Schistosoma mansoni infection cancels the susceptibility to Plasmodium chabaudi through induction of type 1 immune responses in A/J mice

Ayako Yoshida1,2, Haruhiko Maruyama1, Takashi Kumagai1, Teruaki Amano3, Fumie Kobayashi4, Manxin Zhang5, Kunisuke Himeno5 and Nobuo Ohta1

1 Department of Medical Zoology, Nagoya City University Medical School, 1 Azakawasumi, Mizuhocho, Mizuhoku, Nagoya 467-8601, Japan
2 Japan Health Sciences Foundation, Tokyo 103-0001, Japan
3 Department of Parasitology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
4 Department of Tropical Medicine and Parasitology, Kyorin University School of Medicine, Mitaka 181-8611, Japan
5 Department of Parasitology, Tokushima University School of Medicine, Tokushima 770-8503, Japan

Correspondence to: N. Ohta


    Abstract
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Susceptibility to Plasmodium chabaudi depends on the relative dominance of Th1/Th2 responses in host mice. A Th2-dominant response during the early phase of infection in susceptible A/J mice causes a fatal disease course due to severe malaria. Schistosoma mansoni is a potent inducer of a Th2-dominant response not only to the parasite antigens, but also to other antigens concurrently existing in the host animals. In spite of S. mansoni infection, these A/J mice escape death from malaria and showed accompanied enhanced production of IFN-{gamma} to malaria antigens. Treatment with anti-IFN-{gamma} mAb in S. mansoni-infected A/J mice abolished the resistance to malaria, indicating that IFN-{gamma} was responsible for the resistance to P. chabaudi in S. mansoni-infected A/J mice. Results in this study show that under certain circumstances, S. mansoni infection can promote type 1 immune responses in A/J mice that normally develop Th2 responses.

Keywords: HSP 90, IFN-{gamma}, Plasmodium chabaudi, Schistosoma mansoni, Th1/Th2


    Introduction
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 Introduction
 Methods
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 Expression of HSP90 in...
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Protective immunity to the blood stage of the murine malaria parasite, Plasmodium chabaudi, critically requires CD4+ T cells (13). CD4+ T cells are separated into two major subsets based on their cytokine profiles (46). In a resistant strain, such as C57BL/6, the predominance of the two phenotypes changes over the time-course of infection; Th1-regulated mechanisms mediate resolution of a high parasitemia in the early acute phase, while Th2-mediated and antibody-dependent mechanisms correlate with clearance of chronic subpatent infection (1,2). In contrast, susceptible A/J mice, which develop fulminating parasitemia and severe anemia, and succumb to infection, show a predominant Th2 response early in the infection (2,3).

T cells from mice infected with Schistosoma mansoni mount a strong Th2 response at the onset of egg production, with promoted secretion of Th2 cytokines (IL-4, IL-5 and IL-10) when stimulated not only with schistosome antigen, but also with other co-existing antigens (710). Indeed, Th2-dominant immune responses in mice with S. mansoni infection induced resistance against Trichuris muris and Strongyloides venezuerensis (11,12). On the other hand, the S. mansoni-driven Th2 response caused delayed clearance of virus or altered cytokine profiles followed by antigen challenge (9,10). In concomitant S. mansoni/P. chabaudi infections, malaria-resistant C57BL/6 mice developed higher parasitemia than mice with P. chabaudi alone, possibly due to a shift of the Th1/Th2 balance (13).

In this study, we found that S. mansoni induced resistance to P. chabaudi in A/J mice. In vitro analysis of the cytokine profile showed enhanced IFN-{gamma} production even in the presence of Th2-inducing pressure from S. mansoni. Elevated IFN-{gamma} production seems to induce up-regulation of iNOS mRNA expression, which is thought to be an effector tool involved in anti-malaria immunity. Our data suggest that the mechanism determining the Th1/Th2 paradigm is rather complicated and even schistosomes, potent Th2 inducers, are under the control of the host–parasite interaction in determining host phenotypes of the Th response.


