Therapeutic potential of fucoidan in the reduction of hepatic pathology in murine schistosomiasis japonica.

BACKGROUND: Hepatic granuloma formation and fibrosis as the consequence of tissue entrapped eggs produced by female schistosomes characterize the pathology of Schistosoma japonicum infection. It has been proposed that fucoidan, a sulfated polysaccharide existing naturally in brown seaweed Fucus vesiculosus, plays a diversified role to perform immunomodulatory activities. However, whether fucoidan functions in the host hepatic pathology is unknown and identifying the potential mechanism that is responsible for hepatic improvement is still necessary. METHODS: We evaluated the hepatic pathology from S. japonicum-infected mice after treatment with fucoidan. qRT-PCR and immunofluorescence were used to detect the pro- or anti-inflammatory factors and the phosphorylated p65 in the livers. In addition, flow cytometry was also performed to investigate the T cell subsets in the S. japonicum-infected mice after treatment with fucoidan, and functional molecules relatively specific to Treg cells were detected in vitro. Furthermore, macrophages were treated with fucoidan in vitro and to detect the inflammatory cytokines. RESULTS: Treatment with fucoidan significantly reduced the hepatic granuloma size and fibrosis response during S. japonicum infection. The attenuated phospho-p65 protein levels and the mRNA levels of pro-inflammatory cytokines (IL-6, IL-12 and TNF-α) were observed in the livers from fucoidan-treated S. japonicum-infected mice; however, the mRNA levels of anti-inflammatory cytokines (IL-4 and IL-13) were increased. In addition, the infiltration of Treg cells was significantly enhanced both in the livers and spleens from fucoidan-treated S. japonicum-infected mice. Consistent with this, the mRNA levels of IL-10 and TGF-ß were dramatically increased in the livers from S. japonicum-infected mice after fucoidan treatment. Furthermore, in vitro stimulated splenocytes with fucoidan resulted in increasing Treg cells in splenocytes as well as the functional expression of CC chemokine receptor type 4 (CCR4) and CXC chemokine receptor type 5 (CXCR5) in Treg cells. Additionally, fucoidan promoted the mRNA levels of IL-4 and IL-13 in macrophages. CONCLUSIONS: These findings suggest an important role of natural fucoidan in reducing hepatic pathology in the progress of S. japonicum infection with a stronger Treg response, which may reveal a new potential therapeutic strategy for hepatic disease caused by parasitic chronic infection.

Process Development of Sj-p80: A Low-Cost Transmission-Blocking Veterinary Vaccine for Asiatic Schistosomiasis.

Asiatic schistosomiasis caused by Schistosoma japonicum is a neglected tropical disease resulting in significant morbidity to both humans and animals - particularly bovines - in endemic areas. Infection with this parasite leads to less healthy herds, causing problems in communities which rely on bovines for farming, milk and meat production. Additionally, excretion of parasite eggs in feces perpetuates the life cycle and can lead to human infection. We endeavored to develop a minimally purified, inexpensive, and effective vaccine based on the 80 kDa large subunit of the calcium activated neutral protease (calpain) from S. japonicum (Sj-p80). Here we describe the production of veterinary vaccine-grade Sj-p80 at four levels of purity and demonstrate in a pilot study that minimally purified antigen provides protection against infection in mice when paired with a low-cost veterinary adjuvant, Montanide™ ISA61 VG. Preliminary data demonstrate that the vaccine is immunogenic with robust antibody titers following immunization, and vaccination resulted in a reduction of parasite eggs being deposited in the liver (23.4-51.4%) and intestines (1.9-55.1%) depending on antigen purity as well as reducing the ability of these eggs to hatch into miracidia by up to 31.6%. We therefore present Sj-p80 as a candidate vaccine antigen for Asiatic schistosomiasis which is now primed for continued development and testing in bovines in endemic areas. A successful bovine vaccine could play a major role in reducing pathogen transmission to humans by interrupting the parasitic life cycle and improving quality of life for people living in endemic countries.

Sorafenib and praziquantel synergistically attenuate Schistosoma japonicum-induced liver fibrosis in mice.

