Effect of praziquantel treatment during pregnancy on cytokine responses to schistosome antigens: results of a randomised, placebo-controlled trial

doi:10.1086/593215

J Infect Dis. Author manuscript; available in PMC 2010 June 25.

Published in final edited form as:

J Infect Dis. 2008 December 15; 198(12): 1870–1879.

doi: 10.1086/593215

PMCID: PMC2892302

UKMSID: UKMS29620

Effect of praziquantel treatment during pregnancy on cytokine responses to schistosome antigens: results of a randomised, placebo-controlled trial

Robert Tweyongyere,^1,² Patrice A. Mawa,¹ Sophy Ngom-wegi,¹ Juliet Ndibazza,¹ Trinh Duong,³ Birgitte J. Vennervald,⁴ David W. Dunne,⁵ Eli Katunguka-Rwakishaya,² and Alison M. Elliott^1,³

¹Medical Research Council/Uganda Virus Research Institute-Uganda Research Unit on AIDS, Kampala, Uganda

²Faculty of Veterinary Medicine Makerere University, Kampala Uganda

³London School of Hygiene and Tropical Medicine, London, UK

⁴DBL-Institute for Health Research and Development Department of Disease Biology University of Copenhagen, Copenhagen Denmark

⁵Department of Pathology Cambridge University, Cambridge, UK

Correspondence Author: Robert Tweyongyere, MRC/UVRI Uganda Research Unit on AIDS, Uganda Virus Research Institute, P. O. Box 49, Entebbe, Uganda. Tel: +256 712 817220, Fax:+256 41 321137, Email: tmrobert966/at/gmail.com; Email: robert.tweyongere/at/mrcuganda.org; Email: rtweyongyere/at/vetmed.mak.ac.ug

The publisher's final edited version of this article is available free at J Infect Dis

See other articles in PMC that cite the published article.

Abstract

Background:

Praziquantel treatment of schistosomiasis boosts anti-schistosome responses, with ‘type 2 helper T-cell’ bias that may contribute to immunologically mediated killing and to protection against re-infection. Praziquantel treatment during pregnancy was recommended in 2002 but immunological effects of the treatment had not been investigated.

Methods:

A cohort of 387 S. mansoni infected women was recruited within a larger trial of de-worming during pregnancy (ISRCTN32849447; http://www.controlled-trials.com/ISRCTN32849447/elliott). Women were randomised to receive either praziquantel or placebo during pregnancy. Six weeks after delivery all women received praziquantel. Whole blood culture cytokine responses to S. mansoni worm and egg antigens were measured before and six weeks after each treatment.

Results:

Schistosome specific cytokine responses were suppressed during pregnancy. Praziquantel treatment during pregnancy caused significant boosts in gamma interferon (IFNγ), interleukin (IL)-2, IL-4, IL-5 IL-13 and IL-10 responses to schistosome worm antigen and IFNγ, IL-5 and IL-13 to schistosome egg antigen; but these boosts were not as substantial as those seen for treatment after delivery.

Conclusion:

Pregnancy suppresses potentially beneficial boost in cytokine responses associated with praziquantel treatment. Further studies are needed on the long term effect of treating schistosomiasis during pregnancy on morbidity and resistance to reinfection among treated women and their offspring.

Keywords: Schistosomiasis, Schistosoma mansoni, human, praziquantel, treatment, pregnancy, cytokines, immunology, immune responses

Introduction

Praziquantel, the drug of choice against schistosomiasis, [1, 2] has shown excellent safety and therapeutic effect against schistosomiasis morbidity. Praziquantel became available in 1979, but had never been studied in pregnant or lactating women hence, although presumed safe in pregnancy based on animal studies, was widely withheld from pregnant or lactating women during treatment programmes [3]. In 2002, an informal consultation by the World Health Organisation reviewed experience on use of praziquantel in pregnancy. There was little evidence of adverse effects from case reports, or from inadvertent use during pregnancy in mass treatment programmes and it was recommended that pregnant and lactating women with schistosomiasis should be treated [4, 5]. However, the risks and benefits of treatment during pregnancy, and the immunological effects in particular, had not been studied. Thus, in 2005, a WHO scientific working group called for randomised, placebo-controlled trials of treatment during pregnancy for all species of human schistosomes in both low and high transmission areas [6]. We here report immunological findings from such a trial.

Although the precise mode of action of praziquantel is still not clear there is evidence of synergy between praziquantel and the immune system. First, it has been suggested that praziquantel-induced damage to the worm surface tegument may be supplemented by immunologically mediated killing of the exposed schistosome [7-12]. Second, praziquantel treatment has marked effect on anti-schistosome immune responses [13, 14] and leads to a boost in schistosome antigen-specific cytokine responses [15], which may contribute to subsequent immunity to re-infection[16, 17].