    Methods
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 Methods
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 Expression of HSP90 in...
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Mice and parasites
Female A/J and C57BL/6 mice (7 weeks old) were purchased from SRL (Shizuoka, Japan). Mice were infected i.p. with 106 P. chabaudi AS-parasitized erythrocytes [parasitized red blood cells (PRBC)]. Eight weeks prior to the P. chabaudi infection, groups of A/J and C57BL/6 mice were infected with 25 cercariae of S. mansoni of the Puerto Rican strain by percutaneous skin exposure. Tail vein blood was used to measure parasitemia on thin film blood smears stained with Giemsa and to monitor the change of hematocrits as evidence of anemia development.

Detection of heat shock protein (HSP) 90 in P. chabaudi
HSP90 was detected as previously described (1416). In brief, PRBC were prepared from mice 7 days after P. chabaudi infection. PRBC were lysed by saponin treatment and supernatants were applied for Western blotting. Ten micrograms of protein of each sample was loaded. Mouse anti-HSP90 mAb, recognizing protein of ~90 kDa in extract from purified parasites, was the first antibody. Peroxidase-conjugated goat anti-mouse IgG (Zymed, San Francisco, CA) was the second antibody. Binding antibodies were detected by the enhanced chemiluminescence detection method (Amersham, Little Chalfont, UK). Fluorescence emitted from luminol in the presence of peroxidase could be detected by autoradiography film (Fuji Film, Tokyo, Japan). Bands were analyzed by using a density scanner and NIH Image software (National Institutes of Health, Bethesda, MD).

Spleen cell preparation
Spleen cells were aseptically removed 7 days after P. chabaudi infection and perfused with RPMI 1640 (Sigma, St Louis, MO) supplemented with 1% FCS (Gibco/BRL, Rockville, MD). Red blood cells were lysed with cold 0.17 M NH4Cl. The cells were washed twice in fresh medium, and adjusted to 5.0x106 cells/ml in RPMI 1640 supplemented with 10% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin and 20 mM L-glutamine (Gibco/BRL). Aliquots of 2.0 ml in triplicate were incubated in 24-well flat-bottomed tissue culture plates (Nunc, Roskilde, Denmark) with 106/ml PRBC or 10 µg/ml soluble egg antigen (SEA) of S. mansoni as antigen. As controls, cultures were established without any stimulation. Forty-eight hours later, supernatants were collected and stored at –80°C until assay for cytokine.

Cytokine ELISA
IL-4, IL-10 and IFN-{gamma} in culture fluids were assessed by TiterZyme Mouse IL-4, IL-10 and IFN-{gamma} EIA kits (PerSeptive Diagnostics, Framingham, MA) or AN'ALYZATM Mouse IL-4, IL-10 and IFN-{gamma} immunoassay systems (Genzyme, Minneapolis, MN).

Neutralization of cytokines
Neutralizing mAb specific for murine IFN-{gamma} (XMG 1.2) and IL-10 (SXC-1) were purified by precipitation with ammonium sulfate from ascites (17,18). Rat anti-mouse IFN-{gamma} or IL-10 mAb or normal rat {gamma}-globulin (Cappel, Aurora, OH) were injected i.p. at the dose of 0.5 mg on days –2, –1, 0, 1, 2, 4, 7, 10, 14 and 18 after P. chabaudi infection.

mRNA detection by RT-PCR
Spleens were removed 7 days after P. chabaudi infection and stored at –80°C until RNA isolation. RNA was isolated with an RNA extract kit (Gentra Systems, Minneapolis, MN), according to the manufacturer's instruction. cDNA was generated from 1 µg of RNA using an RNA PCR Kit (Perkin Elmer, Branchburg, CA). RT-PCR was performed with oligonucleotide primers as follows (19,20): iNOS sense, 5'-CTGGAGGAGCTCCTGCCTCATG-3' (449 bp); iNOS antisense, 5'-GCAGCATCCCCTCTGATGGTG-3'; ß-actin sense, 5'-TGGAATCCTGTGGCATCCATGAAAC-3' (348 bp); ß-actin antisense, 5'-TAAAACGCAGCTCAGTAAACAGTCCG-3'. The resulting cDNA was subjected to PCR for 35 cycles with each set of primers. Two microliters of cDNA was mixed with 50 µl of 50 mM KCl, pH 8.4, 20 mM Tris–HCl, 1.5 mM MgCl2, 200 mM dNTPs, 2 U Taq DNA polymerase (all Perkin Elmer) and 1.2 mM of primers, and were incubated for 3 min at 94°C. The thermal cycle profile was 94°C for 1 min, 54°C for 1 min and 72°C for 2 min. PCR products were separated in 2% agarose gel.