Liver fibrosis is an important process that occurs in most types of chronic liver diseases and often results in the end stage of liver diseases, such as cirrhosis, portal hypertension, and hepatocellular carcinoma. Sorafenib, a multiple tyrosine kinase inhibitor, has been shown to inhibit liver fibrosis in multiple experimental fibrosis mouse and rat models. The aim of this study was to test the therapeutic effect of sorafenib on liver fibrosis induced by infection with a parasite, Schistosoma japonicum, in mice. Mice were percutaneously infected through the abdomen with Schistosoma cercariae to develop a schistosomula liver fibrosis model. Eight weeks after infection, infected mice were treated with the anti-parasitic agent praziquantel for 2 days and sorafenib for 2 weeks. Hepatic histopathological changes were assessed using hematoxylin and eosin (HE) and Masson's trichome staining. The hepatic expression levels of collagen I, collagen III, alpha-smooth muscle actin (α-SMA), platelet-derived growth factor (PDGF), and PDGF receptor-beta (PDGFR-ß) were analyzed by immunohistochemistry and western blot. Praziquantel administration alone but not sorafenib reduced liver fibrosis, and the combination of praziquantel and sorafenib significantly attenuated liver fibrosis in S. japonicum-infected mice. Moreover, sorafenib plus praziquantel markedly decreased the hepatic deposition of collagen and expression of fibrogenic genes in these mice. In conclusion, the use of sorafenib following praziquantel treatment may represent a potential therapeutic strategy for liver fibrosis induced by S. japonicum in patients.

Transdermal praziquantel administration attenuates hepatic granulomatosis in mice infected with Schistosoma japonicum.

Liver granuloma is a major pathogenic factor responsible for schistosomiasis, and no effective drugs or therapy methods to treat it have been found so far. Praziquantel (PZQ) has shown some anti-schistosomal effect, but little information is available about the effect of PZQ-prolonged administration on granuloma formation around schistosome eggs. Herein, we investigated the effect of PZQ on hepatic granuloma formation by treating the mice infected with Schistosoma japonicum using a long-term PZQ transdermal delivery. The results showed that the mean area of granulomas in the group treated with PZQ transdermal agent was (175.47 ± 116.73) × 10(3) µm(2) at the 49th day postinfection and (71.96 ± 45.99) × 10(3) µm(2) at the 56th day, while that in the control group was (304.51 ± 140.55) × 10(3) µm(2) and (526.44 ± 268.06) × 10(3) µm(2), respectively. The content of hydroxyproline in the livers of mice approached to the normal level on the 154th day in the treatment group, but it continued to increase from the 28th day to the 154th day after infection in the control group and nontreatment group. The ALT activity in serum of mice in the treatment group was also significantly lower than that in the control group (*P ≤ 0.05). Our results suggest that the long-term PZQ transdermal delivery is critical in the therapeutic approach to control the progress of hepatic schistosomiasis induced by egg granulomas.

The molecular characterization and RNAi silencing of SjZFP1 in Schistosoma japonicum.

During development, Schistosoma japonicum undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Proteins containing zinc finger motifs usually play an important role in DNA recognition, RNA packaging, and transcriptional activation. In our current study, we cloned the open reading frame (ORF) of SjZFP1 of S. japonicum, which encodes a zinc finger protein. We analyzed the complementary DNA (cDNA) sequence of SjZFP1 and examined the expression of SjZFP1 messenger RNA (mRNA) at various developmental stages. We also tested the effects of RNA interference (RNAi) silencing on worm burden, spawning, and egg hatching. The ORF in the SjZFP1 cDNA was 1017 bp in length and was predicted to encode a 338-aa protein with a molecular mass of approximately 38.5 kDa and theoretical isoelectric point (pI) of 7.08. Several conserved regions, including a B-box-type zinc-binding domain, two bipartite nuclear localization signal domains, a paired amphipathic helix repeat, and overlapping RING and PHD finger domains, were identified in the predicted amino acid sequence of SjZFP1. Using real-time PCR, we showed that the SjZFP1 mRNA was expressed across all of the developmental stages of the parasite and that the level of transcription was highest in the cercariae, eggs, schistosomula, and mature adult worms. The level of SjZFP1 mRNA expression in cultured schistosomula treated with one of two SjZFP1-specific small interfering RNAs (siRNAs; AY770 and AY546) was reduced by over 80 %, compared with that in the controls. In RNAi experiments in BALB/c mice, the level of SjZFP1 mRNA increased significantly when the mice were treated with the same SjZFP1-specific siRNAs during the early stages of infection. By contrast, the level of SjZFP1 mRNA decreased significantly when the mice were treated with the SjZFP1-specific siRNAs during the middle to late stages of infection. In four independent experiments, fewer worms were recovered from mice treated with the SjZFP1-specific siRNAs, compared with the number of worms recovered from the control mice. Both the average number and hatching rates of liver eggs recovered from mice treated with the SjZFP1-specific siRNAs during the middle to late stages of infection were significantly lower than those of the liver eggs recovered from the control mice. Our results suggest that the SjZFP1 gene might be important for parasite development, spawning in the vertebrate host, and egg hatching.

