Triclabendazole and its two main metabolites lack activity against Schistosoma mansoni in the mouse model

Some have claimed that triclabendazole, a safe and efficacious drug for the treatment of fascioliasis, also exhibits antischistosomal properties, but results are conflicting. We assessed the effect of triclabendazole and its two main metabolites against two different strains of Schistosoma mansoni harbored in mice. Low worm burden reductions (18.6-35.9%) were observed in mice infected with an Egyptian strain of S. mansoni and treated with a single dose of 120 mg/kg 3 days before infection or single/double doses of 120-200 mg/kg 7 weeks after infection. Triclabendazole failed to significantly reduce hepatic and intestinal tissue egg loads, and eggs of all developmental stages were observed. Administration of 400 mg/kg of either triclabendazole, triclabendazole sulphone, or triclabendazole sulfphoxide to mice infected with a Liberian strain of S. mansoni resulted in worm burden reductions < 10%. In comparison, high worm burden reductions (82-100%) were observed in S. mansoni-infected mice treated with single oral doses of 400, 500, or 500 mg/kg twice a day praziquantel, regardless of the S. mansoni strain. We conclude that triclabendazole and its main metabolites display weak and inconsistent schistosomicidal activities.     

Susceptibility and resistance to Schistosoma mansoni reinfection: parallel cellular and isotypic immunologic assessment

Cellular and humoral immune responses to Schistosoma mansoni antigen preparations were evaluated in individuals presumed to be susceptible or resistant to reinfection after chemotherapeutic cure. A consistent proliferative increase in the response to soluble egg antigen (SEA) was observed post-treatment in both the susceptible and resistant groups. However, this change was not related to resistance. Isotype studies showed that IgM antibody levels to soluble worm antigen preparation (SWAP) and cercariae antigens were significantly higher in the resistant group than in the susceptible group. Post-treatment, an increase in IgE anti-SWAP and anti-schistosomular tegument (STEG) responses and a decrease in IgG4 anti-SEA and anti-STEG responses were observed in the resistant group. These finding are similar to those we have reported previously for a putative resistant group termed endemic normals, and are compatible with immunologic studies in different endemic areas. Together, these findings indicate that even on the population level, high IgE specificities coupled with low IgG4 specificities correlate well with documented resistance to reinfection.     

蒿甲醚和青蒿琥酯对感染曼氏血吸虫小鼠治疗作用的初步研究

目的了解蒿甲醚和青蒿琥酯对小鼠曼氏血吸虫作用的效果.方法将小鼠随机分成12个实验组及1个对照组,以皮下注射的方法,每鼠接种约80条尾蚴,接种尾蚴46 d后,分别以蒿甲醚或青蒿琥酯灌胃治疗,第1天,分别以400、300、200 mg/kg的剂量1次灌胃;第2～7天,则每天分别按以上剂量的半量灌胃,7 d灌胃的总剂量分别为1 600、1 200、800 mg/kg.总量1剂组,在第7天,分别按1 600、1 200、800 mg/kg剂量1次灌胃.另设感染阳性对照组,不加治疗. 结果蒿甲醚7日疗法1 600、1 200、800 mg/kg剂量组减虫率分别为53.0%、49.0%和53.0%,减雌率为78.0%～82.0%,总量1剂组效果与7日疗法基本相同.青蒿琥酯7日疗法相应剂量减虫率分别为16.0%、37.0%和49.0%.结论蒿甲醚和青蒿琥酯对小鼠曼氏血吸虫具有一定的杀虫效果,蒿甲醚在疗效和毒性方面稍佳.     

Resistance to reinfection with Schistosoma japonicum in the mouse

BALB/c and outbred mice infected with a Philippine isolate of Schistosoma japonicum for 50 to 60 days expressed strong resistance to reinfection. The extent of this reinfection resistance ranged from 72 to 93% in 5 experiments (mean = 80% resistance) as determined by numbers of immature worms recovered from already infected and age- and sex-matched challenge control mice exposed 20 days previously to cercariae. Determination of numbers of recoverable worms from (the initial) infection suggest that adult worms are lost progressively during the period in which impressive resistance to reinfection is demonstrable. An important unresolved question is whether loss of adult worms is related in any way to expression of resistance to reinfection. Some indirect evidence indicates that the major component of reinfection resistance is expressed prior to day 4 of challenge infection. This evidence derives from analysis of lung petechiae which, in a primary infection, have been shown to provide an indication of number of adult worms which can be detected subsequently (e.g. at 30-40 days of infection). Although anti-parasite immune response have not yet been shown to be responsible for this apparent concomitant immunity, the magnitude of resistance to reinfection in the S. japonicum/mouse system should facilitate identification of any immunological effector mechanisms involved.     