Statistical analysis
Experimental and control values were analyzed for significant differences by Student's t-test.


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Courses of P. chabaudi infection in mice with or without S. mansoni infection
Mice were infected with 106 P. chabaudi AS PRBC. All A/J mice without S. mansoni infection died 8 days post-infection, while the same parasite induced non-lethal effects in A/J mice with S. mansoni infection (Fig. 1aGo). We monitored survival and parasitemia until 11 weeks after S. mansoni infection, because S. mansoni-infected A/J mice started to die 12 weeks post-infection due to schistosome infection. A/J mice have impaired IL-3 responses; therefore, anemic situations should be considered carefully for our data interpretation. The development of anemia during the cause of infection was monitored by determining changes in the hematocrit of peripheral blood. As parasite levels increased, anemia developed progressively as evidenced by the decrease of the hematocrit value. However, hematocrits of A/J mice with or without S. mansoni infection were similar from the start of the experiment to 5 days after P. chabaudi infection (data not shown). The mean peak parasitemia levels, determined as percent PRBC, were 43.85 ± 2.14% for P. chabaudi-infected A/J mice, 44.73 ± 1.75% for co-infected A/J mice, 13.89 ± 2.06% for P. chabaudi-infected C57BL/6 mice and 26.70 ± 2.14% for co-infected C57BL/6 mice on day 8 after P. chabaudi infection (Fig. 1BGo). As described before (13), co-infected C57BL/6 mice showed higher susceptibility; mortality increased in those mice (Fig. 1AGo) and parasitemia was elevated to a significantly higher level than mice with P. chabaudi alone (P < 0.01) (Fig. 1BGo). Co-infected A/J mice showed a significantly higher peak of parasitemia than P. chabaudi-infected C57BL/6 mice but resolution of parasitemia to a low level as in C57BL/6 mice. These data demonstrated that S. mansoni infection made resistant C57BL/6 mice more susceptible to P. chabaudi, while allowing susceptible A/J mice to clear the malaria parasites.



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Fig. 1. Courses of P. chabaudi infection and change in parasitemia in S. mansoni/P. chabaudi-infected A/J (•), P. chabaudi-infected A/J ({circ}), S. mansoni-infected A/J ({blacktriangleup}), S. mansoni/P. chabaudi-infected C57BL/6 ({blacksquare}) and P. chabaudi-infected C57BL/6 ({square}). (A) Percentage survival and (B) course of parasitemia. Results are shown as means ± SEM of five mice. The experiment was repeated 3 times with similar results.

 

    Expression of HSP90 in malaria parasite
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 Expression of HSP90 in...
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In P. yoelii infection, HSP90 expression of malaria parasites disappeared in mice treated with anti-CD4 mAb and SCID mice (14), suggesting HSP90 might be a marker for the strength of host CD4+ cell-related immune response. To decide whether S. mansoni infection affects CD4+ T cell responses against P. chabaudi, HSP90 expression of malaria parasites was investigated in A/J mice 7 days after infection. We observed strong expression of HSP90 in P. chabaudi from the co-infected A/J mice, whereas only slight expression in parasites from P. chabaudi-infected A/J mice was seen (Fig. 2AGo). Each band was analyzed by using a density scanner and NIH Image software (Fig. 2BGo). Differences in the expression level of HSP90 between parasites from co-infected A/J mice and from P. chabaudi (alone)-infected mice were statistically significant (P < 0.05).



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Fig. 2. Expression of HSP90 in P. chabaudi. (A) HSP90 expression in P. chabaudi harvested from individual mice 7 days after infection was detected by Immunoblot analysis. The number of mice tested was n = 3 for co-infection and n = 5 for P. chabaudi only infection. (B) Bands were analyzed by using a density scanner and NIH Image software. Each column represents the mean ± SEM. *P < 0.05.

