Jia-hui Lei

Effect of CD4 + CD25 + regulatory T cells on the immune evasion of Schistosoma japonicum

It has been known that parasites developed sophisticated strategies to escape from the host immune assault. More recently, one strategy to induce immune evasion involved CD4+CD25+ regulatory T cells (Tregs). Mice were infected with Schistosoma japonicum cercariae and then injected intraperitoneally with anti-CD25 monoclonal antibody (anti-CD25 mAb). The results showed that the percentages of CD4+CD25+ Tregs in mice were expanded by S. japonicum infection, and it could be partially blocked by anti-CD25 mAb. Worm burden in anti-CD25 mAb group (23.17 ± 6.94) was significantly lower than that in infected group (30.17 ± 5.85). The level of interferon gamma was increased with anti-CD25 mAb administration; meanwhile, lower concentration of interleukin 10 was observed in the same group. These results suggest that CD4+CD25+ Tregs contribute to the escape of S. japonicum from the host immune responses, while anti-CD25 mAb can partially block CD4+CD25+ Tregs and enhance the protective immunity to the parasite by Th1-type immune response.

Detection of circulating antigen in serum of mice infected with Schistosoma japonicum by immunomagnetic bead ELISA based on IgY

We developed a novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) for detection of circulating antigen (CA) in sera of mice infected with Schistosoma japonicum. The assay involved the use of chicken polyclonal antibodies IgY against soluble egg antigens (SEA) of S. japonicum as a capture antibody and anti-SEA mouse monoclonal antibody NP28-5B labeled horseradish peroxidase (HRP) as a detecting antibody. Two groups of BALB/c mice infected with S. japonicum cercariae were used: lightly infected mice (infected with 10 S. japonicum cercariae) and heavily infected mice (infected with 30 S. japonicum cercariae). The CA was detectable as early as 4 and 5 weeks after infection in the sera of heavily and lightly infected mice, respectively. The CA levels rose rapidly and reached a peak in 8 weeks after infection and then remained a plateau for at least another 6 weeks in both groups. Moreover, the effect of praziquantel on the CA levels was also investigated. The heavily infected mice were treated with praziquantel and the CA levels in sera increased dramatically in the first week post-treatment and then decreased to the control level by 6 weeks after treatment. The novel assay appears to be sensitive for detection of schistosomal antigenemia and valuable to judge the efficacy of chemotherapy in murine schistosomiasis.

Cimetidine enhances the protective effect of GST DNA vaccine against Schistosoma japonicum

Cimetidine (CIM), a histamine-2-receptor antagonist, has a long history of safe use in gastric acid-mediated gastrointestinal disorders. In this study, we used CIM, as an adjuvant, with pEGFP-Sj26 GST (the recombinant plasmid containing enhanced green fluorescent protein gene and the gene encoding 26 kDa glutathione S-transferase of Schistosoma japonicum) DNA vaccine to immunized mice and attempted to enhance the protective effect against S. japonicum. The results showed that the reduction rate of worm and egg burdens in the pEGFP-Sj26GST plus CIM group were 79.0% and 68.4%, respectively, significantly higher than that in pEGFP-Sj26GST alone group (27.0% and 22.5%, P<0.01). Compared with the pEGFP-Sj26GST alone group, mice immunized with pEGFP-Sj26GST plus CIM showed an elevated level of IFN-γ and IL-12 and a low level of IL-10 in splenocytes, while the levels of IL-4 and IL-5 showed no difference between the two groups. Our data also demonstrated that the percentage of CD4(+)CD25(+) regulatory T cells (Tregs) was significantly decreased in the spleens of mice immunized with pEGFP-Sj26GST plus CIM. All these findings suggest that CIM as a potential schistosome DNA vaccine adjuvant can enhance the protective effect of pEGFP-Sj26GST vaccine.

Chronic Schistosoma japonicum Infection Reduces Immune Response to Vaccine against Hepatitis B in Mice

Background Hepatitis B and schistosomiasis are most prevalent in Africa and Asia, and co-infections of both are frequent in these areas. The immunomodulation reported to be induced by schistosome infections might restrict immune control of hepatitis B virus (HBV) leading to more severe viral infection. Vaccination is the most effective measure to control and prevent HBV infection, but there is evidence for a reduced immune response to the vaccine in patients with chronic schistosomiasis japonica. Methodology/Principal Findings In this paper, we demonstrate in a mouse model that a chronic Schistosoma japonicum infection can inhibit the immune response to hepatitis B vaccine (HBV vaccine) and lead to lower production of anti-HBs antibodies, interferon-γ (IFN-γ) and interleukin-2 (IL-2). After deworming with Praziquantel (PZQ), the level of anti-HBs antibodies gradually increased and the Th2-biased profile slowly tapered. At 16 weeks after deworming, the levels of anti-HBs antibodies and Th1/Th2 cytokines returned to the normal levels. Conclusions/Significance The results suggest that the preexisting Th2-dominated immune profile in the host infected with the parasite may down–regulate levels of anti-HBs antibodies and Th1 cytokines. To improve the efficacy of HBV vaccination in schistosome infected humans it may be valuable to treat them with praziquantel (PZQ) some time prior to HBV vaccination.

