BACKGROUND
the increased incidences of autoimmune diseases and atopic diseases found in developed countries  <cit>  have brought the 'hygiene hypothesis' into a hot area of study and debate.
the 'hygiene hypothesis' was first proposed by the british scientist dr. strachan in  <dig> after having observed that having many siblings, especially older ones, correlated with a decreased risk of hay fever  <cit> .
this finding has since been extended to a theory that the changed pattern in or the reduced exposure to microorganisms has led to a dysregulated immune system and hence led to increases in certain disorders like atopy and autoimmune diseases.
indeed, the mutual exclusion relationship between the incidence of immune-mediated disorders with some kinds of microbes infections, especially parasite infections, has repeatedly been reported in epidemiological studies and in animal models <cit> .
however, the requirement of the nature of parasite infection has not been fully elucidated.
worm-like metazoan organisms so called 'helminth', including both nematoda  and platyhelminthes , have been recognized as important infectious agents that can elicit beneficial effects to the infected host in terms of conferring resistance to atopy or autoimmune diseases.
as a representative genus in parasitic platyhelminthes, schistosome or exposure to schistosome derived antigens have been found to offer protection to a range of autoimmune disorders in experimental animal models including type  <dig> diabetes in nonobese diabetic  mice  <cit> , experimental allergic encephalomyelitis    <cit> , graves' disease  <cit> , inflammatory bowel disease  <cit>  and asthma  <cit> .
however, the effect of helminth infection on collagen-induced arthritis, an animal model for human rheumatoid arthritis , is less-well studied <cit> .
the immune response elicited by schistosoma mansoni , the species that is mostly seen in africa and south america, progresses through two phases.
during the first 2- <dig> weeks, called early stage infection, in which the host is exposed to migrating immature parasites, the dominant response is th <dig>  as the parasites mature, mate and begin to produce eggs, the infection enters the acute stage during which the th <dig> response decreases and the th <dig> response emerges and increases.
the th <dig> response decreases after  <dig> weeks of chronic stage of the infection  <cit> .
similar immune response profiles are also found in schistosoma japonicum , the species mostly present in asia  <cit> .
majority of animal studies have found that the protective effects against immune-mediated disorders provided by schistosome infection appeared to be associated with th <dig> immune response induced at egg-stage or acute stage of infection.
only one study done by osade et al on collagen-induced arthritis  model has demonstrated that the early stage of schistosome infection might exert any beneficial effects  <cit> .
they found that protective effects against cia in mice can be provided by  <dig> weeks sm infection  <cit> , an early stage of sm infection in which eggs have not been produced in large quantities and a th2-dominant response is usually not seen  <cit> .
this observed protection against cia offered at early-stage infection lacking association with a th <dig> response prompted us to question whether different stages of schistosome infection would offer unique protection and whether a th2-dominanted cytokine milieu provided by egg-stage of schistosome infection was required to achieve protective effects in cia model.
answers to these questions will help us to better understand the mechanisms involved in parasite immune defense and use them to prevent and treat autoimmune arthritis.
in this study, we selected either a  <dig> or  <dig> weeks sj infection as early or acute stages of infection to study whether different stages of sj infection would affect the development of cia differently.
we found that only the  <dig> weeks infection regimen can offer protective or prophylactic effects against cia, whereas the  <dig> weeks infection failed to provide any beneficial effect and even exacerbated the disease.
further studies indicated that the protective effects were correlated with decreased levels of anti-collagen ii igg especially the igg2a subclass.
cytokine pattern analysis indicated that the presence of the th <dig> cytokine milieu at the initiation period of cia together with its sustained high levels was critical for the acquirement of protective effects.
RESULTS
the protective effects against arthritis were only observed when a pre-established sj infection entered acute stage; whereas an early stage of sj infection exacerbated the arthritis
sm infection can alleviate the development of several autoimmune diseases like type i diabetes  <cit>  and experimental autoimmune encephalomyelitis  <cit>  when the infection enters the acute stage or egg-stage of infection and when a th <dig> response is dominant.
to determine whether different stages of schistosomiasis would affect uniquely autoimmune arthritis in cia model, the most widely accepted animal model for human rheumatoid arthritis , and the role played by a pre-established dominant th <dig> environment, dba/ <dig> mice were infected with the parasitic worm sj either for  <dig> weeks for pre-established early stage of sj infection  or for  <dig> weeks for a pre-established acute stage of sj infection  before the induction of cia by collagen ii  immunization.
as shown in fig.
