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BackgroundPraziquantel (PZQ) has been the first line antischistosomal drug for all species ofSchistosoma, and the only available drug for schistosomiasis japonica, without any alternative drugs since the 1980s. However, PZQ cannot prevent reinfection, and cannot cure schistosomiasis thoroughly because of its poor activity against juvenile schistosomes. In addition, reliance on a single drug is extremely dangerous, the development and spread of resistance to PZQ is becoming a great concern. Therefore, development of novel drug candidates for treatment and control of schistosomiasis is urgently needed.Methodologys/Principal findingsA novel PZQ derivative P96 was synthesized with the substitution of cyclohexyl by cyclopentyl. We investigated thein vitroandin vivoactivities of our drug candidate P96 against different developmental stages ofS. japonicum. Parasitological studies and scanning electron microscopy were used to study the primary action characteristics of P96in vitro. Both mouse and rabbit models were employed to evaluate schistosomicidal efficacy of P96in vivo. Besides calculation of worm reduction rate and egg reduction rate, quantitative real-time PCR was used to evaluate thein vivoantischistosomal activity of P96 at molecular level.In vitro, after 24h exposure, P96 demonstrated the highest activities against both juvenile and adult worm ofS. japonicumin comparison to PZQ. The antischistosomal efficacy was concentration-dependent, with P96 at 50μM demonstrating the most evident schistosomicidal effect. Scanning electron microscopy demonstrated that P96 caused more severe damages to schistosomula and adult worm tegument compared to PZQ.In vivo, our results showed that P96 was effective againstS. japonicumat all developmental stages. Notably, its efficacy against young stage worms was significantly improved compared to PZQ. Moreover, P96 retained the high activity comparable to PZQ against the adult worm ofS. japonicum.ConclusionsP96 is a promising drug candidate for chemotherapy of schistosomiasis japonica, which has broad spectrum of action against various developmental stage, potentially addressing the deficiency of PZQ. It might be promoted as a drug candidate for use either alone or in combination with PZQ for the treatment of schistosomiasis.Author SummarySchistosomiasis is one of the neglected tropical diseases caused by infection ofSchistosoma spp. Currently, in the absence of effective vaccines for schistosomiasis, PZQ is the first line drug chosen for the treatment and control of schistosomiasis in most developing countries. However, after long-term and large-scale administration of PZQ, drug-resistance has been a great concern. Therefore, there is a need for new therapies. In this study, with the aim of preventing the formation of less effective metabolite 4-trans-cyclohexanol, a novel PZQ derivative, P96, is synthesized with the cyclohexyl group substituted by cyclopentyl group. It is this small modification that gives us a big surprise.In vitro, all the biological assessments, including viability reduction rate and morphological properties by scanning electron microscopy, demonstrate that P96 has superior anti-schistosomula activity compared to PZQ, and retains similar or even higher anti-adultS. japonicumactivity to PZQ. The antischistosomal effect of P96 is dose-dependent.In vivo, P96 displays high efficacy against all developmental stages ofS. japonicum, with significantly improved efficacy against young stage worms compared to PZQ. Furthermore, the quantitative detection results of specific circulatory SjR2 DNA prove that P96 has similar activity to PZQ against adult schistosome at molecular level in rabbit sera with infection of schistosomiasis. In conclusion, the novel PZQ derivative, P96 is a promising drug candidate for chemotherapy of schistosomiasis, potentially addressing the deficiency of PZQ, and might be promoted for use either alone or in combination with PZQ for treatment and control of schistosomiasis.

Background Schistosomiasis is a debilitating and neglected tropical disease for which praziquantel (PZQ) remains the first-choice drug for treatment and control of the disease. In our previous studies, we found that the patented compound DW-3-15 (patent no. ZL201110142538.2) displayed significant and stabilized antiparasitic activity through a mechanism that might be distinct from PZQ. Here, we investigated the antischistosomal efficacy of PZQ combined with DW-3-15 against schistosomula and adult worms of Schistosoma japonicum in vitro and in vivo, to verify whether there was a synergistic effect of the two compounds. Methods The antischistosomal efficacy of PZQ combined with DW-3-15 in comparison with an untreated control and monotherapy group against schistosomula and adult worms was assessed both in vitro and in vivo. Parasitological studies, scanning electron microscopy, combination index, and histopathological analysis were used for the assessment. Results The results showed significantly reduced viability of schistosomes, achieving 100% viability reduction for juveniles and males by combination chemotherapy using PZQ together with DW-3-15 in vitro. The combination index was 0.28, 0.27, and 0.53 at the higher concentration of PZQ combined with DW-3-15 against juveniles, males, and females, respectively, indicating that the two compounds display strong synergism. Scanning electron microscopy observations also demonstrated that the compound combination induced more severe and extensive alterations to the tegument and subtegument of S. japonicum than those with each compound alone. In vivo, compared with the single-compound-treated group, the group treated with the higher-dose combination demonstrated the best schistosomicidal efficacy, with significantly reduced worm burden, egg burden, and granuloma count and area, which was evident against schistosomula and adult worms. Conclusions Our study provides a potential novel chemotherapy for schistosomiasis caused by S. japonicum. It would improve the antischistosomal effect on schistosomula and adult worms of S. japonicum, and decrease individual dosages. Graphical Abstract

