Cysteine protease inhibitor of Schistosoma japonicum - A parasite-derived negative immunoregulatory factor.

Studies have shown that cysteine protease inhibitors from some parasites have immunosuppressive effects on the host. We previously have cloned a novel cysteine protease inhibitor from Schistosoma japonicum and purified its recombinant version (protein named rSj-C). Its possible inhibitory effect on the host immune response has not been described.This study shows that rSj-C inhibits lysosomal cysteine protease of murine dendritic cells (DCs). After DCs were incubated with rSj-C and then with soluble adult worm antigen (AWA) of S. japonicum, the mean fluorescence intensity of MHC class II antigens on the surface of DCs decreased significantly by flow cytometry. These results indirectly proved that rSj-C can suppress exogenous-antigen presentation by DCs. The flow cytometric assay revealed that in comparison with control groups, the proportion of CD4+CD25+Foxp3+ T cells among CD4+CD25+ T cells of Schistosom-infected mice increased significantly 8 weeks after the infected mice were injected with rSj-C (p Ë‚ 0.05). Additionally, the expression levels of cytokines IL-4 and TGF-ß produced by T cells increased significantly as compared with these levels in the normal group (p Ë‚ 0.05). These results clearly show that the cysteine protease inhibitor from S. japonicum is a new parasite-derived immunosuppressive factor.

iNOS is essential to maintain a protective Th1/Th2 response and the production of cytokines/chemokines against Schistosoma japonicum infection in rats.

Humans and a wide range of mammals are generally susceptible to Schistosoma infection, while some rodents such as Rattus rats and Microtus spp are not. We previously demonstrated that inherent high expression levels of nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), plays an important role in blocking the growth and development of Schistosoma japonicum in wild-type rats. However, the potential regulatory effects of NO on the immune system and immune response to S. japonicum infection in rats are still unknown. In this study, we used iNOS-knockout (KO) rats to determine the role of iNOS-derived NO in the immune system and immunopathological responses to S. japonicum infection in rats. Our data showed that iNOS deficiency led to weakened immune activity against S. japonicum infection. This was characterized by the impaired T cell responses and a significant decrease in S. japonicum-elicited Th2/Th1 responses and cytokine and chemokine-producing capability in the infected iNOS-KO rats. Unlike iNOS-KO mice, Th1-associated cytokines were also decreased in the absence of iNOS in rats. In addition, a profile of pro-inflammatory and pro-fibrogenic cytokines was detected in serum associated with iNOS deficiency. The alterations in immune responses and cytokine patterns were correlated with a slower clearance of parasites, exacerbated granuloma formation, and fibrosis following S. japonicum infection in iNOS-KO rats. Furthermore, we have provided direct evidence that high levels of NO in rats can promote the development of pulmonary fibrosis induced by egg antigens of S. japonicum, but not inflammation, which was negatively correlated with the expression of TGF-ß3. These studies are the first description of the immunological and pathological profiles in iNOS-KO rats infected with S. japonicum and demonstrate key differences between the responses found in mice. Our results significantly enhance our understanding of the immunoregulatory effects of NO on defensive and immunopathological responses in rats and the broader nature of resistance to pathogens such as S. japonicum.

Soluble egg antigen of Schistosoma japonicum induces pyroptosis in hepatic stellate cells by modulating ROS production.

BACKGROUND: Inflammation-induced dysfunction of hepatic stellate cells (HSCs) is involved in schistosomiasis-associated liver fibrosis, and soluble egg antigen (SEA) is a crucial pathogen-associated molecular pattern associated with liver injury in schistosomiasis. In addition, numerous studies have shown that caspase-1-mediated pyroptosis participates in the development of multiple inflammation-related diseases. However, whether pyroptotic cell death of HSCs is involved in SEA-mediated liver damage is not well understood. METHODS: Primary cultured HSCs and Schistosoma japonicum-infected mouse liver tissue were analysed for histological changes and caspase-1 activation, and the role of pyroptosis in the mechanisms underlying SEA-induced HSC death was investigated. Accumulation of reactive oxygen species (ROS) in infected livers and SEA-stimulated HSCs was measured by flow cytometry and immunofluorescence. RESULTS: Caspase-1 activity was elevated in both liver tissues and HSCs of S. japonicum-infected mice. Furthermore, SEA stimulation increased the proportion of pyroptotic HSCs, as shown by lactate dehydrogenase (LDH) release assays and by flow cytometric analysis of propidium iodide (PI) and caspase-1 double staining in cells. In addition, ROS generation was elevated in infected liver tissues and SEA-stimulated HSCs, and ROS inhibition downregulated SEA-induced caspase-1 activation and pyroptosis in HSCs. CONCLUSIONS: Our present study demonstrates that pyroptotic cell death in HSCs induced by SEA via ROS-mediated caspase-1 activation may serve as a significant mechanism to initiate the inflammatory response and thereby exacerbate liver injury during S. japonicum infection.