<dig>  a significantly induced th <dig> response as evaluated by a significant increase in il- <dig> production by polyclonally activated spleen t cells was found in the  <dig> weeks infection state as compared to the  <dig> weeks infection state and to mice not infected.
however, a slightly reduced th <dig> cytokine ifn-γ production was observed in mice infected with  <dig> weeks compared to those infected with  <dig> weeks and those with no infection but without any statistical significance.
thus, consistent with the profiles found in other strains of mice infected by sm or by sj  <cit> , dba/ <dig> mice also displayed th <dig> dominant response when the infection enters  <dig> weeks but not the  <dig> weeks.
arthritis was induced by application of collagen in complete freund's adjuvant  followed by incomplete freund's adjuvant   <dig> days after first cii injection.
similar as reported  <cit> , dba/ <dig> mice developed signs of arthritis around day  <dig> after cii immunization .
injection of cii in ascia mice which were animals infected with the  <dig> weeks pre-established sj infection before cia induction, disease severity was substantially reduced as assessed by ankle swelling  and cumulative clinical arthritis scores of all four limbs .
while control cia animals reached a cumulative arthritis score of  <dig> at day  <dig> with incidence reaching about 85%, none of the ascia animals developed disease .
in contrast, injection of cii in escia mice which were animals infected with the  <dig> weeks pre-established sj infection before cia induction failed to provide any prophylaxis on cia whether arthritis score , arthritis incidence  or clinical signs  were examined.
in fact, the severity of arthritis was even increased in escia mice measured at day  <dig> .
as a control, non collagen-immunized mice did not develop arthritis regardless they were infected with sj or not .
anti-cii igg and igg2a levels were reduced in protected mice
anti-cii igg has been well known for its important role in the pathogenesis of cia and has become the marker antibody for cia  <cit> .
we compared the anti-cii igg levels in protected ascia mice, unprotected escia mice and uninfected cia mice.
as shown in fig 3a and 3b, significantly decreased levels of anti-cii igg were present in protected ascia mice as compared to uninfected cia mice; whereas no significant difference was observed between unprotected escia mice and uninfected cia mice.
this corresponded well with the prophylaxis effects observed only in ascia mice but not in escia mice.
when profiles of splenic b cells from different groups were examined, b cell percentage was found to be reduced in ascia mice compared to mice only challenged with cia .
however this reduction does not seem to explain the significantly decreased levels of anti-cii igg found in ascia mice, because the total b cell number was not decreased due to the splenomegaly found in ascia mice.
furthermore, a more reduced percentage of b cells was found in escia mice compared to mice only challenged with cia , though anti-cii igg levels remained unaffected in this group.
we further compared the levels of igg <dig> or igg2a subclasses in ascia mice without arthritis with those from arthritic cia or arthritic escia mice.
the level of igg2a was significantly reduced in ascia mice comparing to cia or escia mice , corresponding to lack of arthritis.
in contrast, igg <dig> was reduced in escia mice comparing to cia , although both groups developed arthritis comparably .
these data suggested that igg2a was more relevant to the pathogenesis of the arthritis, and its reduction was associated with lack of arthritis as found in ascia mice.
protective effects in ascia mice were associated with enhanced production of il- <dig> and il- <dig> and reduced production of ifn-γ
many studies have indicated the importance of different th subsets and the balance among them in the pathogenesis of arthritis in the cia model  <cit> .
to understand whether the alleviation of arthritis in ascia mice was associated with altered th responses, splenocytes were isolated at  <dig> weeks after cii immunization and stimulated in vitro with anti-cd <dig> and anti-cd <dig> antibodies.
the production of ifn-γ was reduced and the production of il- <dig> was enhanced in ascia mice compared to escia and cia mice .
furthermore, moderately enhanced il- <dig> levels were also found in ascia mice .
il- <dig> production as induced by polyclonal stimulation of t cells demonstrated no significant differences between any of the groups .
thus, an enhanced th <dig> response and a reduced th <dig> response appeared to correlate with the absence of disease.
the lack of significant il- <dig> production in escia mice  led us to question whether the strong th1-inducing agent complete freund's adjuvant   <cit>  used in cia model would alter the induction of th <dig> responses induced by schistosome infection.