Schistosomiasis liver fibrosis is characterized by the foci of a classical type 2 granulomatous inflammation in response to egg soluble antigen. The inducible costimulator (ICOS)/ICOSL signaling can mediate Th2 polarization and regulate the disease process of chronic schistosomiasis by adaptive immune response. ICOS/ICOSL signaling could regulate innate immune cells, including B cells and macrophage (Mφ), and participate in pulmonary fibrosis. The purpose of the study was to identify whether ICOS/ICOSL signaling could regulate the polarization of Mφ, and to explore the potential role of IL-33 in ICOSL knockout (ICOSL-KO) mice with schistosomiasis. Firstly, the expression level of CD86, CD206 and IL-33 was checked respectively in ICOSL-KO and WT mice infected with Schistosoma japonicum (S. japonicum). Then recombinant IL-33 (rIL-33) was injected into ICOSL-KO mice infected with S. japonicum. Our experiments showed that the liver Mφ was successfully polarized toward to classical activated Mφ (M1) and the expression of IL-33 was inhibited in ICOSL-KO mice. Furthermore, the injection of rIL-33 successfully aggravated liver fibrosis in ICOSL-KO mice, increased the numbers of lymphocyte antigen 6C (Ly6C)hi, enhanced the expression of C-C chemokine ligand (CCL)2,, CCL5 and C-X-C motif chemokine 2 (CXCL2), and promoted polarization of T helper (Th) cells to Th2 cells, as well as induced the autophagy and apoptosis of hepatic stellate cells (HSCs). Overall, the liver fibrosis was alleviated in ICOSL-KO mice along with the reduced expression of IL-33, which could skew the polarization of Mφ toward to M1, induce Th cells activation, HSCs apoptosis and autophagy through Smad2/3 and TGF-β signaling pathway, thus participated in the homeostasis of liver fibrosis of schistosomiasis.

Background This study aims to understand whether there is a seasonal change in the internet search interest for Toxoplasma by using the data derived from Google Trends (GT). Methods The present study searched for the relative search volume (RSV) for the search term ‘Toxoplasma’ in GT within six major English-speaking countries (Australia, New Zealand [Southern Hemisphere] and Canada, Ireland, the UK and the USA [Northern Hemisphere] from 1 January 2004 to 31 December 2019, utilizing the category of ‘health’. Data regarding the RSV of Toxoplasma was obtained and further statistical analysis was performed in R software using the ‘season’ package. Results There were significantly seasonal patterns for the RSV of the search term ‘Toxoplasma’ in five countries (all p Conclusions Overall, our study revealed the seasonal variation for Toxoplasma in using internet search data from GT, providing additional evidence on seasonal patterns in Toxoplasma.

Emerging evidences have highlighted the crucial role of microRNAs (miRNAs) in the liver cirrhosis, but the relationship between miR-130a-3p and liver cirrhosis is not entirely clear. As we all know, schistosomiasis, as one of the zoonoses, can lead to liver cirrhosis when it advances. In this study, we investigated the biological functions of miR-130a-3p on the liver fibrosis of schistosomiasisin vivoandin vitro. The mice infected withSchistosoma japonicum(S. japonicum)were treated with lentivirus vector (LV)-miR-130a-3p by hydrodynamic injection through the tail vein. Our findings showed significantly decreased expression of miR-130a-3p both in the serum of patients with cirrhosis and in the liver of mice infected withS. japonicum. The results showed that LV-miR-130a-3p could effectively enter into the liver and alleviate liver granulomatous inflammation and collagen deposition. Simultaneously, LV-miR-130a-3p-promoted macrophages presented the Ly6Clophenotype, concomitant with the decreased expression of the tissue inhibitor of metalloproteinases (TIMP) 1, and increased the expression of matrix metalloproteinase (MMP) 2, which contributed to the dissolution of collagen. Furthermore, overexpression of miR-130a-3p not only inhibited the activation and proliferation of hepatic stellate cells (HSCs) but also induced the apoptosis of HSCs. In addition, we also confirmed that miR-130a-3p enables to bind with mitogen-activated protein kinase (MAPK) 1 and transforming growth factor-beta receptors (TGFBR) 1 and TGFBR2 genes and inhibit the expressions of these genes. Our findings suggested that miR-130a-3p might represent as the potential candidate biomarker and therapeutic target for the prognosis identification and treatment of schistosomiasis liver fibrosis.

Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite that often infects warm-blooded animals or causes opportunistic infections if exists a suppressed immunity. This study aims to investigate the seroprevalence of T. gondii and its odds ratio (OR) in patients with cancer in compared with healthy individuals, and to find the possible factors. Related literatures reported the seroprevalence of T. gondii in cancer/tumor patients and controls (health individuals) were retrieved from electronic databases PubMed, EMBASE, Chinese Web of Knowledge and The Cochrane Library from inception until Aug 31 2018. The non-weighted prevalence of T. gondii, pooled estimates of OR and its 95% confidence intervals (CI) were calculated through random-effect model. Between-study heterogeneity was tested with Cochrane Q, and statistic I2 was to quantify the results. Funnel plot depiction and Egger’s linear regression test were combined to evaluate the potential of publication bias. The literature identified a total of 2216 potential studies; the final 18 studies were incorporated, with 6001 cancer/tumor patients and 6067 controls. Our results demonstrated that, the cancer/tumor patients had an elevated seroprevalence of T. gondii (18.43% vs 8.19%), and an increased risk of T. gondii infection (OR = 3.18, 95% CI: 2.65–3.82) when compared with the controls. Subgroup analyses suggested that publication year, study sample size and diagnostic options are closely associated with the seroprevalence of T. gondii. Overall, our study indicates that there is an increased risk of T. gondii infection in cancer/tumor patients, suggesting a precautionary monitoring of T. gondii and related risk factors in patients with cancer/tumor.