A boswellic acid-containing extract ameliorates schistosomiasis liver granuloma and fibrosis through regulating NF-κB signaling in mice.

Boswellic acid (BA)-containing extracts such as BSE have anti-inflammatory and immunomodulatory activity. In chronic schistosomiasis, the hepatic granuloma and fibrosis induced by egg deposition in the liver is the most serious pathological manifestations. However, little is known regarding the role of BAs in Schistosoma japonicum (S. japonicum) egg-induced liver granuloma and fibrosis. In order to investigate the effect of a water-soluble complex preparation of BSE, BSE-CD, on S. japonicum egg-induced liver pathology, liver granuloma and fibrosis were induced by infecting C57BL/6 mice with 18-22 cercariae of S. japonicum. S. japonicum cercariae infected mice were injected with BSE-CD at the onset of egg granuloma formation (early phase BSE-CD treatment after 4 weeks infection) or after the formation of liver fibrosis (late phase BSE-CD treatment after 7 weeks infection). Our data show that treatment of infected mice with BSE-CD significantly reduced both the extent of hepatic granuloma and fibrosis. Consistent with an inhibition of NF-κB signaling as evidenced by reduced IκB kinase (IKK) activation, the mRNA expression of VEGF (vascular endothelial growth factor, VEGF), TNF-α (tumor necrosis factor-alpha TNF-α) and MCP-1 (monocyte chemotactic protein 1, MCP-1) was decreased. Moreover, immunohistochemical analysis (IHC) revealed that the content of α-SMA in liver tissue of BSE-CD treated mice was dramatically decreased. Our findings suggest that BSE-CD treatment attenuates S. japonicum egg-induced hepatic granulomas and fibrosis, at least partly due to reduced NF-κB signaling and the subsequently decreased expression of VEGF, TNF-α, and MCP-1. Suppression of the activation of hepatic stellate cells (HSC) may also be involved in the therapeutic efficacy of BSE-CD.

PAMAM-Lys, a novel vaccine delivery vector, enhances the protective effects of the SjC23 DNA vaccine against Schistosoma japonicum infection.

BACKGROUND: Schistosomiasis japonica remains a major public-health concern in China. Praziquantel-based chemotherapy effectively reduces both infections and intensity; however, it can not prevent re-infection. Furthermore, there is an increasing concern about praziquantel resistance following long-term repeated use of the drug in endemic areas. Therefore, development of a schistosomiasis vaccine, as a strategy to prevent and control schistosomiasis japonica, has been given high priority. The present study was conducted to develop PAMAM dendrimers as a novel vaccine delivery vector for a schistosomiasis japonica DNA vaccine and evaluate its ability to enhance protective effects against Schistosoma japonicum infection. METHODOLOGY/PRINCIPAL FINDINGS: Lysine was used to modify 4.0G PAMAM, and the modified product PAMAM-Lys was synthesized. PAMAM-Lys showed both high transfection and low cytotocity for gene delivery in vitro. DNA vaccines combined with PAMAM-Lys produced higher level of protection compare with naked DNA vaccines against S. japonicum infection in a mouse model. Futhermore,antibodies from mice immunized with PAMAM-Lys combined DNA vaccines were significantly higher than those of mice immunized with the naked DNA vaccines. The PAMAM-Lys vector elicited a predominantly IgG2a antibody response and a tremendously increase in the production of IL-2 and IFN-γ. CONCLUSION/SIGNIFICANCE: Lysine-modified PAMAM-Lys is an excellent vector. PAMAM-Lys may enhance the immunoreactivity of DNA vaccine and increase the protective effect of the SjC23 DNA vaccine against S. japonicum infection.

A boswellic acid-containing extract attenuates hepatic granuloma in C57BL/6 mice infected with Schistosoma japonicum.