as shown in fig  <dig>  the il- <dig> production was low at  <dig> weeks of sj infection, elevated at  <dig> to  <dig> weeks of infection and then decreased after  <dig> weeks of infection.
thus the time course of il- <dig> production by sj infection in dba/ <dig> mice is consistent with the time course found in other strains of mice infected by sm or sj  <cit> .
however, the elevated il- <dig> production occurred during  <dig> to  <dig> weeks of sj infection was abrogated if cfa together with collagen was administered at  <dig> weeks of sj infection at which il- <dig> production was still low.
interestingly, cfa and collagen given at  <dig> weeks of infection at which il- <dig> production was high prohibited the il- <dig> production from being decreased when infection entered  <dig> week period.
no differences were observed in spleen size or the appearance of the liver whether cfa was administered or not .
these results indicated that high amount of th1-inducing agent cfa used in cia model per se did not reverse an established th <dig> response in vivo as seen in ascia mice which was in line with the observations made in vitro that an established th <dig> response was irreversible  <cit> .
furthermore, the ability of cfa to prevent an ongoing th <dig> response from being firmly established as seen in escia mice which may reflect the fact that an early phase of the th <dig> response is reversible  <cit> .
more intriguingly and significantly, presence of cfa prolonged th <dig> response which otherwise would be greatly reduced seen in  <dig> weeks infected animals.
taken together, these results indicated that a th <dig> -biased milieu present at the time when cii antigen was introduced as well as during the development of arthritis correlated with lack of arthritis in protected ascia animals.
foxp3+ regulatory t cells were not different between protected and unprotected mice
it is widely accepted that foxp3+ regulatory t cells play critical roles in pathogenesis of various of kinds of autoimmune diseases including human ra <cit> .
both cd4+cd25+ t cells and cd4+cd25+foxp3+ t cells in the spleen were measured and compared.
the correspondence of these two populations of t cells was about 80-90% .
there was a slightly increased representation of foxp3+ treg during infection, whether it was cia mice alone or sj infection with cia mice.
however there was no remarkable increase in foxp3+ treg in ascia mice without arthritis compared to cia or escia mice with arthritis.
the number of mice per group is from  <dig> to  <dig> from two or three separately performed experiments.
each value was derived from the means of individual mouse in the group.
discussion
helminth parasites inhabit immune-competent hosts for long periods of time and appear to develop strategies to induce strong anti-inflammatory responses in the infected host.
loss of natural helminth exposure as a result of improved hygiene and wide-use of anti-helminth drugs result increased incidences of immune-mediated disorders evidenced by epidemiological studies.
on the other hand, the helminths provide the host with more balanced immuno-homeostasis and demonstrate beneficial effects against various kinds of autoimmune diseases in animal studies <cit> .
in fact several clinical trials have attempted to use helminth to treat autoimmune disease in patients and showed that exposure to helminths can reduce disease activity in patients with immune-mediated disease like ulcerative colitis <cit> .
in this study, we investigated the beneficial effect of schistosome infection on less-well studied collagen-induced arthritis mouse model.
the beneficial effect is found to be associated with reduced anti-collagen ii igg production and with a th <dig> cytokine environment induced at egg-stage of infection.
the anti-cii antibodies have been recognized as important pathogenic factors in the initiation and development of cia in mice.
it has been reported that arthritis can be transferred passively to naive animals with cii-reactive serum  <cit> , and by monoclonal antibody to cii  <cit> .
in fact, a different mouse model collagen antibody-induced arthritis  for human ra is established by passively transferring arthritogenic antibodies against collagen.
further studies using the caia model found that the subclass of igg2a appears to be more efficient in inducing ra probably due to its strong capacity to activate complement through the classical pathway  <cit> .
the ability of inhibiting arthritis by egg-stage sj infection in ascia mice but not by early stage infection found in our study was well correlated with reduced levels of anti-cii igg, especially the levels of igg2a.
this alteration in class switch may reflect the significantly repressed igg2a-promoting cytokine ifn-γ production found in ascia group.
on the other hand, the positive association between elevated igg2a and the occurrence of arthritis indicates the pathogenic role played by th <dig> cells and the beneficial role played by th <dig> cells.
consequently, beneficial effects against cia can only be found in the acute or egg-stage of sj infection but not in early stage infection before eggs are produced in large quantity.