Study on histopathology, ultrasonography and some special serum enzymes and collagens for 38 advanced patients of schistosomiasis japonica.

Thirty-eight cases clinically diagnosed as advanced schistosomiaisis were subject to splenectomy in Dongzhi County Special Hospital for Schistosomiasis because of portal hypertension, splenomegaly and/or hypersplenism. Liver biopsy was undertaken in all cases during surgical intervention. Before operation, ultrasonography on the liver and spleen was carried out. Also done was biochemical assay on several indices related to liver damage and fibrosis. Among the 38 cases, 24 were diagnosed as schistosomiasis by the finding of eggs in feces, 13 were diagnosed by positive serological test with IHA or COPT, and only in one case, the diagnosis of schistosomiasis was doubtful before operation. However, the eggs were found in the liver section upon histological examination. All the 38 cases had symptoms and signs of portal hypertension and most of them had general symptoms. Histories of hematemesis and melena were recorded in three cases. The causes of hospitalization were mainly splenomegaly and abdominal distension, and two were suffering from upper gastrointestinal bleeding. Upon histopathological examination, schistosome eggs were found in 33 out of 38 cases. Advanced schistosomaiasis was shown in 18 cases and schistosomiasis associated with hepatitis or cirrhosis was seen in other 20 patients. The main pathological changes were egg granulomas with different degrees of fibrosis and some differences in the pathological changes between schistosomal liver fibrosis (SLF) and mixed liver cirrhosis (both schistosome and hepatitis in origin) were seen. Compared with normal ultrasonography, in all the 38 cases, the length of the left and right liver, and the spleen, and the thickness of the left liver, the width of portal trunk, were all out of normal ranges. The differences between the patients and normal records were significant. However, there were no statistically significant differences in terms of above-mentioned indices as well as liver parenchyma changes on ultrasound between advanced schistosomaiasis and schistosomiasis complicated with hepatitis or cirrhosis (all P>0.5). According to WHO classification criteria on ultrasonography for schistosomiasis, among 20 cases combined with hepatitis or cirrhosis, 11 cases fell in Grade II, and nine cases in Grade III hepatic fibrosis, whereas among 18 cases with schistosomiasis fibrosis, 12 and six were in Grade II and III, respectively. The mean value of serum MAO, PIIIP, IVC and HA in the 38 cases were all significantly higher than normal range. However, no significant differences (all P>0.1) were seen between advanced schistosomiasis and those complicated with hepatitis or cirrhosis in terms of the levels of the four indices. The results showed that ultrasonography has its importance in the diagnosis and evaluation of liver fibrosis. However, in differentiation of the two types of liver damage, ultrasound does not provide important information. Histopathological examination, on the other hand, can provide useful information to identify the hepatic diseases.

Neuronal nitric oxide synthase activity is increased during granulomatous inflammation in the colon and caecum of pigs infected with Schistosoma japonicum.

Neuronal nitric oxide is a non-adrenergic non-cholinergic neurotransmitter in the enteric nervous system and plays a role in a variety of enteropathies including Crohn's and Chagas' diseases, ulcerative colitis, diabetes, atrophy and hypertrophy. The content of neuronal nitric oxide synthase (nNOS) in the colon and the caecum from pigs infected with Schistosoma japonicum was studied using immunohistochemical and histochemical staining for nNOS and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase), respectively. In the infected pigs, lightly, moderately and less severely inflamed tissues showed increased nNOS and NADPH-diaphorase activities in nerve cell bodies and nerve fibres in the enteric plexuses compared to control pigs. There was a significant increase in the nerve cell body density of nNOS immunoreactive nerve cell bodies in the inner submucous plexus, outer submucous plexus and in the myenteric plexus. More intensely stained nerve cell bodies and varicosities were observed in tissue from prenatally infected and prenatally infected, postnatally re-infected pigs compared to postnatally infected pigs. However, the latter showed the highest numerical density of nNOS immunoreactive nerve cell bodies. Marked increases were seen in the inner submucous plexus followed by myenteric plexus, inner circular muscle, outer submucous plexus and mucous plexus. However, in very severe inflamed tissues, the number and staining intensity of nerve cell bodies and nerve fibre varicosities were reduced in plexuses located in the lesions with the inner submucous and mucous plexuses being the most affected. There was no staining in the nervous tissue within the eosinophilic cell abscesses and productive granulomas. The apparent alterations in the activities of enzymes responsible for the generation of nitric oxide (NO) show possible alterations in the NO mediated non-adrenergic non-cholinergic reflexes in the enteric nervous tissue. These alterations might contribute to impaired intestinal motility and absorption, and other pathophysiological conditions seen during S. japonicum infections.