Transmission electron microscopic observations on ultrastructural damage in juvenile Schistosoma mansoni caused by artemether.

Artemether, a derivative of the antimalarial artemisinin, has been shown to induce rapid and extensive alteration to the tegument of juvenile Schistosoma japonicum, S. mansoni and S. haematobium. Less is known with regard to ultrastructural damage caused by artemether; therefore, the present work was designed to assess the damage in juvenile S. mansoni. Mice infected with S. mansoni were treated intragastrically with a single dose of 400 mg/kg artemether 21 days post-infection. Between 8 h and 14 days after treatment groups of two mice were sacrificed, and schistosomula recovered for transmission electron microscopic observations. Ultrastructural damage was seen in the tegument, subtegumental musculature, parenchymal tissues and gastrodermis. It was already apparent 8 h after drug administration and increased gradually to reach a peak, 7 days post-treatment. Tegumental alterations were characterised by swelling, vesiculation and degeneration of sensory structures. Damage in subtegumental musculature, parenchymal tissues and gastrodermis included swelling, focal or extensive lysis, and decrease in granular endoplasmatic reticulum. Fourteen days after treatment ultrastructural damage was still seen in most schistosomula, however, there was partial repair in some specimens. The ability of artemether to cause extensive ultrastructural damage to juvenile S. mansoni correlates with its schistosomicidal effects and confirms earlier findings with S. japonicum.     

血吸虫感染新模型小鼠细胞因子的诱生实验

目的　在已建立了一种血吸虫感染伴随免疫新模型的基础上 ,检测该模型小鼠脾细胞培养诱生白细胞介素 -2 (IL-2 )和白细胞介素 -5(IL-5)的水平 ,初步探讨该新模型抗再感染的细胞免疫机制。方法　小鼠感染日本血吸虫尾蚴 2 0 d后 ,按 3 0 0 mg/ (kg· d)腹腔注射酚酶抑制剂——丙烯基硫脲 ,同时设立未用药对照组和正常小鼠组 ,感染后第 42 d剖杀动物 ,分别用成虫抗原 (SWAP)、虫卵抗原 (SEA)及刀豆素 A(Con A)体外刺激脾细胞诱生细胞因子 ,并检测 IL -2和 IL -5的诱生水平。结果　新模型小鼠经 SWAP诱生 IL -2的水平高于未用药对照组和正常组小鼠 ;经 SEA诱生 IL-2的水平低于对照组小鼠 ,而与正常组小鼠无差异 ;经 Con A诱生 IL-2的水平与对照组小鼠和正常小鼠无差异。此外 ,新模型小鼠经 SWAP、SEA及 Con A 3种抗原刺激诱生 IL -5的水平均明显低于对照组和正常组小鼠。结论　伴随免疫新模型小鼠体内成虫诱导的细胞免疫似以 Th1细胞功能占优势 ,而 Th2细胞功能被抑制     

Oral artemether for prevention of Schistosoma mansoni infection: randomised controlled trial

This randomized, double-blind placebo-controlled trial examined the efficacy of oral artemether for the prevention of Schistosoma mansoni infection among 354 children from Cote d'Ivoire. Stool specimens were screened over 4 consecutive days, followed by two mass treatments with praziquantel 4 weeks apart. All S. mansoni negative children were randomly assigned to placebo (n = 151) or artemether (n = 138). An assessment after 24 hours and examination of blood samples after the 3rd week of initial administration was conducted. Findings revealed that administration of oral artemether showed no adverse reaction, with an observation of a relatively lower incidence of S. mansoni infection (31/128 vs. 68/140; relative risk, 0.50; 95% confidence interval, 0.35-0.71; p = 0.00006). In addition, the geometric mean egg output among positive children in the artemether group was significantly lower in placebo treatment (19 vs. 32 eggs/g stool; p = 0.017). Furthermore, there was a significant reduction in the prevalence of Plasmodium falciparum. The study confirmed the safety and prophylactic effect of oral artemether against S. mansoni, and recommends its use as an additional tool for a more effective schistosomiasis control measure.     

