Loss of natural resistance to schistosome in T cell deficient rat.

Schistosomiasis is among the major neglected tropical diseases and effective prevention by boosting the immune system is still not available. T cells are key cellular components governing adaptive immune response to various infections. While common laboratory mice, such as C57BL/6, are highly susceptible to schistosomiasis, the SD rats are extremely resistant. However, whether adaptive immunity is necessary for such natural resistance to schistosomiasis in rats remains to be determined. Therefore, it is necessary to establish genetic model deficient in T cells and adaptive immunity on the resistant SD background, and to characterize liver pathology during schistosomiasis. In this study we compared experimental schistosomiasis in highly susceptible C57BL/6 (B6) mice and in resistant SD rats, using cercariae of Schistosoma japonicum. We observed a marked T cell expansion in the spleen of infected B6 mice, but not resistant SD rats. Interestingly, CD3e-/- B6 mice in which T cells are completely absent, the infectious burden of adult worms was significantly higher than that in WT mice, suggesting an anti-parasitic role for T cells in B6 mice during schistosome infection. In further experiments, we established Lck deficient SD rats by using CRISPR/Cas9 in which T cell development was completely abolished. Strikingly, we found that such Lck deficiency in SD rats severely impaired their natural resistance to schistosome infection, and fostered parasite growth. Together with an additional genetic model deficient in T cells, the CD3e-/- SD rats, we confirmed the absence of T cell resulted in loss of natural resistance to schistosome infection, but also mitigated liver immunopathology. Our further experiments showed that regulatory T cell differentiation in infected SD rats was significantly decreased during schistosomiasis, in contrast to significant increase of regulatory T cells in infected B6 mice. These data suggest that T cell mediated immune tolerance facilitates persistent infection in mice but not in SD rats. The demonstration of an important role for T cells in natural resistance of SD rats to schistosomiasis provides experimental evidences supporting the rationale to boost T cell responses in humans to prevent and treat schistosomiasis.

FOXP3+ Regulatory T Cells in Hepatic Fibrosis and Splenomegaly Caused by Schistosoma japonicum: The Spleen May Be a Major Source of Tregs in Subjects with Splenomegaly.

Schistosoma eggs cause chronic liver inflammation and a complex disease characterized by hepatic fibrosis (HF) and splenomegaly (SplM). FOXP3+ Tregs could regulate inflammation, but it is unclear where these cells are produced and what roles they play in human schistosomiasis. We investigated blood and spleen FOXP3+ Tregs in Chinese fishermen with lifelong exposure to Schistosoma japonicum and various degrees of liver and spleen disease. FOXP3+ Tregs accounted for 4.3% of CD4+ T cells and 41.2% of FOXP3+CD4+ T cells; they could be divided into CD45RA-FOXP3hi effector (eTregs) and CD45RA+FOXP3low naive Tregs. Blood Treg levels were high in severe HF (+1.3; p = 0.004) and in SplM (+1.03, p = 0.03). Multivariate regression showed that severe HF (+0.85, p = 0.01) and SplM (+0.97; p = 0.05) were independently associated with the higher proportion of Tregs in the blood. This effect was mostly due to an increase in the proportion of eTregs in the blood of HF+++ (+0.9%; p = 0.04) and SplM (+0.9%; p = 0.04) patients. The proportion of eTregs expressing CXCR3 in the blood was lower in the HF+++ patients (37.4 +/- 5.9%) than in those with milder fibrosis (51.7 ± 2%; p = 0.009), whereas proportion were similar for cells expressing CD25hi, CCR7, and CTLA-4. Splenectomy improves symptoms and was associated with decreases in blood FOXP3+ Treg (-2.5; p<0.001) and eTreg (-1.3; p = 0.03) levels. SplM spleens contained a high proportion of eTregs with CXCR3, CCR5 and CTLA4 upregulation and CCR7 downregulation. This, and the strong expression of ligands of CXCR3 and CCR5 in the liver (n = 8) but not in the spleen suggested that spleen eTregs migrated to Th1-infiltrated liver tissues. Such migration may be attenuated in hepatosplenic patients due to lower levels of CXCR3 expression on Tregs (p = 0.009). Thus, higher blood Treg levels are associated with severe liver disease and splenomegaly. Our data are consistent with the hypothesis that the spleen is a major source of Tregs in subjects with splenomegaly. In most cases, Tregs migrate to the Th1-infiltrated liver and the lower levels of CXCR3+ Tregs in the blood of patients with severe schistosomiasis suggest that decreases in Treg migration sites of inflammation may aggravate the disease.