BackgroundHumoral immune responses play an important role in mediating liver granulomatous inflammation and fibrosis in schistosomiasis. Follicular helper T (Tfh) cells have a central role in mediating humoral immune responses. Generation of Tfh cells depends on inducible T cell costimulator (ICOS) signaling, but the underlying molecular mechanisms are incompletely understood in pathogenesis of schistosomiasis.Methodology/Principal FindingsWe used a strain of ICOS-transgenic (Tg) mice to test the degrees of liver granulomatous inflammation and fibrosis, the frequency of splenic Tfh cells and soluble egg antigen-specific cytokine responses longitudinally in mice following Schistosoma japonicum (S. japonicum) infection. In comparison with that in wide-type (WT) mice, significantly severer liver granulomatous inflammation and fibrosis and higher mortality were observed in ICOS-Tg mice. Significantly higher frequency of splenic Tfh cells was accompanied by significantly higher levels of Bcl-6 and CXCR5 expression in the livers of ICOS-Tg mice. Furthermore, significantly higher levels of SEA-specific IL-4, IL-6, IL-10, IL-13, IL-17A, IL-21 and TGF-β1 responses, but lower levels of IFN-γ responses were detected in ICOS-Tg mice, which were abrogated by treatment with ICOS blockers in vitro. In addition, significantly higher levels of serum anti-SEA IgG were detected in ICOS-Tg mice.Conclusions/SignificanceThe ICOS-related signaling may promote the pathogenesis of murine schistosomiasis by polarizing Tfh cells, which may be immune check points for the prevention and intervention of schistosomiasis.Author summaryGranulomatous inflammation and fibrosis in the liver are the major pathogenic characteristics of schistosomiasis. ICOS is crucial for the development of Tfh cells, which are the key modulators of B cell activation and humoral immunity. However, the underlying molecular mechanisms are incompletely understood in pathogenesis of schistosomiasis. Here, our results showed that the ICOS over-expression would significantly induce severer liver inflammation and fibrosis, higher frequency of splenic Tfh, higher levels of anti-SEA IgG as well as imbalanced SEA-specific cytokine responses in ICOS-Tg mice. The findings suggested that ICOS signaling may promote the pathogenesis of murine schistosoma-related liver inflammation and fibrosis by polarizing Tfh cells. Potentially, ICOS signaling and Tfh cells may be immune check points for the prevention and intervention of schistosomiasis.

Background Th1, Th2, Th17, Treg and Tfh cells play important roles in schistosomiasis. Th9 cells secrete IL-9 as a signature cytokine and contribute to several classes of inflammatory disease. However, the effects of Th9 cells in schistosomiasis are unknown. We aimed to explore the dynamic changes and potential roles of Th9 cells in the pathogenesis of hepatic egg granulomatous inflammation in mice infected with Schistosoma japonicum. Methods Twenty mice with S. japonicum infection and five normal controls (NC) were used as models. The average areas of egg granulomas were estimated by hematoxylin-eosin (H & E) staining. Hepatic IL-9 and transcription factor PU.1 levels were detected by immunohistochemistry. Flow cytometry techniques were used to analyze the proportions of Th9 cells. With the help of ELISA, serum levels of IL-9 were examined. Results The egg granulomas began to form from four weeks after infection and continued to develop. In parallel with the development of egg granulomas, the hepatic levels of IL-9 and PU.1 increased very slowly during the first four weeks post-infection and increased rapidly thereafter. Moreover, the proportions of splenic Th9 cells and levels of serum IL-9 had similar developmental trends with the egg granulomas. Conclusion The proliferation of Th9 cells and levels of IL-9 were significantly higher in S. japonicum-infected mice compared to NC. In addition, dynamic changes of Th9 and IL-9 were synchronous with the developmental trend of hepatic egg granulomatous inflammation, suggesting that Th9 cells might be a new subset in the pathogenesis of schistosomiasis.

Hepatic fibrosis induced by schistosomes is regulated by a complex network of cytokines. T helper type 9 (Th9) cells are a new type of effector T helper cells, which mainly secrete the specific cytokine interleukin-9 (IL-9). Interleukin-9 has been shown to contribute to liver fibrosis in patients with chronic hepatitis B and in a mouse model due to carbon tetrachloride. However, the role of IL-9 in schistosomiasis fibrosis remains unknown. In this study, we investigated the roles of IL-9 in schistosomiasis through in vivo and in vitro studies. The in vivo studies found that neutralization of IL-9 reduced liver granulomatous inflammation and collagen deposition around parasite eggs. The in vitro studies found that the treatment of primary hepatic stellate cells with IL-9 induced a significant increase of collagen and α-smooth-muscle actin. Moreover, we also described the dynamics and relevance of IL-9 and IL-4 in mice infected with Schistosoma japonicum. We found that IL-9 might appear more quickly and at higher levels than IL-4. Hence, our findings indicated that IL-9 might play a role in regulating hepatic fibrosis in early-stage schistosomiasis and become a promising approach for regulating hepatic fibrosis caused by S. japonicum.

Accurate discrimination of the Schistosoma japonicum cercariae gender is very important for establishing monosexual infection animal models and for standardizing the real intensity of infection. In this study, a multiplex PCR technique consisting of two pairs of primers, of which one amplifies a 185-bp band specific for the W chromosome and the other amplifies a 420-bp band for the Z chromosome, was established to sex the S. japonicum cercariae. For male cercariae (ZZ), a single 420-bp band is expected, and for female cercariea (ZW), two distinct 185-bp and 420-bp bands can be observed. There was no cross-reaction with S. mansoni, S. haematobium, Clonorchis sinensis, Paragonimus westermani, and Trichinella spiralis. After sexing the cercariae escaped from a single snail, mice in group A were infected with 60 male cercariae and mice of group B were infected with 40 female cercariae. Meanwhile, mice in group C were infected with 10 male and 10 female cercariae that were sexed by multiplex PCR. At 45 days postinfection, male and female adult worms were recovered to verify the accuracy of multiplex PCR for sexing S. japonicum cercariae and to calculate the male and female survival rate and paired worm ratio. Our results showed that the multiplex PCR technique could distinguish male cercariae with 100% accuracy. However, sometimes the discrimination results of multiplex PCR mis-scored mixed sexual cercariae as female cercariae. The mean male adult worm burden in mice of group C was 10.7 ± 2.4, and the mean female adult worm burden was 7.7 ± 2.5. There was a significant difference between the male worm burden and female worm burden in group C. The P value was 0.013. The real paired worm ratio of group C was 74.2% (95%CI 56.6~91.8%). These results demonstrated a male-biased sex ratio in the mice model with equilibrated sex ratio cercariae infection, as predicted by our multiplex PCR technique. In conclusion, our multiplex PCR technique is an effective tool for sexing S. japonicum cercariae, especially for distinguishing male cercariae, which is of great value for establishing monosexual cercariae infection mice models to harvest male adult worms for anti-schistosomal drug screening.