Several factors including age, sex, previous exposure, co-infections and treatment influence an individual's immune response to schistosomiasis [18, 19]. Of particular interest to this study is the influence of pregnancy. Pregnancy is characterised by depression of cell-mediated immunity [20-22] with relative increase in T helper (Th)2-associated and regulatory responses (increased production of interleukin (IL)-4 and IL-10), and decreased Th1 responses (low gamma interferon (IFNγ) and IL-2 production) [23] More particularly, it has been shown that schistosome specific proliferative responses decline with advancing pregnancy in women with schistosomiasis [24].

Thus, within a trial of de-worming during pregnancy, we have explored the effect of pregnancy on immune responses to S. mansoni infection with a hypothesis that praziquantel treatment during pregnancy might be associated with reduced boost in immune responses when compared with treatment of non-pregnant women. Such effect might be important in relation to the efficacy of treatment (reducing the synergy between drug and immunologically mediated killing) and in relation to subsequent resistance to re-infection.

Methods

Study cohort, randomisation to praziquantel treatment and sample collection

A nested cohort of 387 pregnant women infected with Schistosoma mansoni was enrolled within the larger Entebbe Mother and Baby Study (EMABS) on ‘the impact of helminths on the response to immunisation and on susceptibility to infectious diseases in childhood in Uganda’ (ISRCTN32849447; http://www.controlled-trials.com/ISRCTN32849447/elliott) [25].

Details of recruitment, baseline findings, interventions, randomisation and allocation procedure for the main study have been described [25]. Briefly, the study was a randomised, double-blind placebo-controlled trial using praziquantel versus placebo and albendazole versus placebo during pregnancy in a 2×2 factorial design. Participants were recruited between April 2003 and November 2005. Women in the second or third trimester of pregnancy were eligible for inclusion if they were resident in the Entebbe peninsula of Lake Victoria, planned to deliver their baby in Entebbe Hospital and were willing to participate. They were excluded if the pregnancy was not normal, if they had any history of adverse reactions to anthelmintics or evidence of helminth-induced disease requiring immediate treatment, or if they had participated in the study during an earlier pregnancy. Demographic and socio-economic details and clinical history were obtained at enrolment. Women were asked to provide stool samples for examination for helminth ova and blood for diagnostic and immunological analysis. They were then randomised to receive single dose praziquantel (40 mg/kg) or placebo and single dose albendazole (400 mg) or placebo, taken under observation. Six weeks after delivery all mothers received praziquantel and albendazole with additional anthelmintic treatment, if indicated by stool results.

The nested cohort study of immune responses to schistosomiasis began in November 2003, seven months after enrolment to the larger cohort had started, and was confined to women who had a positive stool result for S. mansoni ova. Within this nested study, follow-up samples for immunological assays were obtained at six weeks post-enrolment during pregnancy (if the woman was still pregnant), six weeks after delivery but before post-delivery treatment and 12 weeks after delivery (six weeks after the post-delivery treatment). Follow-up stool samples were obtained six weeks post-enrolment during pregnancy, after delivery and 12 weeks after delivery.

At first, assays for responses to S. mansoni antigens were performed for all enrolling women. Later, to conserve limited stocks of schistosome antigens, single-step rapid test kit strips (BV European Veterinary Laboratory EVL, Woerden, Netherlands) for circulating cathodic antigen (CCA) in urine were performed to screen for S. mansoni infection, allowing assays to be selectively done on enrolment samples likely to be from schistosome-infected participants. Out of 121 women enrolled when we used CCA screening, 78 were CCA positive and had assays while 43 were CCA negative and missed the assays. We compared S. mansoni infection intensity (median epg) between women who were CCA negative, Kato-Katz positive and the women who were positive on both CCA and Kato-Katz and there was no significant difference (median epg: 59.9 and 48.0 respectively, p=0.84) confirming no selection bias.

Ethical clearance was obtained from the Science and Ethics Committee of Uganda Virus Research Institute, the Uganda National Council for Science and Technology and London School of Hygiene and Tropical Medicine.

Parasitological diagnosis

A single stool sample per individual, at each time point, was examined by duplicate Kato-Katz thick smears [26]. Slides were read within 30 minutes for hookworm ova and the next day for S. mansoni ova. S. mansoni infection intensity was expressed in eggs per gram (epg) of stool and categorised as light (1-99epg), moderate (100-399epg) or heavy (≥400epg) [3].