[Effect of Schistosoma japonicum infection on liver drug-metabolizing enzymes of mice].

In this paper, the effect of Schistosoma japonicum infection on liver drug-metabolizing enzymes of mice was studied. The prolongation of hypnosis duration of sodium pentobarbital in vivo and the inhibition of liver aniline hydroxylase (AH), aminopyrine N-demethylase (APD) as well as cytochrome P-450 (Cyt P-450) in vitro were observed in mice infected with 20, 40 and 60 cercariae of S. japonicum after 6 weeks. Meanwhile, with 30 cercariae infection, no significant inhibition of the activity of AH, APD and Cyt P-450 was observed in mice 4 weeks after infection, but significant inhibition of these enzymes was shown by 8 weeks. As compared with control, the activity of AH, APD and Cyt P-450 decreased to 32.6%, 13.7% and 7.7% at 8 weeks. The results indicate that infection of S. japonicum can inhibit the liver drug-metabolizing capacity of mice, and the degree of inhibition depends on the infectivity and the duration of infection.

[Effect of artemether on enzymes involved in carbohydrate metabolism of Schistosoma japonicum].

OBJECTIVE: To study the effect of artemether on several enzymes involved in carbohydrate metabolism of Schistosoma japonicum. METHODS: Mice infected with Schistosoma japonicum cercariae for 4-5 weeks were administered intragastrically with artemether 300 mg/kg and killed 24-72 h after medication. The supernatant fluids of female and male worm homogenates were prepared for determining 9 essential enzymes of carbohydrate metabolism by using horizontal starchgel electrophoresis. RESULTS: The activities of 8 out of 9 enzymes (i.e. hexokinase, aldolase, glucosephophate isomerase, malate dehydrogenase, malic enzyme, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and mannose-6-phosphate isomerase) in female worms from artemether-treated mice were obviously inhibited 24-72 h after treatment. In male worms, only aldolase, mannose-6-phosphate isomerase and glucose 6-phosphate dehydrogenase were slightly inhibited. CONCLUSION: Artemether displayed apparent effects on the carbohydrate metabolism of female schistosomes.

[Expression of matrix metalloproteinase 2 and 9 in liver tissue of patients with schistosomiasis japonica].

OBJECTIVE: To explore the change of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9 (MMP-9) expression in liver tissue of the patients with schistosomiasis japonica. METHODS: Liver biopsy materials were examined pathomorphologically in 26 patients with schistosomiasis in advanced stage and 5 cases without the disease as control. The expression of MMP-2, MMP-9 and C-IV were studied by immunohistochemistry, and result was analyzed by picture quantitative analysis technique. RESULTS: Immnoreactive MMP-2 was mainly expressed in the plasma, the membrane of hepatocytes and hepatic sinusoids, it was also found in myofibroblast cells and endothelial cells of blood vessel. Immnoreactive MMP-9 was observed in hepatic stellate cells, sinusoids and myofibroblast cells, sometimes it was also seen in the plasma of hepatocytes and epithelial cells of bile duct. The expression of MMP-2, MMP-9 and C-IV was significantly stronger in patients with advanced schistosomiasis than that of the control(P < 0.05). The expression of MMP-2 was relevant to the inflammation grade and stage of liver fibrosis(P < 0.01). The expression of MMP-9 did not show significant change(P > 0.05) and the expression of C-IV was consistent with that of MMP-2. CONCLUSION: The study indicates that increased expression of MMP-2 participated in the initiation and development process of liver fibrosis, MMP-9 was related to the initiation of liver fibrosis and pathological change existed in hepatic sinusoids in advanced schistosomiasis japonica.

