQ. Thus, the use of infected snails as an experimental model for development of resistance to S. mansoni is effective, fast, simple and cheap.

Regulation of Schistosoma mansoni Development and Reproduction by the Mitogen-Activated Protein Kinase Signaling Pathway

Background Protein kinases are proven targets for drug development with an increasing number of eukaryotic Protein Kinase (ePK) inhibitors now approved as drugs. Mitogen-activated protein kinase (MAPK) family members connect cell-surface receptors to regulatory targets within cells and influence a number of tissue-specific biological activities such as cell proliferation, differentiation and survival. However, the contributions of members of the MAPK pathway to schistosome development and survival are unclear. Methodology/Principal Findings We employed RNA interference (RNAi) to elucidate the functional roles of five S. mansoni genes (SmCaMK2, SmJNK, SmERK1, SmERK2 and SmRas) involved in MAPK signaling pathway. Mice were injected with post-infective larvae (schistosomula) subsequent to RNAi and the development of adult worms observed. The data demonstrate that SmJNK participates in parasite maturation and survival of the parasites, whereas SmERK are involved in egg production as infected mice had significantly lower egg burdens with female worms presenting underdeveloped ovaries. Furthermore, it was shown that the c-fos transcription factor was overexpressed in parasites submitted to RNAi of SmERK1, SmJNK and SmCaMK2 indicating its putative involvement in gene regulation in this parasites MAPK signaling cascade. Conclusions We conclude that MAPKs proteins play important roles in the parasite in vivo survival, being essential for normal development and successful survival and reproduction of the schistosome parasite. Moreover SmERK and SmJNK are potential targets for drug development.

Praziquantel Treatment Decreases Schistosoma mansoni Genetic Diversity in Experimental Infections

Background Schistosomiasis has a considerable impact on public health in many tropical and subtropical areas. In the new world, schistosomiasis is caused by the digenetic trematode Schistosoma mansoni. Chemotherapy is the main measure for controlling schistosomiasis, and the current drug of choice for treatment is praziquantel (PZQ). Although PZQ is efficient and safe, its repetitive large-scale use in endemic areas may lead to the selection of resistant strains. Isolates less susceptible to PZQ have been found in the field and selected for in the laboratory. The impact of selecting strains with a decreased susceptibility phenotype on disease dynamics and parasite population genetics is not fully understood. This study addresses the impact of PZQ pressure on the genetics of a laboratory population by analyzing frequency variations of polymorphic genetic markers. Methodology Infected mice were treated with increasing PZQ doses until the highest dose of 3×300 mg/Kg was reached. The effect of PZQ treatment on the parasite population was assessed using five polymorphic microsatellite markers. Parasitological and genetic data were compared with those of the untreated control. After six parasite generations submitted to treatment, it was possible to obtain a S. mansoni population with decreased susceptibility to PZQ. In our experiments we also observed that female worms were more susceptible to PZQ than male worms. Conclusions The selective pressure exerted by PZQ led to decreased genetic variability in S. mansoni and increased endogamy. The understanding of how S. mansoni populations respond to successive drug pressure has important implications on the appearance and maintenance of a PZQ resistance phenotype in endemic regions.

Activity of the artemether in experimental schistosomiasis mansoni

The action of the ether of artemisinin (artemether) on Schistosoma mansoni in mice and hamsters experimentally infected with the LE strain was studied. In mice, the drug showed high schistosomicidal activity using a single intramuscular dose of 100 mg/kg/day. By the oral route, this dose showed a low activity. Mice treated with a single intramuscular dose of 200 mg/kg/day, and examined 15 days after treatment, presented 100% alteration of the oogram; when examined 45 days after treatment, the oogram was normal. With doses of 100 mg/kg/day, i.m., during 3 or 5 consecutive days, the death rate of mice was very high. Morphologic analysis of the worms collected by perfusion of mice treated with a single dose of 100 mg/kg/day, i.m., detected a marked decrease in the length of male and female worms, degenerative alterations in the parenchyma and in the reproductive system of the females, with the reduction of vitellinic material and in ovary volume; the intestinal contents presented a marked despigmentation. In the male worms significant alteration was not apparent by optical microscopy.