Schistosome antigens

Soluble S. mansoni adult worm (SWA) and egg (SEA) were prepared as previously described [27]. The endotoxin level in both antigens was below levels that could stimulate detectable production of the cytokines assayed.

Whole blood culture and cytokine assay

Cytokine responses were examined by whole blood culture assays as previously described [28, 29]. Briefly, heparinised blood was diluted to a final concentration of 1 in 4 with serum-free medium (RPMI supplemented with glutamine, penicillin and streptomycin) and 200μl per well added to 96-well, round-bottom plates (TC Microwell, NUNC A/S, Roskelde, Denmark). The blood was stimulated with SWA, SEA or phytohaemagglutinin (PHA- Sigma, UK) at final concentrations of 10μg/ml or left unstimulated. Cultures were incubated at 37°C with 5% CO_2, and supernatants harvested after 6 days. Supernatants were virally inactivated with 0.03% tributyl phosphate and 1% Tween 80 (Sigma) at room temperature for one hour and stored at −80°C. Supernatants were analysed for IFN-γ, IL-2, IL-4, IL-5 and IL-10 using OptEIA ELISA Kits (BD Pharmingen, USA) and for IL-13 using antibody pairs (BD PharMingen, USA) with standards obtained from the National Institute for Biological Standards and Controls (NIBSC), UK. Supernatants from all time points for each participant were assayed in duplicate on the same ELISA plate and a positive control was used for all plates to monitor for inter-assay variations. The sensitivity of each assay, and cut-off for positive response, was the lowest standard concentration (7.8pg/ml, except for IFNγ (8.6pg/ml)). Cytokine concentration in unstimulated wells was subtracted from concentrations in antigen-stimulated wells to obtain the antigen-specific response.

Statistical considerations and analysis

The sample size for the nested study was determined by the proportion of women with schistosomiasis within the main study. We expected to enrol 250 schistosome-infected, pregnant women (125 each in praziquantel and placebo arms). Initial analyses were made using qualitative variables but quantitative analyses were found to be more informative and are presented in this paper. No interim analyses were performed and there were no stopping rules, but serious adverse events were reported to the data monitoring committee to allow stopping if excess of events occurred in any group.

The analysis had four objectives:

To assess the effects of pregnancy on anti-schistosome cytokine responses by comparing responses during pregnancy (at enrolment and six weeks post-enrolment) with responses after delivery in the placebo group. The Wilcoxon signed-rank test was used.
To assess the effect of praziquantel treatment during pregnancy. First, responses before and after treatment were compared in the praziquantel group using the Wilcoxon signed rank test. Then responses at six weeks post-enrolment were compared between the praziquantel group the placebo group using the Wilcoxon ranksum (Mann-Whitney) test.
To examine the influence of pregnancy on the praziquantel-induced boost in cytokine responses at six weeks post-treatment. The boost during pregnancy was compared with the boost after delivery using linear regression based on the change in log₁₀(cytokine concentration+1). The term boost is used to refer to the change in cytokine responses following praziquantel treatment although in some instances decline in the responses could occur.
To examine the influence of pregnancy on cure following praziquantel treatment by comparing post-treatment infection intensity between the group first-treated in pregnancy and the group first-treated after delivery. The Pearson's Chi Square test was used.

Results

During recruitment period for the nested cohort, 2208 women were recruited to the main study of whom 387 had stool samples positive for Schistosoma mansoni. Cytokine response data at enrolment was available on 103 women who received praziquantel and 105 women who received placebo for praziquantel. For logistical reasons (missed appointments, omission of schistosome antigens in assays or insufficient samples) numbers analysed for particular time points varied: results available for the praziquantel and placebo groups, respectively, were 80 and 72 at six weeks post-enrolment (while still pregnant), 91 and 95 six weeks after delivery and 68 and 77 six weeks after post-delivery treatment. At baseline, the praziquantel and placebo groups showed similar characteristics of the women, intensity of schistosome infection, prevalence of hookworm and HIV, and profile of cytokine responses to schistosome antigens (Table 1). At baseline, type 2 cytokine responses to SWA showed positive associations with schistosome infection intensity (IL-4 (rho=0.13 p=0.061), IL-5 (rho=0.14 p=0.036), IL-13 (rho=0.16 p=0.021)). Baseline responses to SEA showed negative associations with infection intensity for IFNγ (rho −0.17 p=0.017),and IL-13 (−0.14 p=0.041), and a positive association for IL-4 (rho=0.14 p=0.05). Cytokine responses to SWA and SEA showed no association with maternal age, estimated gestational age or with other co-infections such as hookworm.