[DNA prime followed by protein boost enhances the protective efficacy against Schistosoma japonicum infection in mice].

Schistosomiasis japonica is an endemic, zoonotic disease of major public health importance in China. Vaccination is needed as a complementary approach to the ongoing control programs. In the present study, we determined if the efficacies of DNA vaccine encoding the SjGST and Sj32 asparaginyl endopeptidase protein could be enhanced by boosting with SjGST-32 protein vaccines. Mice were inoculated with a VR1012-SjGST-32 DNA vaccine followed by boosting with rSjGST-32 at 0, 14 and 28 d. Two weeks after the final boost, mice were challenged percutaneously with cercariae. On day 45 following the challenge, all mice were sacrificed and the numbers of recovered worms and hepatic eggs were counted. Moreover, we analyzed the immune response among various vaccination groups. The results showed that DNA vaccine efficacy was enhanced when mice were boosted with protein vaccine. Adult worm and liver egg burdens were reduced 42.3% and 59.6%, respectively. We further found that DNA vaccine followed by boosting with protein significantly increased the IgG titer and T cell proliferation over those seen in mice vaccinated solely with DNA vaccines. Furthermore, the higher level of IFN-gamma expression in the splenetic CD4+ T cell showed that DNA prime-Protein boosting vaccine induced CD4+ Th1-type responses. Thus, DNA vaccine efficacy was significantly enhanced via boosting protein vaccine which might provide a basis for rational application of the Schistosoma vaccine.

Risk factors for helminth infections in a rural and a peri-urban setting of the Dongting Lake area, People's Republic of China.

Schistosomiasis japonica and soil-transmitted helminthiasis are endemic parasitic diseases in the People's Republic of China (PR China). As very few studies have reported on the distribution and interaction of multiple species helminth infections, we carried out a comparative study of households in a rural village and a peri-urban setting in the Dongting Lake area of Hunan province in November and December 2006 to determine the extent of single and multiple species infections, the underlying risk factors for infection, and the relationships with clinical manifestations and self-reported morbidity. In each household, stool samples were collected and subjected to the Kato-Katz method for identifying Schistosoma japonicum, Ascaris lumbricoides, hookworm and Trichuris trichiura infections. Clinical examinations were performed and questionnaire surveys conducted at both household and individual subject levels. Complete parasitological, clinical and questionnaire data were obtained for 1,298 inhabitants of the two settings. The overall prevalences of single infections of S. japonicum, A. lumbricoides, hookworm and T. trichiura were 6.5%, 5.5%, 3.0% and 0.8%, respectively; the majority of the infections were of light intensity. We found significant negative associations between wealth and infections with S. japonicum and A. lumbricoides. Clinical manifestations of splenomegaly, hepatomegaly and anaemia were prevalent (9.0%, 3.7% and 10.9%, respectively), the latter two being significantly (P<0.05) associated with schistosomiasis. Self-reported symptoms were more common among females but there was considerable under-reporting in both sexes when relying only on spontaneous recall. Our findings may guide the design and targeting of a more equitable, comprehensive and integrated parasitic disease control programme in Hunan province and in other areas of PR China.

Variants of CTGF are associated with hepatic fibrosis in Chinese, Sudanese, and Brazilians infected with schistosomes.