Tegumental changes in 21-day-old Schistosoma mansoni harboured in mice treated with artemether

Alterations in the tegument of 21-day-old Schistosoma mansoni, caused by artemether administered to the infected mice, were studied using scanning electron microscopy (SEM). Mice were infected with S. mansoni cercariae, and after 21 days a single dose of artemether (400 mg/kg) was administered intragastrically. After 24, 72 h and 7 days groups of three mice were killed and the schistosomules collected by perfusion, fixed and processed routinely, and examined by SEM. After 24 h, all male and female worms examined showed alterations in the tegument, characterised by swelling, vesiculation and fusion of tegumental ridges; peeling, erosion and collapse of damaged tegumental surface, and also destruction of the oral sucker and acetabulum. After 72 h, severe damage to the tegument was seen, usually including extensive peeling, swelling and vesiculation, and host leukocytes were adhered to the damaged surface. Some worms were surrounded by clusters of host leukocytes or had even disintegrated. Seven days after treatment, some schistosomules still showed severe tegumental damage, but in some cases the damage was less than at earlier times, which suggested that those schistosomules that had survived were beginning to recover. The ability of artemether to cause severe damage to the tegument correlates with its high efficacy in killing 21-day-old schistosomules.     

Associations between specific antibody responses and resistance to reinfection in a Senegalese population recently exposed to Schistosoma mansoni

We examined associations between schistosome-specific antibody responses and reinfection in Senegalese individuals recently exposed to Schistosoma mansoni. The effects of treatment, age, intensity of infection and duration of exposure on schistosome-specific antibody responses were also investigated by comparing immune responses in individuals exposed for less than 3 years with responses in people exposed for more than 8 years. All individuals were bled before treatment as well as 6 and 12 weeks after. We used a statistical model that included interaction terms between time, age, infection intensity and duration of exposure. The overall patterns of most specific antibody responses by age were similar to those previously published for S. mansoni, Schistosoma japonicum and Schistosoma haematobium infections in different endemic areas. In general, a boost in specific antibody responses against adult worm antigen (SWA) was observed at 6 weeks after treatment whereas the majority of isotype responses against egg antigen (SEA) were not affected by treatment. Our analysis showed that the effect of treatment on schistosome-specific antibody responses is influenced by age, infection intensity and duration of exposure. We found no evidence that treatment matures the specific antibody response of children recently infected with S. mansoni. Our results indicate that the build-up of potentially protective immunoglobulin E (IgE) responses was associated with duration of exposure, or, in other words, experience of infection. Interestingly, in recently exposed individuals there was a significant association between IgA responses to SWA and resistance to reinfection. Resistance to reinfection and production of IgA-SWA was associated with adulthood independently of exposure patterns, suggesting that susceptibility to S. mansoni and the development of protective immune responses is age-dependent.     

Effect of combined treatment with praziquantel and artemether on Schistosoma japonicum and Schistosoma mansoni in experimentally infected animals

Praziquantel and artemether are safe and efficacious antischistosomal drugs that act against different developmental stages of the parasite: praziquantel against adult worms and artemether against schistosomula. A combined treatment has been suggested as a strategy for transmission control. Recent laboratory experiments with rabbits with a mixed infection of Schistosoma japonicum parasites of different ages confirmed the effectiveness of a combination therapy. In the present work, we assessed the effect of a combined treatment on adult worms of S. japonicum and found significantly higher worm reduction rates than with a single dose of praziquantel. In a next step, we extended the study of the combined treatment to Schistosoma mansoni. A combined treatment with 75 mg/kg praziquantel and 150 mg/kg artemether was administered to hamsters infected with juvenile and adult S. mansoni. The two drugs, administered simultaneously or spaced by 6 h, 1, 3 or 7 days, resulted in significantly higher worm reduction rates than a single treatment with praziquantel. A combination therapy with increased doses of 100 mg/kg praziquantel and 300 mg/kg artemether showed very high worm reduction rates of 90% and above, however, some hamsters died in five different combined treatment experiments, suggesting that these drug concentrations were too high. We conclude that a combined treatment with praziquantel and artemether at the lower doses is safe and more effective than praziquantel alone, which forms a foundation for designing respective clinical trials in humans.     