Table 1

Comparison of characteristics at enrolment before treatment between the praziquantel and placebo groups

Cytokine responses to SWA and SEA are suppressed during pregnancy

Results from the placebo group were examined in order to assess effects of pregnancy on anti-schistosome cytokine responses in the absence of treatment (figures (figures1b1b and and2b).2b). At six weeks post-enrolment, IFNγ, IL-5 and IL-13 responses to SWA and SEA were lower than at enrolment (p=0.008 for IFNγ and IL-5 and p=0.006 for IL-13 to SWA; p<0.001 for IFNγ, p=0.002 for IL-5 and p=0.001 for IL-13 to SEA). At six weeks after delivery IFNγ, IL-5 and IL-13 responses to SWA had increased and were higher than at enrolment (p=0.02 for IFNγ, p=0.007 for IL-5 and p<0.001 for IL-13) and at six weeks post-enrolment (p<0.001 for IFNγ, p=0.003 for IL-5 and p=0.001 for IL-13); similarly, IFNγ and IL-13 responses to SEA were significantly higher than at six weeks post-enrolment (p=0.009 for IFNγ and p=0.002 for IL-13). IL-2, IL-4 and IL-10 responses showed no statistically significant changes.

Figure 1

Whole blood culture cytokine levels in response to SWA and effect of praziquantel treatment during pregnancy or after delivery

Figure 2

Whole blood culture cytokine levels in response to SEA and effect of praziquantel treatment during pregnancy or after delivery

Thus IFNγ, IL-5 and IL-13 responses to SWA and SEA were suppressed during pregnancy: they declined with progressing pregnancy and were higher, after delivery, than at either time-point during pregnancy.

Praziquantel treatment during pregnancy causes significant boost in cytokine responses to SWA and SEA

Results during pregnancy were examined in the praziquantel group, and compared with results in the placebo group, in order to assess the effect of praziquantel treatment during pregnancy. Among the women who received praziquantel at enrolment (figure 1a), cytokine production in response to SWA at six weeks post-enrolment was significantly higher than at enrolment (p=0.01 for IFNγ and IL-2; p<0.001 for IL-4, IL-5, IL-13 and IL-10). At six weeks post-enrolment cytokine responses to SWA were significantly higher among the women in the praziquantel group than among the placebo group (p<0.001 for IFNγ, IL-5, IL-13 and IL-10; p=0.003 for IL-2 and IL-4).

The effects of praziquantel treatment during pregnancy on responses to SEA were more limited (figure 2a). Among women who received praziquantel at enrolment, production of IL-2, IL-5 and IL-13 in response to SEA had increased at six weeks post-enrolment (p=0.02 for IL-2, p=0.01 for IL-5 and 0.002 for IL-13). At six weeks post-enrolment, responses to SEA were significantly higher among the women in the praziquantel group than among the placebo group for IL-5 (p=0.003) and IL-13 (p<0.001).

Pregnancy is associated with reduced boost in cytokine responses to SWA and SEA

The magnitude of the praziquantel-induced boost in cytokine responses (between treatment and six weeks after treatment) was compared between the praziquantel group (first-treated at enrolment during pregnancy) and the placebo group (first-treated six weeks after delivery), in order to assess the influence of pregnancy on the boost in responses.

Initial comparisons showed that the boost was lower for treatment during pregnancy than for treatment after delivery for all responses except for IL-5 and IL-13 to SWA (table 2). Several possible sources of bias were considered in relation to this analysis because, at the time of treatment, the group first-treated after delivery (originally the placebo group) differed in several important ways from the group first-treated during pregnancy (originally the praziquantel group). The pre-treatment prevalence of hookworm was lower in the group first-treated after delivery (since 47% had already received albendazole, table 1); and 100% of the group first-treated after delivery received albendazole concurrently with their first praziquantel treatment, compared to 55% of those first-treated during pregnancy. However, analyses during pregnancy showed no effect of hookworm co-infection or of concurrent albendazole on the boost in ant-schistosome responses following praziquantel treatment (data not shown), so these differences were considered unlikely to bias the comparison. Pre-treatment levels of cytokine response were higher in the group first-treated after delivery. Although no major difference in enrolment schistosome infection intensity was noted between the two groups, enrolment schistosome intensity showed positive correlations with post-treatment SWA-specific IL-4 (rho=0.38 p<0.001), IL-5 (rho=0.21 p=0.05), and IL-13 (rho=0.23 p=0.039) responses. Post-treatment responses to SEA showed a tendency to negative correlations with enrolment schistosome intensity which did not attain statistically significant levels. However, after adjustment for pre-treatment cytokine responses and enrolment infection intensity the boost in responses remained markedly higher for women treated after delivery than those treated during pregnancy for all responses except IL-5, IL-13 to SWA and IL-2 to SEA (table 2).

