Schistosoma mansoni: chemotherapy of infections of different ages

Mice were treated with potassium antimony tartrate, hycanthone, oxamniquine, niridazole, or praziquantel at different times after infection with Schistosoma mansoni. The rate of cure was assessed by perfusion of surviving worms approximately 4 weeks after treatment, and the percentage reduction in worm burden was estimated relative to the number of adult worms perfused from control mice, comparably infected but untreated. All six drugs were relatively inactive against S. mansoni between 3 and 4 weeks after infection when compared with treatment at 5 to 6 weeks. However, the drugs differed in the patterns of cure they achieved in the 2-week period after administration of cercariae and in the period around the onset of patency. Worms that had been subjected to amoscanate or hycanthone in the third week after infection showed evidence of this as adults in having a reduced fecundity. Factors such as worm or host physiology, or host immune status may have had roles in the outcome of chemotherapy at different stages of maturation of S. mansoni.     

Effect of chemotherapy on the Schistosoma mansoni eggs

Praziquantel administered to mice with Schistosoma mansoni infection (50 cercarias/8 weeks) was observed to cause death of adult worms and disintegration of the eggs trapped within granulomas, sometimes with calcification, after the 4th day of treatment. Combined administration of oxamniquine/hycanthone to animals similarly infected, although quite effective in killing adult worms, did not interfere with the eggs in the tissue. The miracidium eclosion test was positive up to the 15th day after the curative treatment of these animals. Since praziquantel treatment causes a rapid destruction of eggs, possible serological and pathogenic effects are expected that may enable a faster reabsorption of granulomas by the host tissues than that produced by other equally effective drugs.     

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.     

Effects of praziquantel and oxamniquine on a Saudi Arabian strain of Schistosoma mansoni in mice

The prophylactic and curative effects of praziquantel and oxamniquine on a Saudi Arabian strain of Schistosoma mansoni in MF-1 mice were assessed. The drugs were administered orally. At 240 mg/kg praziquantel, there was a reduction of 89.1% in adult worm recovery and a marked reduction in tissue deposited eggs. The reduction in adult worm recovery after dosing with 50 mg/kg oxamniquine was 89.2%. At low doses (40 mg/kg praziquantel and 30 mg/kg oxamniquine) administered at 11 days, 5 days and 3 h before and 5, 21 and 49 days after infection, the reduction in adult worm recovery was 0.0%, 65.1%, 58.8%, 33.6%, 0.0% and 76.0% for praziquantel and 0.0%, 66.0%, 60.0%, 41.3%, 10.8% and 79.0% for oxamniquine. Numbers of lung schistosomula and the size of hepatic granulomata were also reduced.     

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.     

Properties and drug sensitivity of adenosine triphosphatases from Schistosoma mansoni

The hydrolysis of ATP was measured in the presence of schistosome homogenates and various cations. The enzyme was stimulated strongly by either Ca2+ or Mg2+. Na+ added to the activation by Ca2+. A minor (17%) component was Na+ + K+ + Mg2+-dependent and ouabain-sensitive. Praziquantel, niridazole, oxamniquine, and hycanthone had no direct effect on the ATPase activity of schistosome homogenates. When schistosomes were pretreated with these drugs in vitro, washed thoroughly, and then homogenized, hycanthone, praziquantel, and oxamniquine caused a reduction in ATPase content of the worms. Niridazole did not share this effect. These results suggest that antischistosomal drugs did not directly inhibit ATPase, but did reduce ATPase in whole worms, possibly by removing or damaging the tegument, which is thought to contain most of the ATPase activity. In vitro ATPase measurements may be a useful indicator of pharmacologic activity of some types of drugs.     

In vitro selection of drug resistant Schistosoma mansoni

Schistosomules of Schistosoma mansoni were cultured for 3 days in the presence of schistosomicides and then inoculated intraperitoneally into mice. Drug concentrations killing greater than 99.8% of schistosomules were amoscanate 0.1 p.p.m., oltipraz, 0.5 p.p.m., oxamniquine 240 p.p.m., praziquantel 8 p.p.m. Comparison of drug response of the unselected and selected strains as adult worms in mice showed an increase in tolerance to amoscanate, oltipraz and oxamniquine, but not praziquantel. The oxamniquine tolerant strain did not respond to oxamniquine at 500 mg kg⁻¹. The unselected strain increased in tolerance to three drugs during routine passage in the laboratory. Greater numbers of schistosomules derived from snails exposed to ethyl methane sulfonate appeared to survive culture in metrifonate, suggesting that it may be possible to produce drug resistant schistosomes by mutation and selection.     