Granuloma formation has been shown to be induced and elicited by schistosome egg antigens, and it finally develops into fibrosis in intestine and the liver. Hepatic fibrosis is the main cause of increased morbidity and mortality in humans infected with schistosomes. Boswellic acid (BA)-containing extracts such as extracts of the oleogum resin from Boswellia serrata (BSE) have anti-inflammatory and immunomodulatory activity. However, little is known about the role of such extracts in schistosome egg-induced granulomatous inflammation. In order to investigate the effect of a watersoluble cyclodextrin complex preparation of BSE (BSE-CD) on Schistosoma japonicum (S. japonicum) egg-induced liver granuloma, mice infected with S. japonicum cercariae were injected with BSE-CD during egg granuloma formation. The data showed that BSE-CD significantly reduced the size of liver granuloma and levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST); however, BSE-CD treatment had no effect on worm load and egg burden. The data also showed that BSE-CD significantly decreased the expression of leukotriene B(4) (LTB(4)) and prostaglandin E(2) (PGE(2)), as well as the expression of matrix metallopeptidase 9 (MMP-9) in liver both on the mRNA and protein level. Thus, BSE-CD can significantly attenuate S. japonicum egg-induced hepatic granuloma, which may be partly dependent on the downregulation of some biochemical mediators.

Effect of Mirazid in Schistosoma japonicum-infected mice: parasitological and pathological assessment.

Conflicting reports are found in the literature about the antischistosomal efficacy of Mirazid (MZ), which is a special formulation of myrrh obtained from the stem of the plant Commiphora molmol. This initiated the present study to assess this drug for the first time in experimental schistosomiasis japonicum. Mice were divided into four groups: infected untreated control (I); infected treated with MZ, 500 mg/kg (II); infected treated with MZ, 250 mg/kg (III); and infected treated with praziquantel (PZQ), 200 mg/kg (IV). The drugs were given 7 weeks post-infection for five successive days. All animals were killed 3 weeks posttreatment. Results showed no signs of antibilharzial activity of MZ. Total worms, total tissue egg load, egg developmental stages, and granuloma area were not affected by any of the MZ treatment regimens as compared to the infected untreated group (P > 0.05 for all variables). These results were in contrast to those obtained in PZQ-treated animals in which 82.82 % total worm reduction, 94.62 % egg reduction, and 86.35 % granuloma area reduction were observed. Also, it significantly increased the percentage of dead ova and decreased the percentage of mature ova with complete absence of immature ones in comparison with the control group (P < 0.01 for all variables). In conclusion, the results of the current study raise serious doubts about the antischistosomal activity of MZ.

Cloning and characterization of a novel enzyme: tyrosine hydroxylase from Schistosoma japonicum.

Catecholamines, such as dopamine and noradrenaline, play important roles as neuromuscular transmitters and modulators in all parasitic helminthes, including Schistosoma japonicum. S. japonicum tyrosine hydroxylase (SjTH) was amplified by rapid amplification of cDNA ends polymerase chain reaction that shows strong homology to Schistosoma mansoni tyrosine hydroxylase, the enzyme that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. The SjTH transcripts encoded the protein of 463 amino acids and a predicted size of 54 kDa. Purified recombinant SjTH as an N-terminal histidine fusion protein expressed in Escherichia coli showed catalytic activity that was confirmed with (3)H tyrosine uptake. The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and similar sensitivity to be inhibited by high concentration of the substrate, tyrosine, as the mammalian enzyme. Also, purified SjTH showed characteristic inhibition by catecholamine products. The phosphorylated peptide from SjTH could interact with Sj14-3-3 signal protein. This evidence indicates that SjTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian hosts' enzyme, and its catalytic activity could be regulated by a phosphorylated or dephosphorylated form. This demonstration of SjTH further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously.

Research and development of antischistosomal drugs in the People's Republic of China a 60-year review.