 
Th2 dominant immune responses induced by S. mansoni infection
Spleen cells were collected from A/J mice 8 weeks after S. mansoni infection and stimulated by SEA in vitro. IL-4, IL-10 and IFN-{gamma} were assayed by ELISA (Fig. 3Go). S. mansoni infection induced significantly higher production of IL-4 and IL-10 in A/J mice (P < 0.05), whereas a similar level of IFN-{gamma} production as in normal A/J mice was seen. Thus, we confirmed that immune responses against S. mansoni infection were Th2 dominant in A/J mice.



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Fig. 3. In vitro cytokine production of spleen cells collected from S. mansoni-infected ({blacksquare}) and uninfected ({square}) A/J mice. Spleen cells were prepared from mice 8 weeks after S. mansoni infection, and incubated for 48 h in RPMI 1640 supplemented with 10% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, 20 mM L-glutamine and 10 µg/ml SEA. IL-4, IL-10 and IFN-{gamma} in culture fluids were assessed by ELISA. Each column represents the mean ± SEM of three mice.

 
In vitro cytokine production by spleen cells
In order to assess the influence of S. mansoni infection on malaria antigen-driven cytokine production, we compared the production of IFN-{gamma}, IL-4 and IL-10 in spleen cells at 7 days after P. chabaudi infection (Fig. 4Go). In P. chabaudi-infected C57BL/6 mice, IFN-{gamma} and IL-10 production increased significantly more than in uninfected controls (P < 0.01). In A/J mice, the IL-4 level was significantly increased during P. chabaudi infection; however, there was no significant difference between those with and without S. mansoni infection. Spleen cells from co-infected A/J mice produced significantly higher levels of IFN-{gamma} and IL-10 than P. chabaudi (alone)-infected mice (P < 0.05).





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Fig. 4. In vitro production of IL-4 (A), IL-10 (B) and IFN-{gamma} (C) by spleen cell. Spleen cells were prepared from mice 7 days after P. chabaudi infection, and incubated for 48 h in RPMI 1640 supplemented with 10% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, 20 mM L-glutamine and 106/ml PRBC ({blacksquare}). Medium was added instead of antigen as background control ({square}). IL-4, IL-10 and IFN-{gamma} in culture fluids were assessed by ELISA. Each column represents the mean ± SEM of three mice.

 
In addition, we observed changes of the cytokine profile after stimulation by schistosomal SEA in co-infected A/J mice. The SEA-elicited IL-4 level decreased after P. chabaudi infection, while the SEA-elicited IL-10 level was not changed. IFN-{gamma} production by SEA-stimulated spleen cells from co-infected A/J mice was detected from 3 days after P. chabaudi infection and rose ~10-fold in comparison with S. mansoni-infected mice at 7 days after P. chabaudi infection (data not shown). These data suggested that P. chabaudi infection enhanced the Th1 response against schistosomal antigen in co-infected A/J mice.

In vivo treatment with anti-IL-10 or anti-IFN-{gamma} antibody
We treated S. mansoni-infected A/J mice with anti-IFN-{gamma} or anti-IL-10 antibody. The neutralization of IL-10 did not affect mortality or the level of parasitemia in S. mansoni-infected A/J mice (Fig. 5A and BGo). On the other hand, S. mansoni-infected mice treated with anti-IFN-{gamma} antibody died 9–11 days after P. chabaudi infection (Fig. 5CGo). None of the S. mansoni-infected A/J mice treated with rat {gamma}-globulin died of malaria. S. mansoni-infected mice treated with anti-IFN-{gamma} antibody exhibited a significantly faster development of parasitemia (20.93 ± 0.19 versus 6.17 ± 1.17% on day 5; P < 0.05) and a significantly higher peak of parasitemia than the control mice (62.44 ± 2.88 versus 35.17 ± 5.09%; P < 0.05) (Fig. 5DGo).