Table 2

Comparison of the change in cytokine responses to SWA and SEA at six weeks following praziquantel treatment during pregnancy or after delivery; adjusted for pre-treatment cytokine responses and S. mansoni infection intensity

In keeping with the suppression of responses during pregnancy and the reduced boost in response following treatment during pregnancy, all six-week-post-treatment responses were lower following treatment during pregnancy than following treatment after delivery. However, among women first-treated during pregnancy, the responses had increased at six weeks after delivery and were not significantly different from the responses at six weeks post-treatment among those first-treated after delivery; except IL-4 and IL-10 to SWA which were still lower among women first-treated during pregnancy than among women first-treated after delivery (p=0.002 for IL4 and 0.008 for IL-10).

Pregnancy is not associated with reduced cure rate

To address the hypothesis that suppression of the boost in immune response during pregnancy is associated with reduced cure rate, infection intensity at six weeks after treatment was compared between the group first-treated during pregnancy and the group first-treated after delivery. Of the 80 women in the group first-treated during pregnancy accessed for cytokine responses at six weeks following treatment, 68 (85.0%) did not have egg detected in their stool, eight (10.0%) had light infection, three (3.8%) had moderate infection and one (1.2%) still had heavy infection. Of the 77 women in the group first-treated after delivery, eggs were not detected in 68/76 (89.5%) of the women and 8/76 (10.5%) still had light infection. One of the women did not submit a stool sample at six weeks after the treatment. The difference in post-treatment infection intensity was not statistically significant (p=0.27).

Discussion

The aim of this study was to elucidate the influence of pregnancy on the immune response to schistosome antigens in S. mansoni-infected women and, particularly, on the effects of praziquantel treatment on cytokine responses, with a hypothesis that pregnancy would cause reduced boost in the cytokine responses following treatment.

First, the study showed alterations of cytokine responses to schistosome antigens during pregnancy among women who did not receive praziquantel at enrolment. Schistosome-specific IFNγ, IL-5 and IL-13 responses were depressed during pregnancy. Further, although there was no association between stage of gestation and cytokine responses at enrolment (perhaps due to the variability of responses between individuals and difficulty in estimating the stage of gestation accurately), there was a decline in these three responses within individuals over a six-week period within the pregnancy. This evidence of suppression of responses during pregnancy confirmed and extended the earlier findings by Novato-Silva et al [24] and is consistent with effects reported for other antigens [20, 21]. From the onset of pregnancy, the immunological environment of the uterus is controlled to allow foetal allograft retention [30] and this is associated with a general depression of cellular responses [31, 32]. Schistosome-specific IL-2, IL-4, and IL-10 remained relatively unaffected during pregnancy. IL-2 and IL-4 responses were detected at low frequency, and lack of sensitivity of the assay may have limited our ability to detect any changes. However, these results accord with reports that Th2-associated responses and regulatory activity are generally sustained during pregnancy [33-35], while Th1 responses are suppressed.

Secondly, despite the immunosuppressive effects of pregnancy, praziquantel treatment of S. mansoni during pregnancy caused detectable increase in all measured cytokine responses to SWA and in all responses to SEA except IL-4 (the latter being perhaps below the level of detection of the assay in many cases); thus praziquantel treatment reversed the otherwise declining cytokine profiles observed during pregnancy. The effects were particularly marked for responses to SWA. Other studies with non-pregnant individuals from S. mansoni endemic areas in Uganda have reported that praziquantel treatment cause boosts in IL-4, IL-5, IL-13, and IL-10 responses to SWA, but not in the IFNγ response to SWA or any response to SEA [15, 36].

Responses to SWA and SEA were boosted to different extents following praziquantel treatment during pregnancy. This could have a bearing on morbidity of schistosomiasis during pregnancy, an aspect that was not examined in this study. In both human and experimental murine studies, schistosomiasis morbidity has been closely linked to the host immune responses, with evidence that excess of either type 1 or type 2 responses can cause severe disease [18, 37-39]. Dominance of IFNγ has been associated with acute and childhood-associated hepatosplenic disease [37, 40, 41] while IL-13 together with low IL-10 has been associated with severe hepatic fibrosis [42].