A comprehensive 60-year review of antischistosomal drug discovery and development research in the People's Republic of China (P.R. China) is presented. In the 1950s, three antimonials were developed, which, compared to potassium antimony tartrate-the treatment of choice against schistosomiasis at the time-showed equal efficacy but lower toxicity when administered orally or intramuscularly. Activity of furapromidum against Schistosoma japonicum was reported in the early 1960s, and this drug became the first non-antimonial used in clinical treatment of schistosomiasis japonica. Subsequently, two additional nitrofuran derivatives (furadiamine and fuvinazole) were investigated in the laboratory and clinically. In the late 1960s, niridazole and amoscanate were developed by western scientists, which encouraged Chinese researchers to synthesise and further modify the chemical structures of these compounds. However, the modified compounds were less efficacious but similarly toxic, which impeded their further development. The advent of the safe, highly efficacious broad-spectrum antischistosomal drug praziquantel, in the mid-1970s, eventually resulted in a change of the global schistosomiasis control strategy from a multi-pronged transmission control approach to drug-based morbidity control. Numerous studies were carried out in P.R. China to further the understanding of efficacy, mechanism of action and safety of praziquantel against S. japonicum. Efforts have also been made to develop antischistosomal drugs from Chinese traditional medicine, and research with cucurbitin (an amino acid isolated from pumpkin seeds), among other products, showed interesting in vitro and in vivo activities against S. japonicum. In the 1990s, the artemisinins-originally reserved for malaria treatment-were developed as 'chemoprophylactic' agents against S. japonicum, which in turn led to a series of new developments of artemisinins and related compounds as broad-spectrum antischistosomal and anthelminthic therapies.

Paeoniflorin ameliorates schistosomiasis liver fibrosis through regulating IL-13 and its signalling molecules in mice.

Treatment of liver fibrosis associated with Schistosoma japonicum ova-induced granulomas remains a challenging proposition. Paeoniflorin (PAE, C23H28O11) has anti-inflammatory, anti-allergic, and immunoregulatory effects and it is commonly used in Chinese Herbal prescriptions to treat hepatic disorders. The present study was carried out to investigate the effects of PAE on hepatic fibrosis of mice infected with S. japonicum and to explore its possible mechanism. Upon pathological examination of PAE-treated mice, the size of egg granuloma, fibrosis scores, the concentration of IL-13 and hydroxyproline in liver were significantly reduced compared with the model mice. In the primary culture of hepatic stellate cells (HSCs), PAE inhibited IL-13-induced collagen synthesis. These results suggested that PAE might alleviate the hepatic granulomas and fibrosis caused by S. japonicum and the inhibitory effect of PAE on hepatic fibrosis might be associated with its ability to decrease the level of IL-13 and to interfere with the IL-13 signalling molecule in HSCs.

Expression profile, localization of an 8-kDa calcium-binding protein from Schistosoma japonicum (SjCa8), and vaccine potential of recombinant SjCa8 (rSjCa8) against infections in mice.

Researches on genes expressed in a cercarial stage-specific manner may help us understand the molecular events and functions during schistosome invasion of skin. A genomic clone encoding 8-kDa calcium-binding protein (SjCa8) specifically expressed in cercariae and skin-stage schistosomulum (transformed within 3 h) was obtained from cercariae. Recombinant protein was expressed in vector pET32a (+) and purified using a Ni-NTA purification system. The target protein SjCa8 was determined by matrix-assisted laser desorption/ionization time-of-flight (TOF)/TOF mass spectrometer after thrombin digestion and dialysis. Reverse transcriptase polymerase chain reaction and Western blot revealed SjCa8 can be detected in cercaria and skin-stage schistosomulum but not lung-stage schistosomulum, adult, or egg and was localized to head gland, penetration gland tubes, and penetration glands where Ca(2+) was abundant, and the cercarial tegument (but not tegument of tail) and body-tail junction. Furthermore, SjCa8 was interestingly detected in cercarial secretions. The characterization of SjCa8 indicated that it may undergo structural and physiological modifications, including repair of the surface membrane, changes in permeability that account for the loss of water tolerance, activities of calcium-depending enzymes, and immune signaling, etc. Furthermore, vaccination with rSjCa8 plus adjuvant induced protective effect with 50.39% worm reduction rate and significantly high hepatic and intestine egg reduction rates (54.16%, 50.63%, respectively), which is possibly mediated through an apparent induction of Th1-type immune response for strikingly high level of IgG2a and IgG2b developed in immunized C57BL/6 mice.

[Study on the effect of heluoshugan capsule on liver fibrosis induced by Schistosoma japonicum infection in mice].