Adult worm tegumental damage and egg-granulomas in praziquantel-resistant and -susceptible Schistosoma mansoni treated in vivo

The effect of treatment with praziquantel (PZQ) on the tegument of adult Schistosoma mansoni worms and on liver egg-granulomas has been examined in mice infected with PZQ-resistant and -susceptible parasite isolates. Two PZQ-resistant S. mansoni isolates, one selected by passage in the laboratory under drug pressure and one from Senegal established from eggs excreted by an uncured patient, were compared with PZQ-susceptible control isolates. Scanning electron microscopic observations on the tegument of Schistosoma adult worms treated in vivo with PZQ showed that more severe damage was inflicted by PZQ on susceptible worms than on drug-resistant worms. Observations on the pathology of Schistosoma egg-granulomas in the liver of infected mice after treatment with PZQ indicated that eggs from susceptible control isolates were more sensitive to PZQ than those from drug-resistant isolates.     

Treatment of acute experimental schistosomiasis

A model of acute schistosomiasis of the mouse was used to observe whether curative treatment would be followed by an enhancement of the hepatic and splenic lesions, as a consequence of the massive destruction of worms and eggs within the portal system. Mice infected with 50 cercariae of Schistosoma mansoni were treated with both oxamniquine and praziquantel on the 50th day of infection and submitted to a sequential histologic examination from the 2nd to the 45th day after treatment. Although severe focal lesions due to dead and disintegrating worms were present in the livers of the treated animals, no aggravation of the general changes (reactive hepatitis and splenitis, or periovular granulomas) was seen in comparison with a control non-treated group. Of 50 animals treated during the acute phase of schistosomiasis only one died spontaneously, while 16 out of 30 infected controls died before the end of the experiment. The present investigation indicates that curative treatment during the acute phase of schistosomiasis does not enhance previous lesions at first and results in progressive disappearance of the lesions starting six days following chemotherapy.     

In vitro responses of praziquantel-resistant and -susceptible Schistosoma mansoni to praziquantel

The resistance status of five praziquantel-susceptible and five praziquantel-resistant isolates was confirmed by chemotherapy in CD(1) mice with 3 x 200mg/kg micronised praziquantel. Micronised praziquantel had higher efficacy than two other praziquantel formulations (prepared without milling). The five resistant isolates were less responsive to praziquantel than the five susceptible isolates (59-74% reduction in worm burden in resistant isolates compared with 92-100% in susceptible isolates). Observations were made on the in vitro responses of different stages of 10 isolates to praziquantel. There were different in vitro responses to praziquantel at the egg, miracidial, cercarial and adult stages of Schistosoma mansoni between praziquantel-resistant and praziquantel-susceptible isolates. There were differences in the response of resistant and susceptible isolates following exposure of freshly hatched miracidia to 10(-6)M praziquantel for 1 min and observing the percent change in shape. Using this test it should be possible to determine whether failed therapy in patients infected with S. mansoni is due to the presence of praziquantel-resistant worms. Similarly, by exposing freshly shed cercariae to 4 x 10(-7)M praziquantel and observing the percent of tail shedding over 80 min it should be possible to monitor for the presence of praziquantel-resistant worms in snails collected in the field.     

Effect of Curcuma longa or parziquantel on Schistosoma mansoni infected mice liver--histological and histochemical study

Effect of drug praziquantel (PZQ) and C. longa extract on S. mansoni infected mice is reported. The level of glycogen, alkaline and acid phosphatases (ALP and ACP respectively), and body weight, liver weight and liver weight/body weight ratio were studied in mice infected with S. mansoni. ALP level was increased after infection. C. longa treated mice showed marked reduction in ALP level more than after PZQ-treatment. C. longa enhanced the concentration of glycogen after being reduced by infection, while PZQ-treatment revealed more reduction. C. longa caused enhancement in body weight while PZQ treatment had no effect. The formation of granuloma around schistosome eggs in the liver produced inflammation. C. longa extract and PZQ were effective in reducing granuloma size in infected mice.     

Study of Schistosoma mansoni isolates from patients with failure of treatment with oxamniquine

After three successive treatments with oxamniquine the continuing elimination of Schistosoma mansoni eggs was observed in patients, who came from various regions of Brazil, with different clinical forms of schistosomiasis. The objective of the present study was to determine the experimental behaviour of five different S. mansoni isolates in Swiss Webster mice that were submitted to treatment with the same drug. The experimental group with failure of treatment showed higher mean number of surviving male worms when it was compared to the group without failure of treatment. These date suggest the possibility of resistance to oxamniquine.     