The significant boost in IFNγ responses observed in the current study has not been reported by related studies [15, 43] among non-pregnant individuals in areas of high S. mansoni transmission. The current study involved pregnant women and the relatively low prevalence and intensities of infection indicated low S. mansoni transmission. Furthermore post-treatment follow up time was different. The boost in IFNγ responses observed in this study (following treatment either during pregnancy or after delivery), but not in high transmission areas, suggests that the praziquantel-induced boost profile depends on transmission and intensity of infection. In fact, we noted a negative correlation between the IFNγ boost and the pre-treatment infection intensity in this study, although this effect as not statistically significant (data not shown)

Studies elsewhere have reported that praziquantel treatment of schistosomiasis mansoni and haematobium not only resolves some of the schistosomiasis associated morbidities, but can also delay resurgence of these morbidities after subsequent reinfection [44, 45]. Thus, praziquantel treatment can induce immunological changes that favour type 2 responses that are associated with resistance to reinfection [27, 46-48], and also prevent or delay onset of specific morbidities in reinfection [44, 45]. Our study has shown that boost in cytokine responses to schistosome antigens was suppressed following treatment during pregnancy as compared to treatment after delivery. However the responses among women treated during pregnancy increased substantially after delivery. Moreover, in spite the suppressed boost, the cure rate during pregnancy was not significantly different from that after delivery. This suggests that the long term benefits associated with treatment-induced alterations of the immune responses may not be affected by the observed suppression of the boost in cytokine responses following treatment during pregnancy.

This is first study to demonstrate effects of pregnancy on immune response following praziquantel treatment of schistosomiasis. Further studies are needed to examine the likely impact of the treatment-induced cytokine alterations during pregnancy on morbidity in the women as well as the babies born to the treated women; in order to assess morbidity and immune responses in the children and the long term effect of praziquantel treatment of pregnant women.

Acknowledgements

We thank members of the teams that were involved in the study: the EMaBS women, staff of EMaBS and Entebbe Grade B Hospital maternity ward. We thank the Cambridge Schistosomiasis Immunology Group and in particular Frances Jones for preparation of the schistosome antigens used in this study. We are grateful to the Wellcome Trust, DBL-institute for health Research and Development and Makerere University for funding the study. We thank MRC/UVRI Uganda Research Unit on AIDS and Entebbe Hospital for institutional support.

Sources of funding: The Wellcome Trust, through a senior fellowship held by AME (grant number 064693), Makerere University School of Graduate Studies and DBL-Institute for Health Research and Development.

Footnotes

Conflict of Interest: The authors do not have any commercial or other associations that might pose conflict of interest

Preliminary results of this study have been presented at the following fora:

55^th Annual meeting of the American Society of Tropical Medicine and Hygiene, November 2006, Atlanta, USA.
African International Conference on Immunity, May 2007, Victoria Falls, Zimbabwe
Scientific Meeting of the Wellcome Trust Bloomsbury Centre for Clinical Tropical Medicine, April 2007, Entebbe, Uganda,

References

1. Frenzel K, Grigull L, Odongo-Aginya E, et al. Evidence for a long-term effect of a single dose of praziquantel on Schistosoma mansoni-induced hepatosplenic lesions in northern Uganda. Am J Trop Med Hyg. 1999;60:927–31. [PubMed]

2. Engels D, Chitsulo L, Montresor A, Savioli L. The global epidemiological situation of schistosomiasis and new approaches to control and research. Acta Trop. 2002;82:139–46. [PubMed]

3. WHO Report of the WHO informal consultation on monitoring drug efficacy in the control of schistosomiasis and intestinal nematodes; 8-10 July 1998. WHO; Geneva: 1999.

4. Allen HE, Crompton DW, de Silva N, LoVerde PT, Olds GR. New policies for using anthelmintics in high risk groups. Trends Parasitol. 2002;18:381–2. [PubMed]

5. WHO Report of the WHO Informal Consultation on the use of praziquantel during pregnancy/lactation and albendazole/mebendazole in children under 24 months; 8-9 April 2002. WHO; Geneva: 2003. p. 49.

6. WHO Report of the scientific working group meeting on schistosomiasis; 14-16 November 2005. WHO-TDR; Geneva: 2006. p. 114.