Background Schistosomiasis is a debilitating neglected tropical disease that affects approximately 190 million people around the world. Praziquantel (PZQ) is the only drug available for use against all Schistosoma species. Although PZQ has a high efficacy, recognized concerns have prompted the development of new, alternative drugs for repeated use in endemic areas where PZQ efficacy against strains of Schistosoma is reduced. A hybrid drug containing different pharmacophores within a single molecule is a promising strategy. Our earlier in vivo studies showed the significant antiparasitic activity of a praziquantel derivative, DW-3-15, against Schistosoma japonicum. In the present study, DW-3-15 was synthesized in large amounts by a pharmaceutical company and its schistosomicidal efficacy and stability were further confirmed. Parameters such as parasite viability, pairing and oviposition were evaluated in vitro. An in vivo study was conducted to assess the effect of commercial DW-3-15 on worm burden, egg production and diameter of granulomas. Additionally, to gain insight into the mechanism of action for DW-3-15, morphological changes in the tegument of S. japonicum were also examined. Results The in vitro study showed the antiparasitic activity of DW-3-15 against S. japonicum, with significant reductions in viability of adult and juvenile worms, worm pairings and egg output. Compared to PZQ, DW-3-15 induced similar ultrastructural changes and evident destruction of the tegument surface in male worms. In vivo, the oral administration of DW-3-15 at a dose of 400 mg/kg per day for five consecutive days in mice significantly reduced the total worm burden and number of eggs in the liver. Histological analysis of the livers showed a marked reduction in the average diameter of the egg granuloma. Conclusions Our findings suggest that DW-3-15, a PZQ derivative with the prospect of commercial production, can be developed as a potential promising schistosomicide. Electronic supplementary material The online version of this article (10.1186/s13071-019-3442-7) contains supplementary material, which is available to authorized users.

The pathology of schistosome egg-induced liver granuloma, fibrosis and eventually liver scarring is complicated. CD4+ helper T (Th) cells play critical roles in both host humoral immunity and cellular immunity against parasitic infection and immunopathology in schistosomiasis. Follicular helper T (Tfh) cells are another specialized subset of Th cells and involved in infectious diseases. However, the immune regulatory mechanism of Tfh cells in severe liver pathology of schistosomiasis is still poorly understood. In this study, using a S. japonicum-infected mouse model, we studied the dynamics and effects of Tfh cells in vivo and demonstrated that Tfh phenotype molecules ICOS, PD-1 and functional factor IL-21 were positively correlated with disease development by flow cytometry. Meanwhile, our results also showed that Tfh cells enriched in splenic germinal center (GC) and promoted B cells producing IgM with the progress of hepatic immunopathology by B-T co-culture experiments. More importantly, our data indicated that IL-21 contributed to the formation and development of hepatic egg granuloma and subsequent fibrosis by driving GC responses and activating HSCs by immunohistochemical detection and blocking assay in vitro. Our findings contribute to the better understanding of the immunopathogenesis of schistosomiasis and have implications for therapeutic intervention of hepatic fibrotic diseases.

Currently in China, the schistosomiasis control program has shifted its focus from transmission control to the elimination of the disease. Effective forecast and surveillance systems of schistiosomiasis are of great importance for issuing timely and early warnings on risk of infection, and therefore implementing preventive measures to avoid infection. There is great demand in more sensitive and specific methods to improve the surveillance system for early detection of S. japonicum infection in sentinel mice. In this study, we reported a sensitive nested-PCR assay targeting a 303-bp fragment from highly repetitive retrotransposon SjCHGCS19 to detect the S. japonicum DNA in sera of experimental mice. Meanwhile, detection efficacy of the nested-PCR was compared with two conventional methods for field monitoring schistosomiasis such as ELISA and IHA. The nested-PCR assay could detect the specific DNA at 3-day post-infection in sera of mice with 5 cercariae infection, while for ELISA and IHA, both show negative results even after 2 weeks post-infection in mice with 20 cercariae infection. Our results demonstrated the DNA-based assay was more sensitive to make early diagnosis of S. japonicum infection in sentinel mice models, which will improve the early-warning ability of schistosomiasis surveillance system.

To explore the effect of ICOS signaling on the CD154/CD40 expressions and immunopathology in mice infected with Schistosoma japonicum.ICOS transgenic (ICOS-Tg) mice and wildtype FVB/NJ mice were used as experimental schistosomiasis models. The expressions of CD154 and CD40 on splenocytes and on inflammatory cells around granulomatous infiltration of the liver in the mice infected with S. japonicuin were detected by flow cytometry and im- munohistochemical staining. HE staining was applied to observe the changes on the granulomatous of the mice liver.Compared with the wildtype FVB/NJ mice, the expressions of CD154 on CD4 T splenocytes and of CD40 on CD19' B splenocytes in the ICOS-Tg mice significantly increased in 12 and 16 weeks post-infection (all P0.05). Moreover, the expressions of CD40 and CD154 on inflammatory cells around granulomatous infiltration in the liver of the ICOS-Tg mice were significantly higher than those of the wildtype FVB/NJ mice in 7, 12, 16 and 20 weeks post-infection (all P0.05). The volumes of liver egg granulomas of the ICOS-Tg mice were significantly bigger than those of the wildtype mice (P0.05 or P0.01).In ICOS-Tg mice infected with S. japonicum, the ICOS signaling has a regulatory effect on CD154/CD40 expressions, and may play an important role in the hepatic egg granuloma formation of schistosomiasis.