Evaluation of an IgY-Based Immunomagnetic Enzyme-Linked Immunosorbent Assay System for Detection of Circulating Schistosoma japonicum Antigen in Serum Samples from Patients in China

We have developed a novel egg yolk antibody (IgY)-coated magnetic beads antigen-capture immunoassay for detection of a circulating antigen of Schistosoma japonicum in serum samples of patients in schistosomiasis-endemic areas of China. This IgY-based immunomagnetic bead enzyme-linked immunosorbent assay (IgY-IMB-ELISA) uses polyclonal IgY-coated magnetic beads as a capture antibody, and a monoclonal IgG as a detection antibody. The sensitivity of the magnetic immunoassay was 100% (40 of 40) in cases of acute infection and 91.5% (107 of 117) in chronic cases of schistosomiasis, and no positive reaction was found in 0 of 49 healthy persons. Cross-reactivity was 3.3% (1 of 33) with clonorchiasis and 0% (0 of 20) with paragonimiasis. There was a significant correlation between ELISA absorbance value and egg count (eggs per gram feces) and a correlation coefficient of 0.88 in a small sample of 14 patients. The results demonstrated that the IgY-IMB-ELISA is a sensitive and specific assay for detection of human schistosomiasis japonica.

Detection of Clonorchis sinensis Circulating Antigen in Sera from Chinese Patients by Immunomagnetic Bead ELISA Based on IgY

Background Clonorchiasis, caused by Clonorchis sinensis, is widely distributed in Southeast Asia including China. Clonorchiasis is included in control programs of neglected tropical diseases by World Health Organization (WHO) because it is one of the major health problems in most endemic areas. Diagnosis of clonorchiasis plays a key role in the control programs. However, so far, there is no satisfactory method for clonorchiasis because of low sensitivity, poor practicality and high false positivity of available diagnostic tools. Methodology/Principal Findings We developed an immunomagnetic bead enzyme-linked immunosorbent assay (ELISA) based on IgY (egg yolk immunoglobulin) against cysteine proteinase of C. sinensis for detection of circulating antigen in serum samples of patients infected with C. sinensis. The polyclonal IgY, coated with magnetic beads, was used as a capture antibody and a monoclonal IgG labeled with horseradish peroxidase as a detection antibody in the IgY-based immunomagnetic bead ELISA system (IgY-IMB-ELISA). The results showed that the sensitivity of IgY-IMB-ELISA was 93.3% (14 of 15) in cases of heavy infection (5000 to 9999 eggs per gram feces, i.e, EPG 5000–9999), 86.7% (13 of 15) in cases of moderate infection (EPG 1000–4999) and 75.0% (9 of 12) in cases of light infection (EPG <1000) of clonorchiasis. Together 36 of total 42 (85.7%) serum samples of human clonorchiasis gave a positive reaction. There was a significant correlation between ELISA optical density and egg counts (EPG) with a correlation coefficient of 0.83 in total 42 patients. There were no positive results in patients with trichinosis (n = 10) or cysticercosis (n = 10). Cross-reactivity was 6.7% (2 of 30) with schistosomiasis japonica and 10.0% (3 of 30) with paragonimiasis, respectively. No positive reaction was found in 20 healthy persons. Conclusions Our findings suggest that IgY-IMB-ELISA appears to be a sensitive and specific assay for detection of circulating antigen in human clonorchiasis.

Application of an immunomagnetic bead ELISA based on IgY for detection of circulating antigen in urine of mice infected with Schistosoma japonicum.

Schistosomiasis is an important zoonosis and some livestock especially bovine and swine play a crucial role on the disease transmission in endemic areas. The gold standard for animal Schistosoma japonicum infection is fecal examination although indirect agglutination assay (IHA) is so far mostly used in field survey and laboratory examination. Lack of sensitivity, poor practicality and high false positivity limit the use of those methods for routine veterinary detection as well as human diagnosis. A novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) was developed for detection of circulating schistosomal antigen (CSA) in sera of hosts infected with S. japonicum. To assess the application of this method for diagnosis of domestic animal schistosomiasis with urine sample, the immunomagnetic bead ELISA based on IgY (IgY-IMB-ELISA) was employed in the present study to detect CSA in urine of murine schistosomiasis with either light (10 S. japonicum cercariae infection per mouse) or heavy infection (30 S. japonicum cercariae infection per mouse). The results showed that the CSA levels in urine of heavily and lightly infected mice reached a peak in 8 and 10 weeks after infection, respectively, remaining at a constant plateau in both groups by the end of the experiment (14 weeks after infection). The CSA level in urine of heavily infected mice was much higher than that of lightly infected mice from 8 to 14 weeks after infection. The effect of praziquantel treatment on the CSA level in urine of heavily infected mice was also investigated. It was found that the CSA level in urine of heavily infected mice with treatment was much lower than that of untreated mice at 4 weeks post-treatment, although still higher than that of control mice, and then gradually descended to the background level by 8 weeks after treatment. Our findings suggested that the IgY-IMB-ELISA may be an efficient and practical tool in non-invasive diagnosis of schistosome infection based on antigen detection, and evaluation of the efficacy of chemotherapy as well.