7. Brindley PJ, Sher A. The chemotherapeutic effect of praziquantel against Schistosoma mansoni is dependent on host antibody response. J Immunol. 1987;139:215–20. [PubMed]

8. Brindley PJ, Strand M, Norden AP, Sher A. Role of host antibody in the chemotherapeutic action of praziquantel against Schistosoma mansoni: identification of target antigens. Mol Biochem Parasitol. 1989;34:99–108. [PubMed]

9. Brindley PJ, Sher A. Immunological involvement in the efficacy of praziquantel. Exp Parasitol. 1990;71:245–8. [PubMed]

10. Modha J, Lambertucci JR, Doenhoff MJ, McLaren DJ. Immune dependence of schistosomicidal chemotherapy: an ultrastructural study of Schistosoma mansoni adult worms exposed to praziquantel and immune serum in vivo. Parasite Immunol. 1990;12:321–34. [PubMed]

11. Fallon PG, Cooper RO, Probert AJ, Doenhoff MJ. Immune-dependent chemotherapy of schistosomiasis. Parasitology. 1992;105(Suppl):S41–8. [PubMed]

12. Nyazema NZ, Mutamiri FF, Mudiwa I, Chimuka A, Ndamba J. Immunopharmacological aspects of praziquantel. Cent Afr J Med. 1995;41:284–8. [PubMed]

13. Murphy KM, Reiner SL. The lineage decisions of helper T cells. Nat Rev Immunol. 2002;2:933–44. [PubMed]

14. Mutapi F. Heterogeneities in anti-schistosome humoral responses following chemotherapy. Trends Parasitol. 2001;17:518–24. [PubMed]

15. Joseph S, Jones FM, Walter K, et al. Increases in human T helper 2 cytokine responses to Schistosoma mansoni worm and worm-tegument antigens are induced by treatment with praziquantel. J Infect Dis. 2004;190:835–42. [PubMed]

16. Roberts M, Butterworth AE, Kimani G, et al. Immunity after treatment of human schistosomiasis: association between cellular responses and resistance to reinfection. Infect Immun. 1993;61:4984–93. [PMC free article] [PubMed]

17. Medhat A, Shehata M, Bucci K, et al. Increased interleukin-4 and interleukin-5 production in response to Schistosoma haematobium adult worm antigens correlates with lack of reinfection after treatment. J Infect Dis. 1998;178:512–9. [PubMed]

18. Pearce EJ, MacDonald AS. The immunobiology of schistosomiasis. Nat Rev Immunol. 2002;2:499–511. [PubMed]

19. Remoue F, Rogerie F, Gallissot MC, et al. Sex-dependent neutralizing humoral response to Schistosoma mansoni 28GST antigen in infected human populations. J Infect Dis. 2000;181:1855–9. [PubMed]

20. Gehrz RC, Christianson WR, Linner KM, Conroy MM, McCue SA, Balfour HH., Jr. A longitudinal analysis of lymphocyte proliferative responses to mitogens and antigens during human pregnancy. Am J Obstet Gynecol. 1981;140:665–70. [PubMed]

21. Lederman MM. Cell-mediated immunity and pregnancy. Chest. 1984;86:6S–9S. [PubMed]

22. Chaouat G, Ledee-Bataille N, Dubanchet S. Immune cells in uteroplacental tissues throughout pregnancy: a brief review. Reprod Biomed Online. 2007;14:256–66. [PubMed]

23. Formby B. Immunologic response in pregnancy. Its role in endocrine disorders of pregnancy and influence on the course of maternal autoimmune diseases. Endocrinol Metab Clin North Am. 1995;24:187–205. [PubMed]

24. Novato-Silva E, Gazzinelli G, Colley DG. Immune responses during human schistosomiasis mansoni. XVIII. Immunologic status of pregnant women and their neonates. Scand J Immunol. 1992;35:429–37. [PubMed]

25. Elliott AM, Kizza M, Quigley MA, et al. The impact of helminths on the response to immunization and on the incidence of infection and disease in childhood in Uganda: design of a randomized, double-blind, placebo-controlled, factorial trial of deworming interventions delivered in pregnancy and early childhood [ISRCTN32849447] Clin Trials. 2007;4:42–57. [PMC free article] [PubMed]

26. Katz N, Chaves A, Pellegrino J. A simple device for quantitative stool thick-smear technique in Schistosomiasis mansoni. Rev Inst Med Trop Sao Paulo. 1972;14:397–400. [PubMed]

27. Webster M, Fulford AJ, Braun G, et al. Human immunoglobulin E responses to a recombinant 22.6-kilodalton antigen from Schistosoma mansoni adult worms are associated with low intensities of reinfection after treatment. Infect Immun. 1996;64:4042–6. [PMC free article] [PubMed]

28. Elliott AM, Hurst TJ, Balyeku MN, et al. The immune response to Mycobacterium tuberculosis in HIV-infected and uninfected adults in Uganda: application of a whole blood cytokine assay in an epidemiological study. Int J Tuberc Lung Dis. 1999;3:239–47. [PubMed]