The chemotherapeutic effect of praziquantel against Schistosoma mansoni is dependent on host antibody response

To assess the role of host humoral immune responses in the mechanism of action of praziquantel (PZQ) against Schistosoma mansoni, the efficacy of the drug was compared in infected B cell-depleted (mu-suppressed) vs immunologically intact C3H/HeN mice. We found that PZQ was on the average only 20% as effective in eliminating adult schistosomes from mu-suppressed as compared with control animals. Indeed, in three of four experiments performed, the drug failed to significantly reduce adult worm burdens in the mu-suppressed mice. These results were not due to a delay in parasite death in the infected B cell-depleted animals, because adult worms recovered from these mice as late as 7 wk after chemotherapy were indistinguishable in number and appearance from those recovered from non-drug-treated animals. The efficacy of PZQ against schistosomes in mu-suppressed mice was completely restored by passive transfer of immune serum from donor mice infected for 6 wk and partially restored with IgG purified from the same sera. Moreover, IgG as well as IgM antibodies were detected by immunofluorescence on the surface of adult worms recovered from intact mice as early as 1 hr after administration of the drug in vivo. The tubercles of the male worms appeared to be a major site for antibody binding. These results formally demonstrate that the mechanism of action of PZQ, the most important anti-schistosomal compound in current use, involves a synergy between the drug and the humoral immune response of the host, and suggest that the relevant effector antibodies act directly against parasite antigens which become exposed on the surface of the worms as a consequence of interaction with the drug.     

Schistosoma mansoni: the effect of adrenalectomy on the murine model

Adrenal steroid hormones have been implicated, among others, as one of the most important host factors controlling the onset, establishment, and pathogenesis of schistosomiasis. They appear to inhibit oviposition by Schistosoma mansoni both in vitro and in vivo, and their effect is greatly enhanced when administered in combination with a schistosomicidal drug. Therefore, we hypothesized that adrenalectomy would greatly affect the course of the murine schistosomiasis infection. To test this hypothesis, adrenalectomized mice (Adx) infected with S. mansoni were compared with intact infected and sham-infected controls concerning their mortality rate, numbers of male and female worms, number of eggs, and liver pathology. Compared with controls, Adx infected mice showed an increase of 50% in the mortality rate, as well as 1.7-3 times as many adult worms and twice as many ova per worm pair in their liver. Thus, for the first time, there is evidence that lack of adrenal steroids mediate an increment of the adult worm burden and promote worm fecundity in vivo. The present work was done to test the hypothesis that lack of adrenal steroids enhances adult worm attrition, possibly by their direct effect on the parasite, and by upregulating or downregulating innate and adaptive immune responses.     

Determination of ED50 values for praziquantel in praziquantel-resistant and -susceptible Schistosoma mansoni isolates

The dose of praziquantel required to kill 50% of adult worms in vivo (i.e. the ED50) was estimated for nine different isolates of Schistosoma mansoni in infected mice. Four of the isolates were selected because they had not knowingly been in contact with the drug (i.e. they were putatively praziquantel-susceptible). Five putatively praziquantel-resistant isolates were chosen because they had been selectively bred for drug-resistance in the laboratory and/or had previously been shown to be relatively resistant to praziquantel in the field. The work was performed in three laboratories in different countries using pre-agreed and comparable experimental protocols. All four praziquantel-susceptible isolates had ED50s estimated to be <100 mg/kg (mean=70+/-7 SD; median=68), while all five putatively praziquantel-resistant isolates had estimated ED50s >100 mg/kg (mean=209+/-48 SD; median=192). Thus, the five praziquantel-resistant isolates, including two that had been subjected to drug pressure during more than 20 passages in mice, had drug ED50s that were approximately three times as great as those of the praziquantel-susceptible isolates. Two of the five isolates in the putatively resistant group had previously been passaged 15 or more times in mice without administration of drug-pressure, but had ED50s consistent with the other three isolates in the group, indicating that the trait of praziquantel-resistance did not necessarily impair biological fitness during laboratory passage. The protocols used here to estimate the praziquantel ED50s of S. mansoni isolates should be useful for establishing and monitoring the drug susceptibility/resistance profiles of parasite isolates freshly obtained from endemic areas, particularly those in which increased usage of the drug is likely to occur.     

Enzymatic differences between hycanthone-resistant and sensitive strains of Schistosoma mansoni

1. Hycanthone-sensitive and resistant adult worms of Schistosoma mansoni were found to have generally similar specific activities in ten enzymes of carbohydrate metabolism. 2. Kinetic analyses revealed that pyruvate kinase, glucose-6-phosphate (G6P) dehydrogenase and malate dehydrogenase from both strains possessed similar Michaelis-Menten constants and were not inhibited by hycanthone. 3. Hexokinase and lactate dehydrogenase from the drug-resistant strain were not inhibited by hycanthone and showed three to five times greater Km values than those from the drug-sensitive worms which were also inhibitable by hycanthone. 4. Hycanthone more drastically affected the Vmax of phosphofructokinase from the hycanthone-sensitive parasite. 5. These data showed that the hycanthone inhibitable enzymes were generally from the drug-sensitive strain whereas the enzymes from drug-resistant worms are mostly hycanthone insensitive.     