Use of fluorescent probes as a useful tool to identify resistant Schistosoma mansoni isolates to praziquantel

The use of chemotherapy on a mass scale in endemic areas may lead to the appearance of resistant isolates through the mechanism of selective drug pressure. Studies have demonstrated that praziquantel (PZQ) is able to inhibit the excretory activity and to cause tegumental damage in Schistosoma mansoni adult worms. The use of the probe resorufin to evaluate excretory activity, as well as the probe Hoechst 33258 to detect tegumental damage in adult worms, may represent a method to identify resistant (or less susceptible) isolates. The purpose of the present work was to compare the changes caused by PZQ in the function of the excretory system and in the integrity of the tegument of adult worms from the LE isolate (susceptible to PZQ) and the LE-PZQ isolate (less susceptible to PZQ). Worms from the isolate LE-PZQ showed less severe tegumental lesions, in both in vitro and in vivo experiments, detected by labelling with Hoechst 33258 and continued to have a functional excretory system as shown by labelling with resorufin in vitro.

Avaliação terapêutica do artesunato na infecção experimental pelo Schistosoma mansoni

Abstract Mice experimentally infected with Schistosoma mansoni were treated orally withartesunate (Lactab ® ) in a single dose of 300 or 500mg/kg or over a period of five consecutivedays. The animals were sacrificed 7, 30, 60 or 90 days after treatment. Statistically significantdifferences were found in the distribution and mortality of the worms and in the alterations of theoogram in the treated group when compared to control in all of the tested schemes when theanimals were sacrificed 30 days after treatment. Morphological analysis of female wormsshowed a reduction of ovarian volume and rarefaction of the vitelline follicles. These modificationswere more marked after treatment with the higher dose, explaining the alteration of the oogramwhich reached 100%. However, when the animals were sacrificed 60 or 90 days after treatment,the differences and alterations were smaller, showing that the surviving worms recovered andrestarted oviposition. Key-words: Schistosoma mansoni . Schistosomicide. Artesunate.

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.

Comparison between morphological and staining characteristics of live and dead eggs of Schistosoma mansoni

Schistosoma mansoni eggs are classified, according to morphological characteristics, as follows: viable mature and immature eggs; dead mature and immature eggs, shells and granulomas. The scope of this study was to compare the staining characteristics of different morphological types of eggs in the presence of fluorescent labels and vital dyes, aiming at differentiating live and dead eggs. The eggs were obtained from the intestines of infected mice, and put into saline 0.85%. The fluorescent labels were Hoechst 33258 and Acridine Orange + Ethidium Bromide and vital dyes (Trypan Blue 0.4% and Neutral Red 1%). When labelled with the probe Hoechst 33258, some immature eggs, morphologically considered viable, presented fluorescence (a staining characteristic detected only in dead eggs); mature eggs did not present fluorescence, and the other types of dead eggs, morphologically defined, showed fluorescence. As far as Acridine Orange + Ethidium Bromide are concerned, either the eggs considered to be live, or the dead ones, presented staining with green color, and only the hatched and motionless miracidium was stained with an orange color. Trypan Blue was not able to stain the eggs, considered to be dead but only dead miracidia which had emerged out of the shell. Neutral Red stained both live and dead eggs. Only the fluorescent Hoechst 33258 can be considered a useful tool for differentiation between dead and live eggs.

Atividade moluscicida do extrato butílico de Phytolaca dodecandra (Endod) sobre Biomphalaria glabrata

A buthanol extract of Phytolacca dodecandra (type 44) obtained from Ethiopia berries, was tested as molluscicide in our laboratory and in the field. The lethal dose (LD90) for adult snails, newly hatched and egg-masses of Biomphalaria glabrata, in 24 hours exposure, were of 4.5, 23.0 and 102.0 ppm respectively. The LD90 for the fish Lebistes reticulatus was of 2.0 ppm. These results are similar to those of Lemma (1984) in Ethiopia. In two water ponds treated with 10 ppm of the buthanol extract or 3 ppm of niclosamide the mortality rates of B. glabrata were of 84.6 and 100.0%, respectively. Both treatments were toxic for L. reticulatus in the field trials. The possibility of using molluscicides derived from plants is discussed as an alternative for treatment of schistosomiasis foci in Brazil.