The polymerase chain reaction (PCR) assay has turned out to be one of the most potential tools for diagnosis of schistosomiasis. However, the source and metabolic dynamics of Schistosoma japonicum DNA in the blood of hosts is not clear. In this study, rabbit models with monosexual and mixed sexual cercariae infection were established to interpret the source of the parasite DNA in serum of the hosts. Following administration of praziquantel at 7 weeks postinfection, the metabolic mechanism of S. japonicum DNA in serum of the hosts was studied. The findings showed that, for the monosexual cercariae infection, the parasite DNA was detectable in serum of the host from day 3 to week 3 postinfection, while for the mixed sexual cercariae infection, the detection results were continually positive during the 7 weeks after infection. After treatment with praziquantel, detection of S. japonicum DNA in rabbit sera became positive at the second day posttreatment, and the positive period lasted 3 weeks in the monosexual cercariae infection group. However, with the mixed sexual cercariae infection group, the PCR results remained positive for 16 weeks after treatment. We conclude that the S. japonicum DNA in host serum primarily comes from the residual body of dead schistosomula and/or tegument shedding of worm growing in the first 4 weeks postinfection, while during the spawning stage of the female schistosome, the parasite DNA mainly comes from the disintegration of inactive eggs. The duration from treatment to total elimination of worm origin DNA in serum is not exceeding 3 weeks. However, the DNA release from inactive eggs can last for more than 16 weeks. Further studies are needed to address the sources and metabolic dynamics of S. japonicum DNA in human serum.

To analyze effect on the CD154-CD40 signaling pathway and Th1/Th2 polarization by deficient inducible co-stimulator (ICOS)-ICOS ligand (ICOSL) signaling in mice infected with Schistosoma japonicum.ICOSL knockout (ICOSL-KO) mice and wild-type C57BL/6J mice were used as experimental Schistosomiasis model infected with Schistosoma japonicum. The expressions of CD154 and CD40 on splenocytes and on inflammatory cells around granulomatous infiltration of liver in mice infected with Schistosoma japonicum were analyzed by flow cytometry,immunohistochemical staining, respectively, on the day before infection (0 week)and at the end of 4, 7, 12, 16 and 20 weeks post-infection. The splenocytes of the mice were stimulated with soluble egg antigen(SEA) for 72 hours, then the concentrations of interferon gamma(IFN-γ) and interleukin-4 (IL-4) in the culture supernatants were measured by sandwich enzyme-linked immunosorbent assay (ELISA) kits. The levels of SEA-specific antibodies of IgG and IgG1 and IgG2a were measured in the mice sera by ELISA. The granulomatous pathology in the mice liver was dynamically observed by hematoxylin and eosin (HE) staining.Compared with the wild-type C57BL/6J mice, the expressions of CD154 on CD4+ T splenocytes [(18.62 ± 4.76)% vs.(27.91 ± 3.94)%, (22.44 ± 4.67)% vs.(40.86 ± 5.21)%, (25.50 ± 6.81)% vs.(43.81 ± 8.41)%, (20.22 ± 5.28)% vs.(40.95 ± 7.34)%, (17.87 ± 4.59)% vs.(33.16 ± 6.31)%, all P0.01] and of CD40 on CD19+ B splenocytes [(19.43 ± 3.26)% vs.(24.37 ± 3.59)%, (23.00 ± 4.47)% vs.(31.80 ± 5.86)%, (24.46 ± 5.01)% vs.(35.85 ± 5.32)%, (23.42 ± 4.69)% vs.(33.30 ± 6.14)%, (22.85 ± 3.78)% vs.(30.88 ± 5.94)%, all P0.05] in the ICOSL-KO mice significantly decreased at the end of 4, 7, 12, 16 and 20 weeks post-infection. Moreover, the expressions of CD154[(0.319 ± 0.066) vs.(0.488 ± 0.086), (0.389 ± 0.067) vs.(0.596 ± 0.082), (0.378 ± 0.064) vs.(0.543 ± 0.072), (0.348 ± 0.069) vs.(0.523 ± 0.076), all P0.01] and CD40[ (0.398 ± 0.066) vs.(0.546 ± 0.079), (0.461 ± 0.085) vs.(0.618 ± 0.076), (0.453 ± 0.087) vs.(0.587 ± 0.074), (0.449 ± 0.065) vs.(0.565 ± 0.082), all P0.05] on inflammatory cells around granulomatous infiltration in liver from the ICOSL-KO mice were significantly lower than those of the wild-type C57BL/6J mice at the end of 7, 12, 16 and 20 weeks post-infection. The levels of IFN-γ of the ICOSL-KO mice were significantly higher than those of the wild-type C57BL/6J mice at the end of 4, 7, 12, 16 and 20 weeks post-infection (P0.05). However, the levels of IL-4 of the ICOSL-KO mice were significantly lower than those of the wild-type mice (P0.05). Compared with the wild-type C57BL/6J mice, the levels of SEA-specific antibodies of IgG and IgG1 and IgG2a in the sera of the ICOSL-KO mice significantly decreased (P0.01). Moreover, The Th2 differentiation index of the ICOSL-KO mice was significantly lower than that of the wild-type mice in post-infection (P0.01). Also, the ratio of IgG1/IgG2a of the ICOSL-KO mice were significantly lower than that of the wild-type mice at the end of 7, 12 and 16 weeks post-infection (P0.05). And the volume of liver egg granulomas of the ICOSL-KO mice was significantly smaller than that of the wild-type mice (P0.01).These findings suggest that there is obvious down-regulation in the expressions of CD154 and CD40 and impairment of Th2 immune response in the ICOSL-KO mice infected with Schistosoma japonicum, accompanying with notedly reduced hepatic granulomatous pathology. The ICOS-ICOSL signaling has a regulatory effect on CD154-CD40 signaling pathway, and may play an important role in the hepatic egg granuloma formation of Schistosomiasis.

