RESUMO
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.
Assuntos
Anti-Helmínticos/farmacologia , Resistência a Medicamentos , Corantes Fluorescentes , Praziquantel/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Animais , Bisbenzimidazol/metabolismo , Sistema Digestório/metabolismo , Sistema Digestório/patologia , Corantes Fluorescentes/metabolismo , Oxazinas/metabolismo , Testes de Sensibilidade Parasitária/métodos , Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/parasitologia , Pele/metabolismo , Pele/patologiaRESUMO
The activity of oxamniquine (OXA), praziquantel (PZQ), and a combination of both drugs was evaluated at the intramolluscan phase of Schistosoma mansoni. Biomphalaria glabrata snails infected with S. mansoni were treated with 500 mg/kg OXA, 1000 mg/kg PZQ or with 250 mg/kg OXA and 500 mg/kg PZQ, in association, at the pre-patent and patent phases of infection. The results showed that either treatments with OXA or PZQ, alone, at the pre-patent period, delayed the parasite's development, increasing the pre-patent period by approximately 10 days. At the same pre-patent period, treatment with a combination of OXA/PZQ delayed the parasite's development even more, extending the pre-patent period up to 56 days. At the patent period, treatment with OXA and PZQ, alone, interrupted cercarial shedding. When the snails were treated with 1000 mg/kg PZQ, the cercarial production was re-established 15 days after treatment, but in lower numbers than those obtained before treatment, whereas the snails treated with 500 mg/kg OXA were able to shed cercariae in similar quantities to those observed before treatment. The association 250 mg/kg OXA+500 mg/kg PZQ, at the patent period, not only discontinued cercarial shedding, but also led to the "cure" of the snails, showing a synergistic effect of this combination of drugs. These results suggest that this model will be useful for selection of resistant parasites, as well as for screening new antischistosomal drugs.
Assuntos
Anti-Helmínticos/farmacologia , Biomphalaria/parasitologia , Oxamniquine/farmacologia , Praziquantel/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose mansoni/parasitologia , Animais , Quimioterapia Combinada , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose mansoni/tratamento farmacológico , Estatísticas não ParamétricasRESUMO
Praziquantel (PZQ) is effective against all the evolutive phases of Schistosoma mansoni. Infected Biomphalaria glabrata snails have their cercarial shedding interrupted when exposed to PZQ. Using primary in vitro transformed sporocysts, labeled with the probe Hoechst 33258 (indicator of membrane integrity), and lectin of Glycine max (specific for carbohydrate of N-acetylgalactosamine membrane), we evaluated the presence of lysosomes at this evolutive phase of S. mansoni, as well as the influence of PZQ on these acidic organelles and on the tegument of the sporocyst. Although the sporocyst remained alive, it was observed that there was a marked contraction of its musculature, and there occurred a change in the parasite's structure. Also, the acidic vesicles found in the sporocysts showed a larger delimited area after contact of the parasites with PZQ. Damages to the tegument was also observed, as show a well-marked labeling either with Hoechst 33258 or with lectin of Glycine max after contact of sporocysts with the drug. These results could partially explain the interruption/reduction mechanism of cercarial shedding in snails exposed to PZQ.
Assuntos
Animais , Camundongos , Anti-Helmínticos/farmacologia , Lisossomos/efeitos dos fármacos , Oocistos/efeitos dos fármacos , Praziquantel/farmacologia , Schistosoma/efeitos dos fármacos , Schistosoma/citologia , Schistosoma/crescimento & desenvolvimentoRESUMO
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 percent. The fluorescent labels were Hoechst 33258 and Acridine Orange + Ethidium Bromide and vital dyes (Trypan Blue 0.4 percent and Neutral Red 1 percent). 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.
Assuntos
Animais , Camundongos , Corantes , Corantes Fluorescentes , Óvulo/crescimento & desenvolvimento , Schistosoma mansoni/citologia , Schistosoma mansoni/crescimento & desenvolvimento , Coloração e Rotulagem/métodos , Laranja de Acridina , Óvulo/citologia , Azul TripanoRESUMO
We have been able to label the excretory system of cercariae and all forms of schistosomula, immature and adult worms with the highly fluorescent dye resorufin. We have shown that the accumulation of the resorufin into the excretory tubules and collecting ducts of the male adult worm depends on the presence of extracellular calcium and phosphate ions. In the adult male worms, praziquantel (PZQ) prevents this accumulation in RPMI medium and disperses resorufin from tubules which have been prelabelled. Female worms and all other developmental stages are much less affected either by the presence of calcium and phosphate ions, or the disruption caused by PZQ. The male can inhibit the excretory system in paired female. Fluorescent PZQ localises in the posterior gut (intestine) region of the male adult worm, but not in the excretory system, except for the anionic carboxy fluorescein derivative of PZQ, which may be excreted by this route. All stages of the parasite can recover from damage by PZQ treatment in vitro. The excretory system is highly sensitive to damage to the surface membrane and may be involved in vesicle movement and damage repair processes. In vivo the adult parasite does not recover from PZQ treatment, but what is inhibiting recovery is unknown, but likely to be related to immune effector molecules.