Vitamin E reduces hepatic fibrosis in mice with Schistosoma japonicum infection.

To investigate whether vitamin E protects against hepatic fibrosis in mice with Schistosoma japonicum infection, 24 pathogen-free Kunming mice were selected and randomly divided into four groups: control (uninfected, untreated), model (infected, untreated), low-dose intervention (infected, vitamin E-treated, 30 mg/g bodyweight/day) and high-dose intervention (infected, vitamin E-treated, 60 mg/g bodyweight/day). Mice were infected with Schistosoma japonicum by inoculating abdominal skin with snail hosts. The activities of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were detected in hepatic tissue by colorimetry. The expression levels of laminin (LN), hyaluronic acid (HA), procollagen type Ⅲ (PC-III) and type Ⅳ collagen (IV-C) were detected in the serum by radioimmunoassay. Finally, areas and numbers of granulomas were assessed through histopathology 42 days following treatment. The results revealed that mean areas of granulomas were smaller in the low- and high-dose intervention groups compared to those in the model group. Furthermore, the higher dose of vitamin E resulted in smaller granulomas than the low dose. The levels of LN, HA, PC-III and IV-C in the serum were lower following vitamin E treatment than in the model group. By contrast, activity of SOD, GPx and CAT in hepatic tissue was higher following vitamin E treatment compared to the model group. The activity of MDA was lower in hepatic tissue following vitamin E treatment compared to the model group, but was higher compared to controls. In general, the higher dose of vitamin E affected measurements to a greater extent than the lower dose. In conclusion, vitamin E treatment may reduce the growth of granulomas, slowing the process of hepatic fibrosis, and this effect may be the result of the altered activity of the oxidation-reduction enzyme system.

Infection of Schistosomiasis japanicum is likely to enhance proliferation and migration of human breast cancer cells: mechanism of action of differential expression of MMP2 and MMP9.

OBJECTIVE: To study whether the infection of Schistosomiasis japanicum (S. japanicum) is related to enhanced proliferation and migration of cancer cells, and the molecular mechanism pertains to cancer cell metastasis in human host. METHODS: The gene of S. japanicum glutathione transferase (sjGST) cloned from S. japanicum was expressed, purified and applied in a series of assays to explore the effect of sjGST on proliferation and migration of MDA-MB-435S, and the expression of MMP2 and MMP9. Immunofluorescence assay for the binding of sjGST to MDA-MB-435S was also carried out. RESULTS: Results showed that sjGST enhanced proliferation and migration in human breast cancer cell MDA-MB-435S signifycantly at 50-200 nM, but did not enhance them in human lung cancer cell A549. Immunofluorescence assay for the binding of sjGST to MDA-MB-435S and A549 showed that GST was readily bound to the breast cancer cells, but showed almost no binding to human lung cancer cells. The assays for gelatinase activity showed that both MMP2 and MMP9 activities were increased significantly in the presence of sjGST (50-200 nM) in MDA-MB-435S, but they were not significant in A549. CONCLUSIONS: Our current results show strongly that S. japanicum GST binds to MDA-MB-435S probably via its receptor, and enhances proliferation and migration of the cancer cells by up-regulatory expression of MMP2 and MMP9.

Effect of artemether on glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and pyruvate kinase of Schistosoma japonicum harbored in mice.

AIM: To study the effect of artemether (Art) on glyceraldehyde-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and pyruvate kinase (PK) of S japanicum. METHODS: Mice infected with schistosome cercariae for 32-38 d were treated ig with Art 100-300 mg.kg-1 and killed 24-72 h after medication for collection of schistosomes. The activities of GAPDH, PGK, and PK of the worms were determined by measuring the formation of NADH or consumption of NAD. The lactate content of the worms was also measured. RESULTS: After the infected mice were treated ig with Art 300 mg.kg-1 for 24 h, the inhibition rates of GAPDH were 13% (Male) and 21% (Female), and 48 h later the inhibition rates of the enzyme were 6% (Male) and 28% (Female). When Art 300 mg.kg-1 was given to infected mice for 24 h and 48 h, the inhibition rates of PGK were 60% (Male) and 48% (Female) as well as 75% (Male) and 62% (Female), respectively. Similar results were seen in PK activity. At 72 h after treatment the reduction rate of lactate content in Female worm was 72%, while that of Male was 48%. CONCLUSION: In the glycolytic pathway of both Male and Female schistosomes, PGK and PK activities were inhibited by Art. The GAPDH activity of Female worms was also susceptible to Art, While that of Male worms showed only temporary inhibition after treatment with Art. The Art reduced lactate content more in Female than in Male worms.