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Fig. 5. Neutralization of IL-10 and IFN-{gamma} in vivo. Course of mortality (A) and parasitemia (B) in mice neutralized with IL-10, and course of mortality (C) and parasitemia (D) in mice neutralized with IFN-{gamma} are showed as means. S. mansoni-infected A/J mice were treated with anti-IL-10 antibody ({blacksquare}), anti-IFN-{gamma} antibody ({square}) or normal rat {gamma}-globulin (•), and uninfected A/J mice were treated with normal rat {gamma}-globulin ({circ}) –2, –1, 0, 1, 2, 4 and 7 days after P. chabaudi infection and twice a week thereafter. Results are shown as means ± SEM of five mice.

 
Kinetics of IFN-{gamma} production after P. chabaudi infection
The change of IFN-{gamma} production in spleen cells during the early phase of P. chabaudi infection was monitored by ELISA. Spleen cells were prepared from A/J mice on days 0, 1, 3 and 7 after P. chabaudi infection. In co-infected A/J mice, IFN-{gamma} production evoked by malaria antigen were not detected 24 h after P. chabaudi infection, but amounts increased from 3 days after P. chabaudi infection (Fig. 6Go).



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Fig. 6. Kinetics of IFN-{gamma} production by spleen cells from S. mansoni/P. chabaudi-infected (A) or P. chabaudi-infected A/J mice (B). Spleen cells were prepared from mice 0, 1, 3 and 7 days after P. chabaudi infection, and incubated for 48 h in RPMI 1640 and 106/ml PRBC ({blacksquare}). Medium was added instead of antigen as background control ({square}) IFN-{gamma} in culture fluids was assessed by ELISA. Each column represents the mean ± SEM of three mice.

 
Splenic iNOS mRNA levels in S. mansoni-infected mice
We assessed the influence of S. mansoni infection on NO production by testing iNOS mRNA in spleens detected by RT-PCR. As shown in Fig. 7Go, S. mansoni infection enhanced iNOS mRNA expression comparable to that in the resistant C57BL/6 mice.



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Fig. 7. mRNA expression of iNOS in spleens. (A) Spleens were removed from mice with or without S. mansoni infection 7 days after P. chabaudi infection. Expression of mRNA for iNOS was examined by RT-PCR. (B) Bands were analyzed by using a density scanner and NIH Image software. The density of each band corresponding to iNOS mRNA expression was normalized against the bands corresponding to ß-actin expression. Each column represents the mean ± SEM.

 

    Discussion
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 Expression of HSP90 in...
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As a model system to define innate and acquired immune mechanisms of clinical non-cerebral malaria, two inbred mouse strains, A/J and C57BL/6, which are susceptible or resistant to blood-stage P. chabaudi, have provided important information (2123). The basic concept obtained from those studies indicates that the susceptibility is determined by Th1/Th2 balance in the infected hosts and Th1 cells during early phase of infection are essential for protection. Our present data are consistent with the concept and susceptible A/J mice became resistant to P. chabaudi accompanied by a change in the profile of the cytokine pattern. Our present results are, thus, quite surprising because A/J mice infected with S. mansoni, which is a potent Th2 inducer, showed resistance to P. chabaudi possibly through enhanced production of IFN-{gamma}, a typical Th1 cytokine.

Mechanisms responsible for such phenomena are still not clear; however, there is enough reason to assume that CD4+ T cells activated by schistosome eggs are involved. First, enhanced expression of HSP90 by the malaria parasites from co-infected hosts was observed. HSP of malaria have been shown to be evolutionarily conserved to the HSP family identified from mammalian cells (24,25) and have important roles as immunodominant antigens (26, 27). HSP90 expression in other malaria parasites, P. falciparum and P. yoelii, was extensively investigated (16,28). The lethal strain of P. yoelii harvested from C57BL/6 mice showed strong expression of HSP90, while only slight expression was seen in the non-lethal strain. HSP90 expression in the lethal strain was diminished when mice were treated with anti-CD4 mAb (14), so that malaria parasites might express HSP90 to escape from the attack of host CD4+ T cells. Together with this, our findings indicate the presence of strong CD4+ T cell responses against P. chabaudi in co-infected A/J mice. Second, a Th2-dominant CD4+ T cell response appears just after exposure of the host immune system to S. mansoni eggs (7,8). Effects of concurrent S. mansoni infection in our study are consistent with results observed for malaria-resistant C57BL/6 mice (13). S. mansoni infection provided such more susceptible conditions for P. chabaudi in C57BL/6 mice by activated Th2 cells, whereas the opposite effects were observed in A/J mice. In addition, the resistance to malaria in S. mansoni-infected A/J mice was not sufficient before schistosomal egg lying (data not shown). These findings strongly suggest that the immunomodulation by S. mansoni is not uni-directional, but rather depends on the circumstances in the host mice, and on interactions among host and concomitant parasites.