Assuntos
Animais , Feminino , Masculino , Anti-Helmínticos/farmacologia , Polilisina/farmacologia , Praziquantel/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Corantes Fluorescentes , Oxazinas , Schistosoma mansoni/fisiologiaRESUMO
Damage to the surface membrane of adult Schistosoma mansoni, and the activity of the excretory system, as shown by resorufin fluorescence, was observed following treatment with praziquantel and incubation with other molecules. Praziquantel treatment induced damage to the surface membrane as measured by the use of a variety of fluorescent compounds. The excretory system of the male worm was inhibited immediately after praziquantel treatment, but fully recovered after culture for 2 h following removal of praziquantel. The excretory system of the female, observed to be minimally active in untreated worm pairs, was often greatly activated in paired females, as shown by intense resorufin labelling, after praziquantel treatment, and this continued during recovery of the male excretory system. In experiments with normal worm pairs, the female could be activated by inhibiting the metabolic rate of the pair by a cooling procedure. The effects on the excretory system of changes in culture conditions (such as changes in pH, concentrations of bacterial lipopolysaccharide, cytokines, reactive oxygen species, compounds which remove cholesterol, such as beta-methyl cyclodextrin, and damaging basic poly-L-lysine) were also assessed. It is concluded that the extensive excretory system of the adult worm is responsive to drug treatment and to certain changes in environmental conditions. Its activity seems to be strongly linked to the integrity of the surface membrane.
Assuntos
Anti-Helmínticos/farmacologia , Praziquantel/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/fisiologia , Animais , Bisbenzimidazol , Temperatura Baixa , Escherichia coli , Feminino , Corantes Fluorescentes , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Oxazinas/metabolismo , Polilisina/farmacologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologia , beta-Ciclodextrinas/farmacologiaRESUMO
Praziquantel (PZQ) is effective against all the evolutive phases of Schistosoma mansoni. Infected Biomphalaria glabrata snails have their cercarial shedding interrupted when exposed to PZQ. Using primary in vitro transformed sporocysts, labeled with the probe Hoechst 33258 (indicator of membrane integrity), and lectin of Glycine max (specific for carbohydrate of N-acetylgalactosamine membrane), we evaluated the presence of lysosomes at this evolutive phase of S. mansoni, as well as the influence of PZQ on these acidic organelles and on the tegument of the sporocyst. Although the sporocyst remained alive, it was observed that there was a marked contraction of its musculature, and there occurred a change in the parasite's structure. Also, the acidic vesicles found in the sporocysts showed a larger delimited area after contact of the parasites with PZQ. Damages to the tegument was also observed, as show a well-marked labeling either with Hoechst 33258 or with lectin of Glycine max after contact of sporocysts with the drug. These results could partially explain the interruption/reduction mechanism of cercarial shedding in snails exposed to PZQ.
Assuntos
Anti-Helmínticos/farmacologia , Lisossomos/efeitos dos fármacos , Oocistos/efeitos dos fármacos , Praziquantel/farmacologia , Schistosoma/efeitos dos fármacos , Animais , Camundongos , Schistosoma/citologia , Schistosoma/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Corantes , Corantes Fluorescentes , Óvulo/crescimento & desenvolvimento , Schistosoma mansoni/citologia , Schistosoma mansoni/crescimento & desenvolvimento , Coloração e Rotulagem/métodos , Laranja de Acridina , Animais , Camundongos , Óvulo/citologia , Azul TripanoRESUMO
We have been able to label the excretory system of cercariae and all forms of schistosomula, immature and adult worms with the highly fluorescent dye resorufin. We have shown that the accumulation of the resorufin into the excretory tubules and collecting ducts of the male adult worm depends on the presence of extracellular calcium and phosphate ions. In the adult male worms, praziquantel (PZQ) prevents this accumulation in RPMI medium and disperses resorufin from tubules which have been prelabelled. Female worms and all other developmental stages are much less affected either by the presence of calcium and phosphate ions, or the disruption caused by PZQ. The male can inhibit the excretory system in paired female. Fluorescent PZQ localises in the posterior gut (intestine) region of the male adult worm, but not in the excretory system, except for the anionic carboxy fluorescein derivative of PZQ, which may be excreted by this route. All stages of the parasite can recover from damage by PZQ treatment in vitro. The excretory system is highly sensitive to damage to the surface membrane and may be involved in vesicle movement and damage repair processes. In vivo the adult parasite does not recover from PZQ treatment, but what is inhibiting recovery is unknown, but likely to be related to immune effector molecules.
Assuntos
Anti-Helmínticos/farmacologia , Polilisina/farmacologia , Praziquantel/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Animais , Feminino , Corantes Fluorescentes , Masculino , Oxazinas , Schistosoma mansoni/fisiologiaRESUMO
A fluorescent dye monochlorobimane (MCB) that binds glutathione (GSH) was used as a tool for measuring the concentration of GSH in skin and mechanically-transformed schistosomula. The specificity of MCB binding to GSH was confirmed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The MCB binding to GSH is an energy-dependent process since no labelling could be seen at low temperature. When 24-h-old schistosomula were depleted of GSH by buthionine sulfoximine (a specific inhibitor of GSH synthesis) for 18 h, a significant decrease (P < 0.001) in fluorescence was observed. PZQ treatment of the schistosomula after first labelling the parasites with MCB did not greatly affect MCB binding to GSH. However, when the 24-h-old schistosomula were first PZQ treated and afterwards labelled with MCB, the pattern of labelling was identical to that of those of the non-labelled parasites. When 24-h-old schistosomula were first PZQ treated, washed and labelled in the presence of 1 mM GSH, the level of fluorescence was recovered. These results suggest that PZQ depletes GSH from schistosomula, and may render them susceptible to the host's immune system.