In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Schistosoma japonicum DNA in faecal and serum samples of rabbits, and serum samples of humans infected with S. japonicum. This LAMP assay was based on the sequence of highly repetitive retrotransposon SjR2, and was able to detect 0.08 fg S. japonicum DNA, which is 10(4) times more sensitive than conventional PCR. The LAMP assay was also highly specific for S. japonicum and able to detect S. japonicum DNA in rabbit sera at 1 week p.i. Following administration of praziquantel, detection of S. japonicum DNA in rabbit sera became negative at 12 weeks post-treatment. These results demonstrated that LAMP was effective for early diagnosis of, and evaluation of therapy effectiveness for, S. japonicum infection. Both PCR and LAMP assays were then used to detect S. japonicum DNA in 30 serum samples from S. japonicum-infected patients and 20 serum samples from healthy persons. The percentage sensitivity of LAMP was 96.7%, whereas that of PCR was only 60%, indicating that LAMP was more sensitive than conventional PCR for clinical diagnosis of schistosomiasis cases in endemic areas. The established LAMP assay should provide a useful and practical tool for the routine diagnosis and therapeutic evaluation of human schistosomiasis.

To investigate the in vitro effect of photoactivated hypericin on anti-Schistosoma japonicum adult male worms.Kunming mice were infected with 60-80 Schistosoma japonicum single-sex cercariae. At 6 weeks post-infection, the mice were sacrificed and adult male worms of S. japonicum were collected. The worms were incubated in DMEM medium containing different concentrations of hypericin (0.1, 0.2, 0.5, 1.0, 1.5, 2.0, and 2.5 micromol/L) in the presence or absence of light. In photoactivated hypericin groups, after 6 h of dark incubation the worms were exposed to LED light irradiation (590 nm) for 30, 60, 90, and 120 min, respectively, and then cultured overnight in darkness (16h). In the next morning, the parasites were washed, resuspended in drug-free medium, and incubated in the dark for 48 h. These worms were observed with stereomicroscopy and scanning electron microscopy (SEM).Photoactivated hypericin showed the ability to kill Schistosoma japonicum in vitro. The death rate was 20% in 0.1 micromol/L photoactivated hypericin group under 30 min irradiation, and 100% in 2 micromol/L under 90 min irradiation and 2.5 micromol/L under 60 min irradiation, respectively. In blank control group, DMSO control group, and hypericin groups without light irradiation, worms were alive. After 60 min irradiation, the worms in 1.0, 2.5, 5.0 micromol/L photoactivated hypericin groups showed spastic paralysis characterized by reduced body length, pronounced tight curl, body stiffness, and complete cessation of movement. Surface tegumental damages of adult worms in 2.0 micromol/L photoactivated hypericin group for 60 min irradiation were observed under SEM, such as vacuole formation, erosion and peeling of the tegument, collapse of the sensory papillae, and even the normal structure disappeared completely. Both death rate and morphological damage of the worms treated by photoactivated hypericin were positively correlated with hypericin dose and light irradiation time.Photoactivated hypericin has anti-Schistosoma japonicum adult male worms effect in vitro.

To investigate the effect of Th2 polarization mediated by ICOS signaling pathway on hepatic fibrosis in mice infected with Schistosoma japonicum.ICOS transgenic (ICOS-Tg) mice and wild-type FVB/NJ mice were used as experimental schistosomiasis model. The sera, livers and spleen lymphocytes of mice were collected, and spleen lymphocytes were stimulated with SEA for 72 h on the day before infection (0 week), and at 4, 7, 12, 16 and 20 weeks post-infection. The concentrations of Th1 cytokines (IFN-gamma and IL-12) and Th2 cytokines (IL-4 and IL-13) in the culture supernatants were measured with sandwich ELISA kit. The levels of SEA-specific antibodies of IgG and its subtypes (IgG1 and IgG2a) in mice sera were measured by ELISA. The concentrations of hyaluronic acid (HA) and hydroxyproline (HYP) in mice sera were measured with sandwich ELISA kit. The expression of alpha-SMA, TGF-beta1 and collagen-I in livers from ICOS-Tg/wild-type mice were assessed by immunohistochemical staining. Liver granulomatous pathology and fibrosis level in ICOS-Tg/wild-type mice was dynamically observed with hematoxylin-eosin (HE) staining and Masson trichrome staining, respectively.The levels of Th2-type cytokines (IL-4 and IL-13) of ICOS-Tg mice were significantly higher than that of wild-type FVB/NJ mice on 7, 12, 16, and 20 weeks post-infection (P0.05). However, Th1 cytokines IFN-gamma and IL-12 showed no significant difference between the two groups (P0.05). Th2 differentiation index of ICOS-Tg mice was significantly higher than that of wild-type mice on 7, 12, 16 and 20 weeks post-infection (P0.05 or P0.01). Compared with wild-type mice, the levels of SEA-specific antibodies of IgG and its subtypes (IgG1 and IgG2a) in ICOS-Tg mice increased significantly (P0.05 or P0.01, except IgG on 4 and 7 weeks post-infection). Moreover, the ratio of IgG1/IgG2a in ICOS-Tg mice (5.75 +/- 0.94, 4.96 +/- 0.98) were significantly higher than that of wild-type mice (4.31 +/- 0.81, 3.41 +/- 0.83) on 12 and 16 weeks post-infection (P0.05). The levels of HA on 7, 12, 16, and 20 weeks post-infection (P0.05 or P0.01) and HYP on 12, 16, and 20 weeks post-infection (P0.05) in ICOS-Tg mice were significantly higher than that of wild-type mice. Immunohistochemical staining showed, from 7 to 20 weeks post-infection, alpha-SMA and TGF-beta1 expression in liver of ICOS-Tg mice was significantly higher than that of wild-type mice (P0.05 or P0.01); collagen-I level was also higher than wild-type mice. However, there was a significant difference in collagen-I level between the two groups on 20 weeks post-infection (P0.05). Furthermore, HE staining showed, on 7, 12, and 16 weeks post-infection, single-egg granuloma volume of ICOS-Tg mice [(28.72 +/- 6.68) x 10(6), (20.47 +/- 5.09) x 10(6) and (12.77 +/- 4.86) x 10(6) microm 3] was significantly higher than that of wild-type mice [(18.04 +/- 6.21) x 10(6), (15.28 +/- 4.87) x 10(6) and (11.24 +/- 4.38) x 10(6) microm 3]. Masson staining showed that level of hepatic fibrosis in ICOS-Tg mice were higher than that of wild-type mice, but the fibrosis scores showed no statistically significant difference between the two groups (P0.05).Th2 immune response is up-regulated in ICOS-Tg mice infected with S. japonicum, and the degree of hepatic fibrosis and related indicators increase. These findings suggest that Th2 polarization mediated by ICOS signaling plays a role in hepatic fibrosis formation in mice infected with S. japonicum.