Genetic analysis of praziquantel resistance in Schistosoma mansoni

To determine the genetic basis of the resistance of Schistosoma mansoni to praziquantel (PZQ) and to understand whether the resistance is dominant or recessive trait, a schistosome cross was undertaken between a PZQ-susceptible and a PZQ-resistant isolate using infections of the single-sex cercariae which were identified by a direct W1-specific PCR technique. The resistances of F1 and F2 generation to PZQ were evaluated using in vitro egg, miracidial and cercarial tests. The F1 hybrid progeny from crosses between the susceptible and resistant isolates were resistant to PZQ. The resistant phenotype reappeared in the F2 progeny. It can thus be considered that the PZQ resistance behaves like a dominant trait.     

Genetic analysis of hycanthone resistance in Schistosoma mansoni

Interbreeding between hycanthone-resistant and hycanthone-sensitive schistosomes was achieved using a worm transfer technique which considerably reduced the length and the complexity of the operations generally involved in performing schistosome genetic crosses. A mouse was considered to harbor resistant schistosomes if, three weeks or more after a single intrasmuscular injection of 80 mg/kg hycanthone schistosome eggs were still excreted in the feces, at least one normal worm pair was obtained by perfusion, or miracidia could be seen hatching from the liver. The F1 hybrid progeny from crosses between sensitive and resistant schistosomes proved to be sensitive to hycanthone, irrespective of whether the resistant parent was the male or the female. The resistant phenotype reappeared in back-crosses and in the F2 progeny. These results could be confirmed using the traditional technique of single sex infections. It can thus be concluded that hycanthone resistance behaves like an autosomal recessive trait. These results suggest that hycanthone-resistant schistosomes are deficient in some factor, possibly an enzymatic activity which transforms hycanthone into a biologically active molecule, as suggested in a recent hypothesis on the mode of action of hycanthone.     

Characterization of monoclonal antibodies against Schistosoma mansoni

Monoclonal antibodies directed against Schistosoma mansoni antigens were produced by the in vitro fusion of B lymphocytes, obtained from mice infected with S. mansoni, and SP2/0 myeloma cells. Antibody reactivity was assessed by ELISA binding, utilizing 4 M KCl extracts of cercariae and adult worms, soluble egg antigen (SEA), and purified antigenic preparations, and by indirect immunofluorescence using living schistosomula. The monoclonal antibodies recognized a wide spectrum of antigenic determinants. The specificity of the monoclonal reactivities ranged from high cross-reactivity to extreme restriction, vis-a-vis the distribution of the recognized determinants within genus, species, stages, and purified antigenic preparations. The specificity of reactivity of monoclonal antibodies for a given determinant was greater than that of immune mouse serum. These studies establish the feasibility of the production of large numbers of monoclonal antibodies and of their use of antigen identification. The monoclonal antibodies are available to interested investigators upon request.     

Biochemical and immunologic predictors of efficacy of treatment or reinfection risk for Schistosoma mansoni

Most Schistosoma mansoni infections are egg-negative after a single dose of oxamniquine. A cohort of 661 infected children was treated at 6-month intervals and assessed for nutritional and parasitological status. Initial biochemical and immunologic markers were measured in a subset of 84 children. All were treated at the start of therapy and at 6 months. Immunoglobulins only served as markers for active infection. No markers were predictive of cure or reinfection, except initial infection intensity and serum low-density lipoprotein. Ten percent were persistently infected and had no change in infection intensity at any time-point. Several factors suggest that this group was biologically different. In addition to failing to reduce their worm burden, they had significantly higher initial intensity of infection (100 versus 65 eggs/g, P = 0.001) and significantly lower initial serum low-density lipoprotein (72 versus 104 mg/dL, P = 0.045). The biologic plausibility of this observation is discussed.     