Abnormal fibrosis occurs during chronic hepatic inflammations and is the principal cause of death in hepatitis C virus and schistosome infections. Hepatic fibrosis (HF) may develop either slowly or rapidly in schistosome-infected subjects. This depends, in part, on a major genetic control exerted by genes of chromosome 6q23. A gene (connective tissue growth factor [CTGF]) is located in that region that encodes a strongly fibrogenic molecule. We show that the single nucleotide polymorphism (SNP) rs9402373 that lies close to CTGF is associated with severe HF (P = 2 x 10(-6); odds ratio [OR] = 2.01; confidence interval of OR [CI] = 1.51-2.7) in two Chinese samples, in Sudanese, and in Brazilians infected with either Schistosoma japonicum or S. mansoni. Furthermore, SNP rs12526196, also located close to CTGF, is independently associated with severe fibrosis (P = 6 x 10(-4); OR = 1.94; CI = 1.32-2.82) in the Chinese and Sudanese subjects. Both variants affect nuclear factor binding and may alter gene transcription or transcript stability. The identified variants may be valuable markers for the prediction of disease progression, and identify a critical step in the development of HF that could be a target for chemotherapy.

A randomized, double-blind, placebo-controlled trial of safety and efficacy of combined praziquantel and artemether treatment for acute schistosomiasis japonica in China.

OBJECTIVE: To evaluate the safety and efficacy of combining artemether (AM) and praziquantel (PZQ) in different regimens for treating acute schistosomiasis japonica. METHODS: We undertook a randomized, double-blind, placebo-controlled trial within four specialized schistosomiasis hospitals in the Dongting Lake region, Hunan province, China, between May 2003 and December 2005. Study participants were randomized into one of four treatment regimes: group A received 60 mg/kg PZQ + 6 mg/kg AM; group B received 60 mg/kg PZQ + AM placebo; group C received 120 mg/kg PZQ + 6 mg/kg AM; and group D received 120 mg/kg PZQ + AM placebo. All participants were followed up over a 45-day period. The primary endpoint of the trial was human infection status (determined by positive stool examination). Secondary endpoints involved clinical observations and blood biochemistry, including monitoring haemoglobin and alanine aminotransferase levels over time. FINDINGS: Treatment efficacies of the four different treatment regimens were 98.0%, 96.4%, 97.7% and 95.7% for group A, B, C, and D respectively (P > 0.05). The group B had a greater treatment efficacy (96.4%) than the group D (95.7%) (P > 0.05). Group A treatment was better for clearance of fever (P < 0.05) and resulted in a shorter hospitalization time (P < 0.05). CONCLUSION: This is the first report of a randomized, double-blind, placebo-controlled trial for evaluating combined chemotherapy with AM and two different dosages (60 mg/kg and 120 mg/kg) of PZQ in the treatment of acute schistosomiasis japonica in China. The combination of AM and PZQ chemotherapy did not improve treatment efficacy compared with PZQ alone. PZQ given as a dosage of 60 mg/kg (1 day, 3 x 20 mg/kg doses at 4-5 hour intervals) may be as effective as a dosage of 120 mg/kg (6 days, 20 mg/kg for each day split into 3 doses at 4-5 hour intervals).

Regulatory role of interleukin-10 and interferon-gamma in severe hepatic central and peripheral fibrosis in humans infected with Schistosoma japonicum.

BACKGROUND: Schistosoma japonicum is the most pathogenic agent of hepatosplenic schistosomiasis. It causes fibrosis of the central (CentF) and peripheral (PerF) portal areas. We investigated whether CentF and PerF in Chinese fishermen infected with S. japonicum were associated with an abnormal production of cytokines and chemokines that, in experimental models, have been implicated in the regulation of fibrosis. METHODS: Cytokines were measured by enzyme-linked immunosorbent assay in cultures of peripheral blood mononuclear cells from 127 patients, after stimulation with S. japonicum egg antigens. Data were analyzed by logistic regression that included age, sex, number of treatment episodes, alcohol use, and exposure as covariates. RESULTS: CentF was associated with low levels of interleukin (IL)-10 (P= .0004), regulated on activation normally T cell expressed and secreted (P= .0004), and macrophage inflammatory protein-1alpha (P= .007). In a multivariate analysis, only IL-10 was associated with CentF (odds ratio [OR], 10.8; 95% confidence interval [CI], 3.2-38; P= .0004). Splenomegaly was also associated with low IL-10 production and, independently, with CentF. In multivariate analysis, PerF was associated with low production of interferon (IFN)-gamma (OR, 8.2; 95% CI, 2-33; P= .0035) but not with production of IL-10. CONCLUSIONS: IL-10 is associated with protection against central fibrosis, because of its anti-inflammatory and antifibrosis effects. IFN-gamma is associated with protection against PerF, which depends more on egg load and egg-associated toxicity.