schistosomiasis is a well-characterized th <dig> response-dominated disease  <cit> .
shortly after the beginning of egg deposition, a strong egg-specific th <dig> response develops characterized by high levels of il- <dig>  il- <dig>  and il- <dig> which decreases when the infection enters the chronic stage  <cit> .
not surprisingly the negative effect of schistosome infection on the induction and development of type i diabetes in nod model  <cit> , multiple sclerosis in eae model  <cit>  and in mouse model of graves' hyperthyroidism  <cit>  has repeatedly been reported in mice infected by schistosme for 6- <dig> weeks before autoimmune diseases were induced.
by comparison between the beneficial effects of sj infection and the il- <dig> production profiles, our study clearly demonstrated that the presence of th <dig> at the beginning stage of autoimmune attack was important in conferring the protection.
in addition, our study also demonstrated a positive correlation between the prolonged presence of elevated th <dig> levels during the development stage of autoinflammatory response and the absence of disease.
we noticed the different result obtained by osada et al  <cit>  in which  <dig> weeks prior schistosome infection was shown to offer protection in cia.
an obvious difference between our study and that of osada's was that different strains of schistosoma were used, ie, sm in their study and sj in ours.
since the amount of eggs along with the levels of th <dig> response induced are low after  <dig> weeks sm infection  <cit> , osada's study seems to suggest that the th <dig> milieu present at initiation phase of autoimmune attack is not crucial to achieve the protection.
in addition, elevated il- <dig> production was still found to be produced by activated splenocytes from infected and cfa injected mice, suggesting that the injection of the th1-promoting agent cfa in their study has little impact on the development of the th <dig> response.
it is not clear at present that if different strains of worms used can fully explain the different results obtained.
another possible protective mechanism involved in our study was likely associated with the enhanced production of regulatory cytokine il- <dig> by activated t cells found in protected animals.
th <dig> cells, foxp3+ cd4+ treg cells and foxp3- il-10-producing cd4+ so called tr <dig> cells <cit>  may all contribute to this induced production of t cell-derived il- <dig>  although no significant increase in treg numbers was found in protected group, the possibility that the activities of treg and tr <dig> cells were enhanced can not be excluded.
no significant difference on production of il- <dig> was found among protected mice, unprotected mice and cia mice, even though th <dig> appears to be accepted as the more critical pathogenic th cells in the cia model than th <dig>  <cit> .
our study also provided an approach to explore the plasticity of th subsets in vivo.
in vitro studies have demonstrated that th <dig> is an unstable subset  <cit> , whereas th <dig> and th <dig> subsets, once they are well established, are not reversible  <cit> .
with the application of th1-promoting agent cfa at different time point, we found that cfa together with collagen is able to inhibit the th <dig> response only at  <dig> weeks but not at  <dig> weeks of sj infection when th <dig> development is at an early stage but not well-established.
thus our results for the first time in vivo indicated that a well established th <dig> response can not be reversed, whereas an early stage th <dig> response can be reversed in the presence of cfa treatment.
this phenomenon may serve as the basis for the beneficial effects by sj infection in maintaining immune-homeostasis in infected host hence providing protection against immune-mediated diseases.
CONCLUSIONS
in summary, we demonstrated in this study that schistosoma japonicum infection like schistosoma masoni can inhibit the incidence and severity of autoimmune arthritis in the mouse cia model.
however this effect is absolutely infection stage-dependent, ie, it occurs at acute stage of infection but not at early stage of infection.
furthermore this beneficial effect is positively associated with the presence of an irreversibly established th <dig> response at the initiation of autoimmune response and with its sustained high levels during the development of arthritis.
thus our study provides more insight into the complex mechanisms regarding to the "hygiene hypothesis" and the potential difficulties faced when egg or egg-derived materials are used for therapeutic agents in th1-associated autoimmune diseases.