A mouse model of tuberculosis reinfection

Recent clinical observations shows that individuals treated with chemotherapy for tuberculosis who live in endemic areas are four times more likely to develop secondary disease, often as not caused by exogenous reinfection. In a mouse model described here, we show that mice infected with the virulent W-Beijing Mycobacterium tuberculosis strain HN878, then given chemotherapy to clear the infection, were resistant to re-challenge with the same organism thereafter. This resistance, which was mediated by rapid expression of CD4 T cells expressing markers consistent with both central and effector memory immunity, was only transient however. After 20-30 days of the reinfection the numbers of these cells steadily declined, the bacterial load in the lungs surged up, and the lung tissues became increasingly consolidated. No evidence was found for a regulatory T cell response in these mice, but many T cells harvested from the lungs showed evidence of increased PD-1 expression, indicating exhaustion. These data indicate that the memory T cell response to reinfection may not be as stable and long lived as previously thought, a finding with obvious implications for vaccine development.     

Autoradiographic analysis of resistance to reinfection with Schistosoma mansoni in mice. Evidence that the liver is a major site of worm elimination

Migration and elimination of 75Se-labeled Schistosoma mansoni were studied in previously infected mice by means of autoradiography and worm recovery. As in control mice, there appeared to be little if any worm elimination in the skin of previously infected mice. In previously infected mice, there appeared to be some worm elimination in the lungs and in migration sites between the lungs and liver, though much less than would have been expected from previous studies. In two of three experiments most of the challenge worm elimination in previously infected mice, above the normal attrition level occurring in the controls, appeared to take place in the liver. In the third experiment, it appeared that all of the challenge worm elimination occurred after migration to the lungs, and at least one-third of it in the liver. It was also observed that the lung chop procedure recovered viable schistosomula much less efficiently from previously infected mice than from controls indicating that the reduction in lung schistosomulum recovery overestimates the amount of lung and prelung killing that occurs in reinfected mice.     

Studies on experimental mixed infection of Schistosoma haematobium and Schistosoma mansoni in the albino mouse

Swiss albino mice have been infected with S. haematobium and challenged with S. mansoni. No apparent disturbances as regards worm load and oviposition were observed. Yet certain deviations in the egg-distribution sites for both species were found. They were attributed to the possibility of earlier de-development of collateral circulation. Cross-pairing between the two species was met with, which might explain the extra number of S. haematobium eggs found in the small intestine. Although no major effect has been revealed, no conclusion at this stage could be put forward as regards complete absence of cross-immunity.     

Induction of resistance to Schistosoma mansoni by immunization with subfractions of worms

Induction of resistance to a Schistosoma mansoni infection was analyzed following injections of glutaraldehyde-fixed parasites or of subfractions prepared from nonfixed parasites killed by repeated freeze-thaw cycles. The parasites were isolated from mice infected 4 weeks previously and the subfractions were prepared by extraction in buffered saline or in detergent. A variety of immunization protocols were conducted in rats and mice. These included different sites of injection, dose size, and number of booster injections; different adjuvants; and the effects of boosting with live infections. Induction of a partial resistance is observed in rats, but has not been achieved in mice immunized by the same procedures. The alum-precipitated detergent-solubilized fraction was superior at lower doses. Exposure of rats to a low dose cercarial infection 3 weeks prior to challenge did not boost the resistance if the rats were already partially resistant through vaccination with worm subfractions in adjuvant. Serum from vaccinated rats transfers partial protection to a challenge infection of naive rats.     

Induction of protective immunity against Schistosoma mansoni by a non-living vaccine. VI. Antigen recognition by non-responder mouse strains

Previous studies have shown that many strains of mice develop partial resistance to Schistosoma mansoni as a result of intradermal vaccination with soluble schistosome antigens plus BCG. However, P and BALB/c mice are non-responsive to this intradermal vaccination protocol. In this study, humoral and cellular responses to schistosome antigens in vaccinated P and BALB/c mice were compared to those in protected C57BL/6 mice to identify an immune correlate to resistance in this model. Levels of circulating IgG and IgM antibodies to soluble adult worm antigens, as measured by ELISA, were comparable between strains. Moreover, Western blot analysis revealed no qualitative differences in antibody reactivity, with sera from vaccinated animals of all three strains recognizing the antigen previously identified as Sm-97 (paramyosin). However, vaccinated P and BALB/c mice showed specific defects in cell-mediated immunity to schistosome antigens, including decreased production of macrophage-activating lymphokines and an inability to produce activated macrophage effector cells in vivo at the site of antigen challenge. These observations strengthen our hypothesis that the intradermal vaccine acts through induction of T-cell-mediated immune resistance mechanisms.