A series of chiral praziquantel analogues were synthesized and evaluated against Schistosoma japonicum both in vitro and in vivo. All compounds exhibited low to considerable good activity in vivo . Remarkably, worm reduction rate of R- 3 was 60.0% at a single oral dose of 200 mg/kg against juvenile stage of Schistosoma japonicum . The target compounds displayed in vivo antischistosomal activity against both Schistosoma japonicum and Schistosoma mansoni. Furthermore, all R-isomers displayed stronger antischistosomal activity than S-isomers in vivo, indicating R-isomers were the active enantiomers, while S-isomers were less active ones. This structure activity relationship (SAR) could have important implications in further drug development for schistosomiasis.

Analogues of pyrrolo-[1,2,5]benzothiadiazepine were prepared and evaluated against Schistosoma japonica. The biological data revealed that most benzothiazepine derivatives show anti-schistosomal activity to some extent, while α-chloronation of the title compound and another bioisosteric derivative pyrrolo-[1,2,5]benzodiazepine displayed the most distinct worm killing activity. This study proved that benzodiazepine may serve as a novel structural skeleton for the development of anti-schistosomal agents.

Background Schistosomiasis japonica is a serious debilitating and sometimes fatal disease. Accurate diagnostic tests play a key role in patient management and control of the disease. However, currently available diagnostic methods are not ideal, and the detection of the parasite DNA in blood samples has turned out to be one of the most promising tools for the diagnosis of schistosomiasis. In our previous investigations, a 230-bp sequence from the highly repetitive retrotransposon SjR2 was identified and it showed high sensitivity and specificity for detecting Schistosoma japonicum DNA in the sera of rabbit model and patients. Recently, 29 retrotransposons were found in S. japonicum genome by our group. The present study highlighted the key factors for selecting a new perspective sensitive target DNA sequence for the diagnosis of schistosomiasis, which can serve as example for other parasitic pathogens. Methodology/Principal Findings In this study, we demonstrated that the key factors based on the bioinformatic analysis for selecting target sequence are the higher genome proportion, repetitive complete copies and partial copies, and active ESTs than the others in the chromosome genome. New primers based on 25 novel retrotransposons and SjR2 were designed and their sensitivity and specificity for detecting S. japonicum DNA were compared. The results showed that a new 303-bp sequence from non-long terminal repeat (LTR) retrotransposon (SjCHGCS19) had high sensitivity and specificity. The 303-bp target sequence was amplified from the sera of rabbit model at 3 d post-infection by nested-PCR and it became negative at 17 weeks post-treatment. Furthermore, the percentage sensitivity of the nested-PCR was 97.67% in 43 serum samples of S. japonicum-infected patients. Conclusions/Significance Our findings highlighted the key factors based on the bioinformatic analysis for selecting target sequence from S. japonicum genome, which provide basis for establishing powerful molecular diagnostic techniques that can be used for monitoring early infection and therapy efficacy to support schistosomiasis control programs., Author Summary Schistosomiasis remains a serious parasitic disease worldwide. Adequate case-finding is important and crucial for the effective control programs as schistosomiasis control programs are chiefly based on treatment of infected populations. However, the currently available diagnostic assays are not ideal, and DNA detection can provide useful alternative approaches for the sensitive and specific diagnosis of schistosomiasis, provided that reliable genetic markers are employed in the tests. However, different target sequences used for DNA detection of samples showed different sensitivity. In previous studies, we identified a 230-bp sequence of high sensitivity and specificity from the retrotransposon SjR2 of Schistosoma japonicum. Here, new primers based on 25 novel retrotransposons were designed and their sensitivities and specificities for detecting S. japonicum DNA were compared. Of these, a new 303-bp DNA sequence from the non-LTR retrotransposon (SjCHGCS19) had high sensitivity and specificity in detecting S. japonicum DNA. More importantly, we also found that both the SjCHGCS19 (303-bp sequence) and SjR2 (230-bp sequence) have high repetitive copies, higher genome proportions and active ESTs in the genome of S. japonicum. Our findings provide new insights into selecting suitable target sequences which may play a key role for the sensitive and specific detection of S. japonicum DNA.

The article summarizes the newest researches of antischistosomal drugs and discusses the possible alternatives to praziquantel from three aspects, so as to provide the evidence for the development of antischistosomal drugs.

A praziquantel analog 10-hydroxy praziquantel and eight praziquantel/peroxide conjugates were synthesized. The biological activity of these compounds was evaluated against juvenile and adult stages of Schistosoma japonicum. Unlike praziquantel, 10-hydroxy praziquantel exhibits activity against both juvenile and adult Schistosoma japonicumin. All hybrid compounds displayed modest to significant worm killing activity. The present study has important significance for the development of hybrid antischistosomal drugs.