29. Weir RE, Morgan AR, Britton WJ, Butlin CR, Dockrell HM. Development of a whole blood assay to measure T cell responses to leprosy: a new tool for immuno-epidemiological field studies of leprosy immunity. J Immunol Methods. 1994;176:93–101. [PubMed]

30. Robertson SA. Control of the immunological environment of the uterus. Rev Reprod. 2000;5:164–74. [PubMed]

31. Mellor AL, Munn DH. Extinguishing maternal immune responses during pregnancy: implications for immunosuppression. Semin Immunol. 2001;13:213–8. [PubMed]

32. Guleria I, Sayegh MH. Maternal acceptance of the fetus: true human tolerance. J Immunol. 2007;178:3345–51. [PubMed]

33. Wilder RL. Hormones, pregnancy, and autoimmune diseases. Ann N Y Acad Sci. 1998;840:45–50. [PubMed]

34. Al-Shammri S, Rawoot P, Azizieh F, et al. Th1/Th2 cytokine patterns and clinical profiles during and after pregnancy in women with multiple sclerosis. J Neurol Sci. 2004;222:21–7. [PubMed]

35. Doria A, Iaccarino L, Arienti S, et al. Th2 immune deviation induced by pregnancy: the two faces of autoimmune rheumatic diseases. Reprod Toxicol. 2006;22:234–41. [PubMed]

36. Dunne DW, Vennervald BJ, Booth M, et al. Applied and basic research on the epidemiology, morbidity, and immunology of schistosomiasis in fishing communities on Lake Albert, Uganda. Trans R Soc Trop Med Hyg. 2006;100:216–23. [PubMed]

37. de Jesus AR, Silva A, Santana LB, et al. Clinical and immunologic evaluation of 31 patients with acute schistosomiasis mansoni. J Infect Dis. 2002;185:98–105. [PubMed]

38. Hoffmann KF, Wynn TA, Dunne DW. Cytokine-mediated host responses during schistosome infections; walking the fine line between immunological control and immunopathology. Adv Parasitol. 2002;52:265–307. [PubMed]

39. Wynn TA, Thompson RW, Cheever AW, Mentink-Kane MM. Immunopathogenesis of schistosomiasis. Immunol Rev. 2004;201:156–67. [PubMed]

40. Booth M, Mwatha JK, Joseph S, et al. Periportal fibrosis in human Schistosoma mansoni infection is associated with low IL-10, low IFN-gamma, high TNF-alpha, or low RANTES, depending on age and gender. J Immunol. 2004;172:1295–303. [PubMed]

41. Mwatha JK, Kimani G, Kamau T, et al. High levels of TNF, soluble TNF receptors, soluble ICAM-1, and IFN-gamma, but low levels of IL-5, are associated with hepatosplenic disease in human schistosomiasis mansoni. J Immunol. 1998;160:1992–9. [PubMed]

42. Oliveira LFA, Moreno EC, Gazzinelli G, et al. Cytokine production associated with periportal fibrosis during chronic schistosomiasis mansoni in humans. Infect Immun. 2006;74:1215–21. [PMC free article] [PubMed]

43. Fitzsimmons CM, Joseph S, Jones FM, et al. Chemotherapy for schistosomiasis in Ugandan fishermen: treatment can cause a rapid increase in interleukin-5 levels in plasma but decreased levels of eosinophilia and worm-specific immunoglobulin E. Infect Immun. 2004;72:4023–30. [PMC free article] [PubMed]

44. Hatz CF, Vennervald BJ, Nkulila T, et al. Evolution of Schistosoma haematobium-related pathology over 24 months after treatment with praziquantel among school children in southeastern Tanzania. Am J Trop Med Hyg. 1998;59:775–81. [PubMed]

45. Vennervald BJ, Booth M, Butterworth AE, et al. Regression of hepatosplenomegaly in Kenyan school-aged children after praziquantel treatment and three years of greatly reduced exposure to Schistosoma mansoni. Trans R Soc Trop Med Hyg. 2005;99:150–60. [PubMed]

46. Dunne DW, Butterworth AE, Fulford AJ, et al. Immunity after treatment of human schistosomiasis: association between IgE antibodies to adult worm antigens and resistance to reinfection. Eur J Immunol. 1992;22:1483–94. [PubMed]

47. Dunne DW, Pearce EJ. Immunology of hepatosplenic schistosomiasis mansoni: a human perspective. Microbes Infect. 1999;1:553–60. [PubMed]

48. Mutapi F, Ndhlovu PD, Hagan P, et al. Chemotherapy accelerates the development of acquired immune responses to Schistosoma haematobium infection. J Infect Dis. 1998;178:289–93. [PubMed]