 the increased incidences of autoimmune diseases and atopic diseases found in developed countries have brought the'hygiene hypothesis' into a hot area of study and debate. the'hygiene hypothesis' was first proposed by the british scientist dr. strachan in after having observed that having many siblings, especially older ones, correlated with a decreased risk of hay fever. this finding has since been extended to a theory that the changed pattern in or the reduced exposure to microorganisms has led to a dysregulated immune system and hence led to increases in certain disorders like atopy and autoimmune diseases. indeed, the mutual exclusion relationship between the incidence of immune-mediated disorders with some kinds of microbes infections, especially parasite infections, has repeatedly been reported in epidemiological studies and in animal models. however, the requirement of the nature of parasite infection has not been fully elucidated. worm-like metazoan organisms so called'helminth', including both nematoda and platyhelminthes, have been recognized as important infectious agents that can elicit beneficial effects to the infected host in terms of conferring resistance to atopy or autoimmune diseases. as a representative genus in parasitic platyhelminthes, schistosome or exposure to schistosome derived antigens have been found to offer protection to a range of autoimmune disorders in experimental animal models including type diabetes in nonobese diabetic mice, experimental allergic encephalomyelitis, graves' disease, inflammatory bowel disease and asthma. however, the effect of helminth infection on collagen-induced arthritis, an animal model for human rheumatoid arthritis, is less-well studied. the immune response elicited by schistosoma mansoni, the species that is mostly seen in africa and south america, progresses through two phases. during the first 2- weeks, called early stage infection, in which the host is exposed to migrating immature parasites, the dominant response is th as the parasites mature, mate and begin to produce eggs, the infection enters the acute stage during which the th response decreases and the th response emerges and increases. the th response decreases after weeks of chronic stage of the infection. similar immune response profiles are also found in schistosoma japonicum, the species mostly present in asia. majority of animal studies have found that the protective effects against immune-mediated disorders provided by schistosome infection appeared to be associated with th immune response induced at egg-stage or acute stage of infection. only one study done by osade et al on collagen-induced arthritis model has demonstrated that the early stage of schistosome infection might exert any beneficial effects. they found that protective effects against cia in mice can be provided by weeks sm infection, an early stage of sm infection in which eggs have not been produced in large quantities and a th2-dominant response is usually not seen. this observed protection against cia offered at early-stage infection lacking association with a th response prompted us to question whether different stages of schistosome infection would offer unique protection and whether a th2-dominanted cytokine milieu provided by egg-stage of schistosome infection was required to achieve protective effects in cia model. answers to these questions will help us to better understand the mechanisms involved in parasite immune defense and use them to prevent and treat autoimmune arthritis. in this study, we selected either a or weeks sj infection as early or acute stages of infection to study whether different stages of sj infection would affect the development of cia differently. we found that only the weeks infection regimen can offer protective or prophylactic effects against cia, whereas the weeks infection failed to provide any beneficial effect and even exacerbated the disease. further studies indicated that the protective effects were correlated with decreased levels of anti-collagen ii igg especially the igg2a subclass. cytokine pattern analysis indicated that the presence of the th cytokine milieu at the initiation period of cia together with its sustained high levels was critical for the acquirement of protective effects.  the protective effects against arthritis were only observed when a pre-established sj infection entered acute stage; whereas an early stage of sj infection exacerbated the arthritis sm infection can alleviate the development of several autoimmune diseases like type i diabetes and experimental autoimmune encephalomyelitis when the infection enters the acute stage or egg-stage of infection and when a th response is dominant. to determine whether different stages of schistosomiasis would affect uniquely autoimmune arthritis in cia model, the most widely accepted animal model for human rheumatoid arthritis, and the role played by a pre-established dominant th environment, dba/ mice were infected with the parasitic worm sj either for weeks for pre-established early stage of sj infection or for weeks for a pre-established acute stage of sj infection before the induction of cia by collagen ii immunization. as shown in fig. a significantly induced th response as evaluated by a significant increase in il- production by polyclonally activated spleen t cells was found in the weeks infection state as compared to the weeks infection state and to mice not infected. however, a slightly reduced th cytokine ifn-γ production was observed in mice infected with weeks compared to those infected with weeks and those with no infection but without any statistical significance. thus, consistent with the profiles found in other strains of mice infected by sm or by sj, dba/ mice also displayed th dominant response when the infection enters weeks but not the weeks. arthritis was induced by application of collagen in complete freund's adjuvant followed by incomplete freund's adjuvant days after first cii injection. similar as reported, dba/ mice developed signs of arthritis around day after cii immunization. injection of cii in ascia mice which were animals infected with the weeks pre-established sj infection before cia induction, disease severity was substantially reduced as assessed by ankle swelling and cumulative clinical arthritis scores of all four limbs. while control cia animals reached