A specific PCR assay for the detection of Schistosoma japonicum DNA in rabbit fecal and serum samples was developed by amplifying a 230-bp fragment from the sequence information of the clone G55A of the highly repetitive retrotransposon SjR2. The minimum amount of DNA detectable using the PCR assay was 0.8pg, and the expected PCR product was amplified when DNA equivalent of 1.1 egg from feces was used as template. In the meantime, serum anti-worm IgG was examined by ELISA. ELISA gave positive results at 4-6 weeks post-infection depending on the cercarial doses. The parasite eggs were detected in feces at 7 weeks post-infection. In contrast, S. japonicum DNA was detected in sera at first week post-infection, and it became negative at 10 weeks post-treatment, whereas the anti-worm IgG was still at high levels at 23 weeks post-treatment. These data demonstrated that the PCR assay established provides a potential tool for the early diagnosis and therapy evaluation for S. japonicum infection in humans.

To establish the ICOS transgenic mice schistosomiasis japonica model and to observe the immune response and immunopathology of the model.The transgenic mice were infected with Schistosoma japonicum. Spleen cells and sera of mice were harvested at week 4, 6, and 8 after infection. The cytokines IFN-gamma and IL-4 were measured in culture supernatans by ELISA. The serum IgG, IgGI and IgG2a were measured by ELISA at different period of infection. Liver tissue sections were prepared with HE staining. Liver granuloma formation was observed under microscope.The expression level of IFN-y showed no significant difference between ICOS transgenic mice and control, while that of IL-4 in ICOS transgenic mice was significantly up-regulated to 20.81+/-1.95 and 25.31+/-3.37 pg/ml at week 6 and 8 respectively (P0.01). The serum IgG and IgGl in ICOS transgenic mice were also significantly higher than those in control. Th2 differentiation index and lgC1/IgG2a were used to evaluate the immune regulation balance of Thl/Th2, and results showed that Th2 response in ICOS transgenic mice was significantly stronger than that of the control. The egg granuloma response in ICOS transgenic mice was also significantly stronger than that in control (P0.01). The rate of egg granuloma enlargement was 24.48% and 26.37% at week 6 and 8 respectively.The findings suggest that there is stronger Th2 type response in ICOS transgenic mice infected with Schistosoma japonicum and ICOS may play an important role in the egg granuloma formation of Schistosoma japonicum.

Background Schistosomiasis has decreased significantly in prevalence and intensity of infection in China, thus more accurate and sensitive methods are desperately needed for the further control of schistosomiasis. The present work aimed to assess the utility of the loop-mediated isothermal amplification (LAMP) for detection of light intensity infection or false-negative patients and patients post-treatment, targeting the highly repetitive retrotransposon SjR2 of Schistosoma japonicum. Methodology/ Principal Findings LAMP was first assessed in rabbits with low intensity infection (EPG, Author Summary Accurate diagnostic tests play a key role in patient management and control of schistosomiasis, especially in China where the prevalence and intensity of Schistosoma japonicum infection is low in recent years. The present study aimed to assess the utility of the loop-mediated isothermal amplification (LAMP) assay for detection of light intensity infection or false-negative patients and for the evaluation of chemotherapy effectiveness in patients, targeting the highly repetitive retrotransposon SjR2 of S. japonicum, using 110 serum samples of schistosomiasis patients. The results showed that the LAMP assay had high sensitivity of 95.1% for the diagnosis of S. japonicum infection with the lowest intensity (EPG

Background Granulomatous and fibrosing inflammation in response to parasite eggs is the main pathology that occurs during infection with Schistosoma spp. CD4+ T cells play critical roles in both host immune responses against parasitic infection and immunopathology in schistosomiasis,and coordinate many types of immune cells that contribute to fibrosis. ICOSL plays an important role in controlling specific aspects of T cell activation, differentiation, and function. Previous work has suggested that ICOS is essential for Th17 cell development. However, the immunopathogenesis of this pathway in schistosomiasis fibrosisis still unclear. Methodology/Principal Findings Using models of schistosomiasis in ICOSL KO and the C57BL/6 WT mice, we studied the role of the ICOSL/ICOS interaction in the mediation of the Th17 response in host granulomatous inflammation, particularly in liver fibrosis during S. japonicum infection, and investigated the immune responses and pathology of ICOSL KO mice in these models. The results showed that ICOSL KO mice exhibited improved survival, reduced liver granulomatous inflammation around parasite eggs, markedly inhibited hepatic fibrosis development, lower levels of Th17-related cytokines (IL-17/IL-21), Th2-related cytokines (IL-4/IL-6/IL-10), a pro-fibrotic cytokine (IL-13), and TGF-β1, but higher level of Th1-related cytokine (IFN-γ) compared to wild-type (WT) mice. The reduced progression of fibrogenesis was correlated with the down-regulation of Th17 and Th2 and the elimination of ICOSL/ICOS interactions. Conclusions/Significance Our findings suggest that IL-17-producing cells contribute to the hepatic granulomatous inflammation and subsequent fibrosis. Importantly, there was a clearly positive correlation between the presence of IL-17-producing cells and ICOS expression in ICOSL KO mice, and additional results indicated that Th17 was involved in the pathological tissue remodeling in liver fibrosis induced by schistosomiasis., Author Summary The full activation and differentiation of T cells into Th1, Th2 or Th17 cells requires costimulatory molecules and cytokines. ICOS has also been implicated in chronic inflammation and is critical for Th17 cell development. CD4+ IL-17-secreting T cells have been shown to contribute to pathology in some models of liver fibrosis. However, neither the significance nor the immunopathogenesis of this pathway have been elucidated in schistosomiasis fibrosis. The present study used the ICOSL KO mice to assess the role of the ICOSL/ICOS interaction in the mediation of the Th17 response in host granulomatous inflammation, particularly in liver fibrosis during S.japonicum infection. This study further clarifies the immune regulatory mechanism of fibrosis and sheds light on the understanding of the immunopathogenesis of Schistosoma-induced fibrosis. It might reveal new therapeutic targets that interfere with Th17 cell migration or differentiation in granulomas and the subsequent fibrosis following infection with S. japonicum.