Schistosoma japonicum: susceptibility of neonate mice born to infected and noninfected mothers following subsequent challenge

This study was to investigate the differences between neonate mice born to Schistosoma japonicum‐infected mothers and those born to noninfected mothers in subsequent challenge. The intensity of infection (evidenced by worm burden and liver egg burden) and liver immunopathology (number and size of liver granulomas) were significantly reduced in neonates from infected mothers (I.M.) compared with neonates from noninfected mothers (N.M.). Anti‐soluble worm antigen of S. japonicum (SWA) IgG could be detected in sera of neonates from I.M. (N.N./I.M.) at 1 week after delivery, remained a plateau for 2 weeks and gradually decreased until 8 weeks of age. Parasite‐specific IgM was not detected in sera from N.N./I.M. at any time after delivery. At 6 weeks after infection, the level of anti‐SWA IgG in infected neonates from I.M. (I.N./I.M.) was significantly higher than that of infected neonates from N.M. (I.N./N.M.). In addition, production of IFN‐γ, IL‐12 and TGF‐β by cultured splenocytes from I.N./I.M. was significantly increased, while the level of IL‐4 was significantly decreased when compared to those from I.N./N.M.. These data demonstrate that congenital exposure to schistosomiasis japonica may render neonatal mice born to I.M. less susceptible to subsequent challenge and result in down‐regulation of both infection intensity and immunopathology.

Enhanced Wnt Signalling in Hepatocytes is Associated with Schistosoma japonicum Infection and Contributes to Liver Fibrosis

Liver fibrosis is the most serious pathology caused by Schistosoma japonicum infection, which arises when schistosome eggs are deposited in the liver. Eosinophils, macrophages and hepatic stellate cells (HSCs) have been identified as major cellular contributors to the development of granulomas and fibrosis, but little is known about the effects of hepatocytes on granuloma formation. Here, we found that the levels of Wnt signalling-related molecules, transforming growth factor β (TGF-β) and connective tissue growth factor (CTGF) in hepatocytes were markedly elevated after S. japonicum infection. Liver fibrosis was exacerbated when exogenous Wnt3a was introduced, but was alleviated when Wnt signalling was suppressed by DKK1, accompanied by the reduced expression of TGF-β and CTGF in hepatocytes. These results indicate that the hepatocytic expression of TGF-β and CTGF is mediated by Wnt signalling. Additionally, the hepatocytes isolated from infected mice promoted the activation of primary HSCs in vitro, however, this effect was not observed when hepatocytes from DKK1 treated S. japonicum-infected mice was employed in the co-culture system. Our findings identify a novel pro-fibrogenic role of hepatocytes in schistosomiasis-induced liver fibrosis that is dependent on Wnt signalling, which may serve as a potential target for ameliorating hepatic fibrosis caused by helminths.

Effect of cytotoxic T-lymphocyte-associated protein 4 on CD4 + CD25 + regulatory T cells in murine Schistosomiasis japonica

In a previous study we demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) contributed to the escape of Schistosoma japonicum (S. japonicum) from the hosts immune responses. In this paper, we studied the effect of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on CD4(+)CD25(+) Tregs in murine Schistosomiasis japonica and its corresponding role in the immune evasion of S. japonicum in mice. The results showed substantial reductions of worm burden and egg production in worm groups treated with anti-CD25 or anti-CTLA-4 monoclonal antibodies (mAb) compared to an infected but untreated control. The reduction effect was even enhanced in an experimental group co-treated with both mAbs. Compared to the control group, the percentage of CD4(+)CD25(+) Tregs was very much lower in the anti-CD25 mAb group as determined by FACS analyses and higher in the anti-CTLA-4 mAb group. ELISA analyses showed that both the anti-CTLA-4 mAb and the co-treated groups had higher levels of cytokines compared to the control group as well as larger egg granuloma sizes as determined by microscopical analyses of liver sections of infected mice. These results suggest that treatment with an anti-CTLA-4 mAb allows the host to clear S. japonicum, but at the cost of elevated pathological damage. The latter indicated a role of CTLA-4 in granuloma formation. Moreover, CD4(+)CD25(+) Tregs and CTLA-4 may exert synergistic effects during immune evasion processes by enhancing Th1-type immune response.