OBJECTIVE: To investigate the effect of Chinese traditional medicine heluoshugan capsule on liver fibrosis induced by Schistosoma japonicum infection in mice. METHODS: Liver fibrosis in mice was established by Schistosoma japonicum infection in 6 weeks. Suspension of heluoshugan prepared with normal saline was given orally to the mice, 2 capsules for 20 mice daily for 8 weeks. The level of vascular endothelial growth factor (VEGF), focal adhesion kinase (FAK) and type I, III collagen in liver tissue were detected by immuno-histochemistry. RESULTS: The results showed that heluoshugan improved the pathological change of the liver tissue, decreased the level of type I, III collagen, especially type III collagen (P < 0.01). The level of VEGF and FAK expression was inhibited after the administration of heluoshugan, though the level usually increased in liver fibrosis due to the infection. CONCLUSIONS: The result suggests that heluoshugan capsule might have therapeutic effect on liver fibrosis induced by the infection of Schistosoma japonicum in mice, by inhibiting the activation of hepatic stellate cells and the pathological change of liver blood vessel.

Immunity to schistosomiasis in animals: an update.

The present paper reviews the available literature on the development of immunity to animal Schistosoma infections. The majority of the studies on animal schistosomiasis were performed in cattle and pigs and only Schistosoma mattheei, S. bovis and S. japonicum received particular attention, mainly because of their recognized veterinary significance or zoonotic aspect. Although it is an accepted fact that acquired resistance to Schistosoma is of major importance in the regulation of infection intensity in the field, almost nothing is yet known of either the nature of the antigens or of the immune mechanisms involved. The recent studies on immunity development focus in particular on the occurrence of maternal to foetal transfer of immunological substances related to animal Schistosoma infections and possible effects of these transfers on the immunity development of the foetus/newborn. Since congenital infections for Schistosoma species other than S. japonicum are extremely rare, the most plausible route for foetal contact is the transplacental or postnatal transfer of immunological substances. Prenatal transfers of specific antibodies and antigens via placental lesions and postnatal transfers via the colostrum were observed in cattle and pigs, and subsequent modifications of the immune response of the newborn were observed. Placental lesions induced by Schistosoma eggs could allow other pathogens to cross the placenta.

The effect of colostrum on pigs pre-natally or post-natally exposed to Schistosoma japonicum.

Pre-natal infection of Schistosoma japonicum in pigs may prove to be a useful model in shedding light on human pre-natal schistosomiasis. This study describes the effects of immune colostrum on worm burdens, tissue egg counts, liver pathology and crude worm or egg antigen-specific IgG and IgA responses, in groups of pigs pre-natally, pre-natally + post-natally or post-natally exposed to S. japonicum. Results suggest that pre-natal exposure and immune colostrum did not affect the establishment of a post-natal challenge infection. However, immune colostrum seemed to increase the levels of septal fibrosis in pre-natally exposed pigs. These findings indicate that further investigations will prove valuable, elucidating the influence of the parasitological and immunological status of the sow, on pre-natally exposed pigs, and on the ability of these pigs to develop resistance against S. japonicum later in life.

Research on calpain of Schistosoma japonicum as a vaccine candidate.

Vaccine development by the use of calpain of Schistosoma japonicum has been tried in our laboratory. We cloned cDNA encoding the heavy chain of S. japonicum calpain, and prepared recombinant molecule of a possible vaccine region of the heavy chain. When BALB/c mice were immunized with our recombinant calpain of S. japonicum with Freund's complete adjuvant, we observed significant reduction in worm burden (41.2% reduction, P<0.05), and also significant anti-fecundity effects. In this sense, calpain of S. japonicum seems to have infection control as well as anti-disease effects. Mechanisms of vaccine effects of calpain remain to be clarified, however, several effector mechanisms are suspected. In immunized mice, raised level of iNos expression was observed, while adhesion of peritoneal exudates cells were also observed in the presence of calpain-immunized sera, suggesting the possibilities of both cellular and humoral protective mechanisms. We examined tissue distribution of calpain in various developmental stages of S. japonicum. Strong signal was observed around excretory grand of cercariae, and they secreted calpain during their migratory movement tested in vitro. Together with the findings, calpain seems to induce larvicidal effects in the immunized mice. We observed time-course kinetics of antibody production against vaccine candidates in experimental S. japonicum infection in pigs. Although significant levels of antibody production were observed for paramyosin and GST, no significant antibody production was observed for calpain. This suggests that calpain is less immunogenic, and route of immunization and/or choice of adjuvant are important in future trials of calpain vaccine.

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

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