The protective efficacy against Schistosoma japonicum infection by immunization with DNA vaccine and levamisole as adjuvant in mice.

Levamisole (LMS) as an adjuvant enhances cell-mediated immunity in DNA vaccination; we investigated the efficacy and liver immunopathology alleviation of a DNA vaccine, VR1012-SjGST-32, in a LMS formulation in the murine challenge model. Compared to controls, the VR1012-SjGST-32 plus LMS can reduce worm and egg burdens, as well as, immunopathological complications associated chronic inflammation significantly in liver, which were apparently associated with Th1-type response. Together, these results suggest that the LMS as a potential Schistosome DNA vaccine adjuvant can enhance both worm killing and disease prevention, which is possibly mediated through the induction of a strong Th1-dominant environment in immunized mice.

[Study on the relationship between the degree of periportal fibrosis, hepatic parenchymatous fibrosis and diameter of portal vein in Schistosoma japonicum infection].

The degree of periportal fibrosis, hepatic parenchymatous fibrosis and the diameter of portal vein in fishermen from highly endemic area of schistosomiasis japonica in Dongting Lake region were measured. The results showed a significant correlation between the degree of periportal fibrosis and parenchymatous fibrosis and the portal venous diameter with a correlation coefficient of 0.375 and 0.332 respectively. The authors consider that the diameter of the portal vein can be used to assess the hepatic morbidity of patients.

[Protective effects of co-immunization with SjCTPI-Hsp70 and interleukin-12 DNA vaccines against Schistosoma japonicum challenge infection in water buffalo].

OBJECTIVE: To induce protective effect of co-immunization with S. japonicum triose-phosphate isomerase fused to heat shock protein 70 (SjCTPI-Hsp70) plasmid and interleukin-12 (IL-12) DNA vaccines against Schistosoma japonicum (Chinese strain) infection in water buffalo. METHODS: Forty-five 8-10 months-old water buffalo from a nonendemic area were divided into three treatment groups each with fifteen buffalo: experimental group A (SjCTPI-Hsp70+IL-12, 300 microg), experimental group B (SjCTPI+IL-12, 300 microg), and control group C (pVAX+IL-12, 300 microg). All buffalo were immunized with a series of 3 intramuscular injections administered once every four weeks. Twenty-eight days postvaccination, water buffalo were percutaneously challenged with 1000 S. japonicum cercariae. Fecal examinations were conducted two days prior, one day prior, and on perfusion day, and the number of hatching miracidia and eggs per gram feces were recorded. Fifty-six days post-infection, the buffalo were sacrificed and perfused via the descending aorta. The recovered adult worms and eggs in liver tissue were counted. RESULTS: Groups A and B showed a worm reduction rate of 51.2% and 41.5% (chi2=1.89, P>0.05)), female worm reduction of 48.9% and 44.7% (chi2=0.35,P>0.05), fecal egg reduction of 52.1% and 38.3% (chi2=3.84,P<0.05), a reduction of miracidia-hatching rate by 52.1% and 33.2% (chi2=7.30, P<0.01), and liver egg reduction of 61.5% and 42.0% (chi2=7.61 , P<0.01), respectively. CONCLUSION: Co-immunization with SjCTPI-Hsp70 and IL-12 DNA vaccines induces protective immunity against S. japonicum in water buffalo.