It is still not conclusive whether other cell population(s) could be the sources of IFN-{gamma}, like type 1 CD8+ T cells and NK cells in S. mansoni-infected A/J mice. Schistosomiasis was shown to induce concurrent Th2 as well as type 1 CD8+ T cell responses (29,30). Moreover, IL-12 treatment of susceptible A/J mice induced Th1-mediated protective immunity against lethal P. chabaudi infection (31). In that case, IFN-{gamma} secreted by NK cells contributes to early resistance mechanisms against blood-stage P. chabaudi (32). In S. mansoni infection, NK cells were demonstrated within circumoval granulomas and could be a source of the initial IFN-{gamma} (33,34). These cell populations activated by S. mansoni infection could be the source of high IFN-{gamma} in co-infected A/J mice. More detailed analysis is needed.

Tumor necrosis factor (TNF)-{alpha} and NO are also involved in protection against P. chabaudi (31,3537). The effects of TNF-{alpha} during malaria infection are presumed to be both protective and pathogenic (3840). In the murine malaria model, TNF-{alpha} seems to mediate anemia and cerebral symptoms, which are associated with major causes of mortality (39,40). On the other hand, mice treated with recombinant TNF-{alpha} had reduced parasitemia and mortality (38). In addition, the increase of NO during early infection is correlated with resistance against P. chabaudi (3,36,41). Although the difference in TNF-{alpha} production between co-infected and only P. chabaudi-infected A/J mice was not significant (data not shown), we detected elevated iNOS mRNA expression in spleens of co-infected A/J mice. As a consequence of up-regulated IFN-{gamma} secretion, activated macrophages might provide resistance against P. chabaudi through raised NO levels.

Even though IFN-{gamma} is shown to be critical for the mortality of P. chabaudi-infected A/J mice, another essential question remained to be answered. In co-infected A/J mice, IFN-{gamma} production elicited by malaria antigen was detected 3 days post-infection. The Th1 response is essential for the protection in the early phase. In our study, parasitemia on day 7 in A/J mice showed no significant difference between mice with and without S. mansoni infection. This might suggest that the strength and/or timing of IFN-{gamma} production in co-infected A/J mice was not enough to prevent high parasitemia or, alternatively, there could be another factor determining parasitemia during P. chabaudi infection. Further analyses are needed.

In conclusion, S. mansoni infection protected A/J mice from lethal P. chabaudi infection through the induction of increased IFN-{gamma} production. Multiple parasitic infection is common in people living in schistosomiasis endemic area. In these areas, it is probable that the host response to pathogens might be determined by complex interactions among host and concomitant parasites. The clinical course or effects of vaccination could be modulated to beneficial or harmful situations by such interactions. Our data, thus, suggest that mass treatment of a particular parasitic disease might change the immunological status of the population and even produce unexpected `side effects' by enhancing pathogenicity of unrelated microbes.


    Acknowledgments
 
The authors thank Dr Kazuyuki Tanabe, Osaka Institute of Technology, for his gift of the P. chabaudi AS strain. This study was supported in part by a grant-in Aid for Scientific Research on Priority Areas `Molecular Basis for Malaria Control' (08281104) and for Scientific Research (09490025 and 09309010) from the Ministry of Education, Science, Culture and Sports, Japan, a grant from the Ministry of Health and Welfare, Japan (H10-Shinko-26 and 9Kou-3), and a grant from the Japan–US Cooperative Medical Science Program (1998–1999).


    Abbreviations
 
HSP heat shock protein
PRBC parasitized red blood cells
SEA soluble egg antigen
TNF tumor necrosis factor

    Notes
 
Transmitting editor: K. Okumura

Received 17 November 1999, accepted 10 April 2000.


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 Introduction
 Methods
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 Discussion
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