Effect of nitric oxide synthase inhibition on Schistosoma japonicum egg-induced granuloma formation in the mouse liver.

Nitric oxide (NO) plays diverse roles in a variety of pathological processes. We investigated the role of NO in Schistosoma japonicum egg-induced granuloma formation in a mouse hepatic model. Immunohistological analysis revealed that there is the most intense and extensive inducible nitric oxide (iNOS) expression 2 weeks after egg implantation, and thereafter it decreased considerably with time. Treatment with nitric oxide synthase inhibitors, NIL (L-N6- (iminoethyl)-lysine) or N(omega)-nitro-L-arginine methyl ester (L-NAME), resulted in two different types of unusual granulomas at 2 weeks. One type showed suppressed fibrosis, while another showed foreign body-type multinuclear cell formation which frequently appeared particularly when 50 microg/ml NIL was given. At 3 weeks following treatment, fibrotic granulomas with scanty peripheral cellularity was obvious. However, there were no apparent changes after this period (at 4 weeks). Cytokine analysis in NIL-treated mice showed a significant increase of IL-4 and IL-13 production at 2 weeks. These findings indicated that nitric oxide contributes to granuloma development during the early stages, probably through the regulation of Th2 cytokine production.

Infection induces antibodies against the cercarial secretions, but not against the cercarial elastases of Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum and Trichobilharzia ocellata.

Cercarial secretions from different species of the parasite Schistosoma and from Trichobilharzia ocellata contain a proteolytic activity, cercarial elastase, which was demonstrated by a 30 kDa band in gelatin gels. Sera of patients infected with Schistosoma mansoni, Schistosoma haematobium or Schistosoma japonicum contain immunoglobulin G which react in ELISA with cercarial secretions from all schistosomes and cross-react among the different parasite species. In Western blots, however, infection sera from patients, as well as heavily infected mice or rabbits, did not react with a 30-kDa protein. Moreover, when sections from infected snails (Biomphalaria, Bulinus and Lymnaea) were analysed by immunofluorescence using the same infection sera, only the tegument of the developing cercariae was recognized, but not the acetabular glands. In contrast, when antisera against purified cercarial elastase from either S. mansoni or S. haematobium were tested with sections of infected Biomphalaria or Bulinus, fluorescence was strong in the preacetabular glands of the cercariae of either species, but undetectable with the tegument. Cross-reactivity of both antisera extended to T. ocellata-infected Lymnaea, but not to S. japonicum-infected Oncomelania. In conclusion, although immunization with purified cercarial elastase results in antibody production, the enzyme does not induce an apparent antibody response following natural infection.

Schistosoma japonicum: effect of artemether on glutathione S-transferase and superoxide dismutase.

Glutathione S-transferase (GST) and superoxide dismutase (SOD) are major antioxidant enzymes of schistosomes that are involved in detoxification processes. To study the effect of artemether on these enzymes, mice infected with adult Schistosoma japonicum, were treated with artemether either at a subcurative (100 mg/kg) or a curative dose (300 mg/kg). Schistosomes were recovered 24-72 h post-treatment separated by sex and used for GST and SOD activity measurements. Female worms showed consistently higher GST inhibitions than males. For instance, 24 h after administration of 100 mg/kg artemether, GST activities of female worms were inhibited by 23.3%, as compared to 12.7% in males. Both activities were significantly lower when compared to worms recovered from untreated mice. Slightly higher inhibitions were observed at the higher dose of artemether, which gradually increased to levels of 52.5-55.1%, 72 h post-treatment. GST inhibitions could be reversed by application of 1,4-dithiothreitol at a concentration of 10 mmol/L. Adding L-cysteine also reduced GST inhibitions, but in female worms, GST activities remained significantly higher than in worms from untreated animals. Administration of 300 mg/kg artemether resulted in significant reductions of SOD activities in both sexes. In conclusion, these results suggest that the inhibition of GST and, to a lesser extent also SOD enzymes, could lead to increased schistosome susceptibility to oxidant attacks and might be linked with the antischistosomal action of artemether.