a cumulative arthritis score of at day with incidence reaching about 85%, none of the ascia animals developed disease. in contrast, injection of cii in escia mice which were animals infected with the weeks pre-established sj infection before cia induction failed to provide any prophylaxis on cia whether arthritis score, arthritis incidence or clinical signs were examined. in fact, the severity of arthritis was even increased in escia mice measured at day. as a control, non collagen-immunized mice did not develop arthritis regardless they were infected with sj or not. anti-cii igg and igg2a levels were reduced in protected mice anti-cii igg has been well known for its important role in the pathogenesis of cia and has become the marker antibody for cia. we compared the anti-cii igg levels in protected ascia mice, unprotected escia mice and uninfected cia mice. as shown in fig 3a and 3b, significantly decreased levels of anti-cii igg were present in protected ascia mice as compared to uninfected cia mice; whereas no significant difference was observed between unprotected escia mice and uninfected cia mice. this corresponded well with the prophylaxis effects observed only in ascia mice but not in escia mice. when profiles of splenic b cells from different groups were examined, b cell percentage was found to be reduced in ascia mice compared to mice only challenged with cia. however this reduction does not seem to explain the significantly decreased levels of anti-cii igg found in ascia mice, because the total b cell number was not decreased due to the splenomegaly found in ascia mice. furthermore, a more reduced percentage of b cells was found in escia mice compared to mice only challenged with cia, though anti-cii igg levels remained unaffected in this group. we further compared the levels of igg or igg2a subclasses in ascia mice without arthritis with those from arthritic cia or arthritic escia mice. the level of igg2a was significantly reduced in ascia mice comparing to cia or escia mice, corresponding to lack of arthritis. in contrast, igg was reduced in escia mice comparing to cia, although both groups developed arthritis comparably. these data suggested that igg2a was more relevant to the pathogenesis of the arthritis, and its reduction was associated with lack of arthritis as found in ascia mice. protective effects in ascia mice were associated with enhanced production of il- and il- and reduced production of ifn-γ many studies have indicated the importance of different th subsets and the balance among them in the pathogenesis of arthritis in the cia model. to understand whether the alleviation of arthritis in ascia mice was associated with altered th responses, splenocytes were isolated at weeks after cii immunization and stimulated in vitro with anti-cd and anti-cd antibodies. the production of ifn-γ was reduced and the production of il- was enhanced in ascia mice compared to escia and cia mice. furthermore, moderately enhanced il- levels were also found in ascia mice. il- production as induced by polyclonal stimulation of t cells demonstrated no significant differences between any of the groups. thus, an enhanced th response and a reduced th response appeared to correlate with the absence of disease. the lack of significant il- production in escia mice led us to question whether the strong th1-inducing agent complete freund's adjuvant used in cia model would alter the induction of th responses induced by schistosome infection. as shown in fig the il- production was low at weeks of sj infection, elevated at to weeks of infection and then decreased after weeks of infection. thus the time course of il- production by sj infection in dba/ mice is consistent with the time course found in other strains of mice infected by sm or sj. however, the elevated il- production occurred during to weeks of sj infection was abrogated if cfa together with collagen was administered at weeks of sj infection at which il- production was still low. interestingly, cfa and collagen given at weeks of infection at which il- production was high prohibited the il- production from being decreased when infection entered week period. no differences were observed in spleen size or the appearance of the liver whether cfa was administered or not. these results indicated that high amount of th1-inducing agent cfa used in cia model per se did not reverse an established th response in vivo as seen in ascia mice which was in line with the observations made in vitro that an established th response was irreversible. furthermore, the ability of cfa to prevent an ongoing th response from being firmly established as seen in escia mice which may reflect the fact that an early phase of the th response is reversible. more intriguingly and significantly, presence of cfa prolonged th response which otherwise would be greatly reduced seen in weeks infected animals. taken together, these results indicated that a th -biased milieu present at the time when cii antigen was introduced as well as during the development of arthritis correlated with lack of arthritis in protected ascia animals. foxp3+ regulatory t cells were not different between protected and unprotected mice it is widely accepted that foxp3+ regulatory t cells play critical roles in pathogenesis of various of kinds of autoimmune diseases including human ra. both cd4+cd25+ t cells and cd4+cd25+foxp3+ t cells in the spleen were measured and compared. the correspondence of these two populations of t cells was about 80-90%. there was a slightly increased representation of foxp3+ treg during infection, whether it was cia mice alone or sj infection with cia mice. however there was no remarkable increase in foxp3+ treg in ascia mice without arthritis compared to cia or escia mice with arthritis. the number of mice per group is from to from two or three separately performed experiments. each value was derived from the means of individual mouse in the group. discussion helminth parasites inhabit immune-competent hosts for long periods of time and appear to develop strategies to induce strong anti-inflammatory responses in the infected host. loss of natural helminth exposure as a result of improved hygiene and wide-use of anti-helminth drugs result increased incidences of immune-mediated disorders evidenced by epidemiological studies. on the other hand, the helminths provide the