[Study on the relationship of immune status with severity of schistosomiasis japonica in fishermen in the Dongting Lake].

OBJECTIVE: To investigate the relationship of the immune status and the intensity of infection or the severity of the hepatosplenic pathology among fishermen with schistosomiasis japonica in the Dongting Lake region. METHODS: Inquiring and physical examination (IPE), stool examination, B-ultrasonography of the liver and spleen, flow cytometry, turbidimetry and ELISA were undertaken to acquire or determine the intensity of infection (EPG in stool), pathological change in the liver and spleen and the level of cellular and humoral immunity. Data were analyzed with SPSS 10.0 statistics software. RESULTS: Compared with subjects from non-endemic area, the CD4+ T cells and the CD4+/CD8+ ratio in fishing population in the endemic area significantly decreased. The decrease of the CD4+/CD8+ ratio was more significant among population with positive stool exam and with the increase of EPG and/or severity of pathological change in the liver and spleen. Contrarily, the level of the total IgM and the anti-SEA IgG in serum from fishing population in the endemic area was significantly higher than those from non-endemic area. High level serum antibodies in those stool positives were remarkable with the increase of EPG and/or the severity of hepatosplenic pathological change. The total IgA increased considerably in the subjects with significant pathological change of the liver and spleen. A high total IgG was only detected in those stool positives. CONCLUSION: The immune status in fishermen with schistosomiasis in the Dongting Lake showed a suppressed cellular immunity and a hyper functioning humoral immune response. The imbalance of the immunity was related to the increase of the intensity of infection and the progress of the hepatosplenic pathology.

[Analysis on morbidity and chemotherapy effects of Schistosoma japonicum infection in fishermen on Dongting Lake].

OBJECTIVE: To clarify and evaluate the morbidity of schistosome infection and the effectiveness of chemotherapy among fishermen on East Dongting Lake. METHODS: Information on water-contact, history of infection and of praziquantel (PZQ) treatment among fishermen was collected. Kato-Katz method and miracidium hatching test were applied to detect the pathogens in stool specimen. Serum antibodies against soluble egg antigen (SEA) were detected with ELISA and IHA. B-ultrasonic examination was used to determine the pathological changes of liver and spleen. Chemotherapy [PZQ 40 mg/(kg x d)] was given to the fishermen followed by a re-examination after a transmission season. RESULTS: The first investigation (six months before chemotherapy) showed that among 268 people inquired, 90.7% were frequently or intermittently contacting water, 24.0% were treated with PZQ each year, 39.4% had never been treated in the recent five years. Stool positive rate was 68.1% (111/163) and the geometric mean eggs per gram feces (EPG) were 48.77. Males had a higher infection rate (76.0%) and intensity (62.97 EPG) compared with that of females (58.7% infection rate and 30.42 EPG). The highest positive rate (83.3%) was in the age group of 11 to 20 years old. The prevalence of those who frequently or intermittently contacted water and were never treated before was 76.3% (106/139) and 79.7% (51/64), respectively. Serological positive rate was 88.0% (IHA) or 78.7% (ELISA). B-ultrasound revealed 77.4% (82/106) of the fishermen showing pathological changes in liver and/or spleen due to schistosomiasis. 37.7% of the patients showed II-III stage liver fibrosis (male: 53.0%, female: 15%), 58.5% hepatomegaly and 19.8% splenomegaly. The second investigation (six months after chemotherapy with PZQ) showed a stool positive rate of 35.4% and an average EPG 36.13 in the treatment group which were considerably lower than 56.5% infection rate and 68.47 EPG in the group without treatment. In 39 patients treated, the reversion rate from egg positive to negative was 48.7%, pathological change in liver and spleen declined by 40.6%. CONCLUSION: The prevalence and morbidity of schistosomiasis in fishermen on Dongting Lake were high due to frequent exposure to the affected water, and chemotherapy can effectively reduce the prevalence, the intensity of infection and morbidity of the fishermen.