Lack of correlation between schistosomicidal and anticholinergic properties of hycanthone and related drugs

Visual observation of the motor activity of Schistosoma mansoni kept in vitro showed an increase of activity in the presence of hycanthone (HC). In addition, HC caused a delay in the paralytic effects of carbachol. Similar results were observed in the presence of oxamniquine (OXA). The same pattern of motor activity, however, was shown by HC-resistant worms, by Schistosoma japonicum, and by worms exposed to drug precursors (lucanthone and UK-3883), which are not schistosomicidal in vitro. Other analogs with in vitro killing activity (IA-4 and IA-4 N-oxide) showed minimal anticholinergic effects. The anticholinergic effects of HC and OXA were quickly reversible in vitro and in vivo, whereas their antischistosomal effects are irreversible and delayed. Incubation of schistosomes with high concentrations of carbachol or with anticholinergic drugs failed to compete with the schistosomicidal effects of HC. These results are viewed as contradictory to the hypothesis that HC kills schistosomes by blocking their acetylcholine receptors.     

Altered response of strain of Schistosoma mansoni to oxamniquine and praziquantel

The susceptibility of a fourth generation Ouh strain (Paranapanema Valley, S?o Paulo, Brazil) of Schistosoma mansoni to oxamniquine (OXA) and praziquantel (PZQ) was studied. Ten groups of 13 female albino mice each were infected with 70 cercariae per animal. These mice were medicated orally on the 50th day after infection. Five groups were given OXA doses of 0, 100, 200, 300 and 400 mg/kg (single doses) and the rest were treated with PZQ doses of 0, 100, 200, and 250 mg/kg/5 days. Each group was sub-divided: 8 animals underwent perfusion after 15 days treatment, 5 mice followed up for oviposition and their feces were tested every 15 days for miracidia hatching. The efficacy of the OXA doses of 100 and 200 mg/kg was 66% and 91.4%, respectively and for the 100 mg/kg PZQ dose it was 90.1%. The follow-up groups with 100 and 200 mg/kg of OXA and PZQ, 100 and 150 mg/kg, showed that they re-established the oviposition after a period of 60 to 75 days of treatment. The ED50 was 69.6 mg/kg OXA and 39.4 mg/kg PZQ. The results show the tolerance of the Ouh strain to a dose of 100 mg with both drugs and they appoint the need for a dose review during the follow up of the oviposition and in monitoring phenomena in the field.     

Mice infected with Schistosoma mansoni generate antibodies to LacdiNAc (GalNAc beta 1-->4GlcNAc) determinants.

Schistosoma mansoni is a parasitic trematode infecting humans and animals. We reported previously that adult S. mansoni synthesizes complex type biantennary N-glycans bearing the terminal sequence GalNAc beta 1-->4GlcNAc-R (lacdiNAc or LDN). We now report that mice infected with S. mansoni generate antibodies to LDN, as assessed by ELISA using a synthetic neoglycoconjugate containing LDN sequences. Sera of infected mice, but not uninfected mice, contained primarily IgM and low levels of IgG toward LDN. Interestingly, these antibodies also recognize bovine milk glycoproteins, which are known to express LDN sequences. The anti-LDN in sera of infected mice were affinity purified on immobilized bovine milk glycoproteins and shown to specifically bind LDN. An IgM monoclonal antibody (SMLDN1.1) was derived from the spleens of S. mansoni infected mice and shown to specifically bind LDN determinants. Immunoblots with affinity purified anti-LDN and SMLDN1.1 demonstrate that LDN sequences occur primarily on N-glycans of numerous glycoproteins of adult S. mansoni. LDN sequences are also expressed in many glycoproteins from S. japonicum and S. haematobium. The availability of antibody to LDN determinants should aid in defining the roles of these glycans in helminth and vertebrate biology.     

Action of colchicine on hepatic schistosomal granuloma

Amorphous material and altered collagen fragments within dilated secretory vesicles and cisternae of fibroblast cytoplasm were the main ultrastructural changes seen in hepatic periovular granulomas formed in mice infected with Schistosoma mansoni and treated with colchicine. Despite promoting ultrastructural changes in the fibroblasts found in hepatic periovular granulomas, colchicine administration to infected mice did not significantly change the light microscopic appearance of the hepatic schistosomal lesions, did not diminish the amount of total hepatic collagen, and did not change the collagen isotypes in the granulomas, as observed after a comparative study with non-colchicine treated infected control mice. When administered to mice two weeks after curative treatment of schistosomiasis with praziquantel, colchicine did not seem to increase extracellular collagen degradation or to induce a more rapid resorption of hepatic periovular granulomas, although still promoting ultrastructural alterations in fibroblasts.