host with more balanced immuno-homeostasis and demonstrate beneficial effects against various kinds of autoimmune diseases in animal studies. in fact several clinical trials have attempted to use helminth to treat autoimmune disease in patients and showed that exposure to helminths can reduce disease activity in patients with immune-mediated disease like ulcerative colitis. in this study, we investigated the beneficial effect of schistosome infection on less-well studied collagen-induced arthritis mouse model. the beneficial effect is found to be associated with reduced anti-collagen ii igg production and with a th cytokine environment induced at egg-stage of infection. the anti-cii antibodies have been recognized as important pathogenic factors in the initiation and development of cia in mice. it has been reported that arthritis can be transferred passively to naive animals with cii-reactive serum, and by monoclonal antibody to cii. in fact, a different mouse model collagen antibody-induced arthritis for human ra is established by passively transferring arthritogenic antibodies against collagen. further studies using the caia model found that the subclass of igg2a appears to be more efficient in inducing ra probably due to its strong capacity to activate complement through the classical pathway. the ability of inhibiting arthritis by egg-stage sj infection in ascia mice but not by early stage infection found in our study was well correlated with reduced levels of anti-cii igg, especially the levels of igg2a. this alteration in class switch may reflect the significantly repressed igg2a-promoting cytokine ifn-γ production found in ascia group. on the other hand, the positive association between elevated igg2a and the occurrence of arthritis indicates the pathogenic role played by th cells and the beneficial role played by th cells. consequently, beneficial effects against cia can only be found in the acute or egg-stage of sj infection but not in early stage infection before eggs are produced in large quantity. schistosomiasis is a well-characterized th response-dominated disease. shortly after the beginning of egg deposition, a strong egg-specific th response develops characterized by high levels of il- il- and il- which decreases when the infection enters the chronic stage. not surprisingly the negative effect of schistosome infection on the induction and development of type i diabetes in nod model, multiple sclerosis in eae model and in mouse model of graves' hyperthyroidism has repeatedly been reported in mice infected by schistosme for 6- weeks before autoimmune diseases were induced. by comparison between the beneficial effects of sj infection and the il- production profiles, our study clearly demonstrated that the presence of th at the beginning stage of autoimmune attack was important in conferring the protection. in addition, our study also demonstrated a positive correlation between the prolonged presence of elevated th levels during the development stage of autoinflammatory response and the absence of disease. we noticed the different result obtained by osada et al in which weeks prior schistosome infection was shown to offer protection in cia. an obvious difference between our study and that of osada's was that different strains of schistosoma were used, ie, sm in their study and sj in ours. since the amount of eggs along with the levels of th response induced are low after weeks sm infection, osada's study seems to suggest that the th milieu present at initiation phase of autoimmune attack is not crucial to achieve the protection. in addition, elevated il- production was still found to be produced by activated splenocytes from infected and cfa injected mice, suggesting that the injection of the th1-promoting agent cfa in their study has little impact on the development of the th response. it is not clear at present that if different strains of worms used can fully explain the different results obtained. another possible protective mechanism involved in our study was likely associated with the enhanced production of regulatory cytokine il- by activated t cells found in protected animals. th cells, foxp3+ cd4+ treg cells and foxp3- il-10-producing cd4+ so called tr cells may all contribute to this induced production of t cell-derived il- although no significant increase in treg numbers was found in protected group, the possibility that the activities of treg and tr cells were enhanced can not be excluded. no significant difference on production of il- was found among protected mice, unprotected mice and cia mice, even though th appears to be accepted as the more critical pathogenic th cells in the cia model than th. our study also provided an approach to explore the plasticity of th subsets in vivo. in vitro studies have demonstrated that th is an unstable subset, whereas th and th subsets, once they are well established, are not reversible. with the application of th1-promoting agent cfa at different time point, we found that cfa together with collagen is able to inhibit the th response only at weeks but not at weeks of sj infection when th development is at an early stage but not well-established. thus our results for the first time in vivo indicated that a well established th response can not be reversed, whereas an early stage th response can be reversed in the presence of cfa treatment. this phenomenon may serve as the basis for the beneficial effects by sj infection in maintaining immune-homeostasis in infected host hence providing protection against immune-mediated diseases.  in summary, we demonstrated in this study that schistosoma japonicum infection like schistosoma masoni can inhibit the incidence and severity of autoimmune arthritis in the mouse cia model. however this effect is absolutely infection stage-dependent, ie, it occurs at acute stage of infection but not at early stage of infection. furthermore this beneficial effect is positively associated with the presence of an irreversibly established th response at the initiation of autoimmune response and with its sustained high levels during the development of arthritis. thus our study provides more insight into the complex mechanisms regarding to the "hygiene hypothesis" and the potential difficulties faced when egg or egg-derived materials are used for therapeutic agents in th1-associated autoimmune diseases. 
following schistosoma japonicum infection, collagen ii was administered to induce collagen-induced arthritis, an animal model for human rheumatoid arthritis. arthritis susceptible strain dba/ male mice were infected with schistosoma japonicum for either weeks resulting in early stage infection or for weeks resulting in acute or egg stage infection. however, since schistosome infection undergoes different stages, each associated with a specific induction of th responses, the requirements for the ability of the different stages of schistosome infection to protect against autoimmune disease has not been elucidated. the present study was designed to study whether different stages of schistosome infection offer unique protection in collagen-induced arthritis and its mechanisms. in contrast, in unprotected mice minimal levels of il- were present at the initial stage of collagen ii challenge together with lack of il- induction following schistosoma japonicum infection. the protective effect against collagen-induced arthritis provided by schistosoma japonicum infection is infection stage-dependent. a long-term existing schistosome infection can aid in maintaining immuno-homeostasis, thus providing protection against various types of autoimmune diseases to the infected host. infection by schistosoma japonicum significantly reduced the severity and the incidence of experimental autoimmune collagen-induced arthritis. the protection against collagen-induced arthritis correlated with reduced levels of anti-collagen ii igg, especially the subclass of igg2a. however, this beneficial effect can only be provided by a pre-established acute stage of infection but not by a pre-established early stage of the infection.
 a long-term existing schistosome infection can aid in maintaining immuno-homeostasis, thus providing protection against various types of autoimmune diseases to the infected host. such benefits have often been associated with acute or egg stage infection and with the egg-induced th response. however, since schistosome infection undergoes different stages, each associated with a specific induction of th responses, the requirements for the ability of the different stages of schistosome infection to protect against autoimmune disease has not been elucidated. the present study was designed to study whether different stages of schistosome infection offer unique protection in collagen-induced arthritis and its mechanisms.  arthritis susceptible strain dba/ male mice were infected with schistosoma japonicum for either weeks resulting in early stage infection or for weeks resulting in acute or egg stage infection. following schistosoma japonicum infection, collagen ii was administered to induce collagen-induced arthritis, an animal model for human rheumatoid arthritis. infection by schistosoma japonicum significantly reduced the severity and the incidence of experimental autoimmune collagen-induced arthritis. however, this beneficial effect can only be provided by a pre-established acute stage of infection but not by a pre-established early stage of the infection. the protection against collagen-induced arthritis correlated with reduced levels of anti-collagen ii igg, especially the subclass of igg2a. moreover, in protected mice increased levels of il- were present at the time of collagen ii injection together with sustained higher il- levels during the course of arthritis development. in contrast, in unprotected mice minimal levels of il- were present at the initial stage of collagen ii challenge together with lack of il- induction following schistosoma japonicum infection.  the protective effect against collagen-induced arthritis provided by schistosoma japonicum infection is infection stage-dependent. furthermore, the ability of schistosomiasis to negatively regulate the onset of collagen-induced arthritis is associated with a dominant as well as long-lasting th response at the initiation and development of autoimmune joint and systemic inflammation. 