Small change, big difference: A promising praziquantel derivative designated P96 with broad-spectrum antischistosomal activity for chemotherapy of schistosomiasis japonica.

BACKGROUND: Praziquantel (PZQ) has been the first line antischistosomal drug for all species of Schistosoma, 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 FINDINGS: One of the PZQ derivative christened P96 with the substitution of cyclohexyl by cyclopentyl was synthesized by School of Pharmaceutical Sciences of Shandong University. We investigated the in vitro and in vivo activities of P96 against different developmental stages of S. japonicum. Parasitological studies and scanning electron microscopy were used to study the primary action characteristics of P96 in vitro. Both mouse and rabbit models were employed to evaluate schistosomicidal efficacy of P96 in vivo. Besides calculation of worm reduction rate and egg reduction rate, quantitative real-time PCR was used to evaluate the in vivo antischistosomal activity of P96 at molecular level. In vitro, after 24h exposure, P96 demonstrated the highest activities against both juvenile and adult worm of S. japonicum in 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 against S. japonicum at 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 of S. japonicum. CONCLUSIONS: P96 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.

Synergistic effect of combination chemotherapy with praziquantel and DW-3-15 for Schistosoma japonicum in vitro and in vivo.

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.

MiR-130a-3p Alleviates Liver Fibrosis by Suppressing HSCs Activation and Skewing Macrophage to Ly6Clo Phenotype.

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 schistosomiasis in vivo and in vitro. The mice infected with Schistosoma 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 with S. 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 Ly6Clo phenotype, 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.

Seasonal variations and public search interests in Toxoplasma: a 16-year retrospective analysis of big data on Google Trends.

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<0.05), except for the UK. A peak in December-March and a trough in July-September (Canada, Ireland, the UK and the USA) were observed, while a peak in June/August and a trough in December/February (Australia, New Zealand) were also found. Moreover, the presence of seasonal patterns regarding RSV for 'Toxoplasma' between the Southern and Northern Hemispheres was also found (both p<0.05), with a reversed meteorological month. 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.

Increased risk of Toxoplasma gondii infection in cancer patients: A meta-analysis of current evidence based on case-control study.

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.

Early detection of circulating DNA of Schistosoma japonicum in sentinel mice models.

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.

[Dynamic alteration of CD154/CD40 and its effects on Th1/Th2 polarization in inducible co-stimulator ligand knockout mice infected with Schistosoma japonicum].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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 P<0.01] 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 P<0.05] 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 P<0.01] 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 P<0.05] 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 (P<0.05). However, the levels of IL-4 of the ICOSL-KO mice were significantly lower than those of the wild-type mice (P<0.05). 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 (P<0.01). Moreover, The Th2 differentiation index of the ICOSL-KO mice was significantly lower than that of the wild-type mice in post-infection (P<0.01). 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 (P<0.05). And the volume of liver egg granulomas of the ICOSL-KO mice was significantly smaller than that of the wild-type mice (P<0.01). CONCLUSION: 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.

DNA detection of Schistosoma japonicum: diagnostic validity of a LAMP assay for low-intensity infection and effects of chemotherapy in humans.

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<10). Then 110 patient sera from Hunan Province, China, and 47 sera after treatment by praziquantel were used to evaluate the diagnostic validity of LAMP. Meanwhile, 42 sera from healthy individuals in a non-endemic area, and 60 sera from "healthy" residents who were identified as being negative for feces examination and immuno-methods in an endemic area were also examined. The results showed that LAMP could detect S. japonicum DNA in sera from rabbits at 3rd day post-infection. Following administration of praziquantel, the S. japonicum DNA in rabbit sera became negative at 10 weeks post-treatment. Of 110 sera from patients, LAMP showed 95.5% sensitivity, and even for 41 patients with less than 10 EPG, the sensitivity of LAMP still reached to 95.1%. For 47 patients after treatment, the negative conversion rate of S. japonicum DNA in patient sera increased from 23.4%, 61.7% to 83.0% at 3 months, 6 months and 9 months post-treatment, respectively. No false-positive result was obtained for 42 human sera from non-endemic area, while for the 60 "healthy" individuals from endemic area, 10 (16.7%) individuals were positive by LAMP, which suggested that these individuals might be false-negative patients. CONCLUSIONS/ SIGNIFICANCE: The present study demonstrated that the LAMP assay is sensitive, specific, and affordable, which would help reduce schistosomiasis transmission through targeted treatment of individuals, particularly for those with negative stool examinations who may yet remain infected. The LAMP assay may provide a potential tool to support schistosomiasis control and elimination strategies.

Th17 down-regulation is involved in reduced progression of schistosomiasis fibrosis in ICOSL KO mice.

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-ß1, 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.

[Effect of ICOS signaling on CD154/CD40 expressions in mice infected with Schistosoma japonicum].

OBJECTIVE: To explore the effect of ICOS signaling on the CD154/CD40 expressions and immunopathology in mice infected with Schistosoma japonicum. METHODS: 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. RESULTS: 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 P < 0.05). 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 P < 0.05). The volumes of liver egg granulomas of the ICOS-Tg mice were significantly bigger than those of the wildtype mice (P < 0.05 or P < 0.01). CONCLUSIONS: 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.

An artemisinin derivative of praziquantel as an orally active antischistosomal agent.

BACKGROUND: Schistosomiasis is a major health problem in tropical and sub-tropical areas caused by species of trematode belonging to the genus Schistosoma. The treatment and control of this disease has been relying on the use of a single drug praziquantel. However, the drug resistance concern urged the development of new drugs against schistosoma. Here, we report our systematic biological evaluation of DW-3-15, a new lead compound developed based on our conjugation design rationale as an effective anti-schistosomal agent. METHODOLOGY/PRINCIPAL FINDINGS: The antischistosomal activity of DW-3-15 was systematically evaluated in S. japonicum infected mouse model for its stage-sensitivity and dose response. The results revealed that DW-3-15 exhibited 60-85% worm reduction rate against different development stage of worm. Scanning electron microscopy (SEM) observation indicated that DW-3-15 may damage to the tegument of male schistosomes. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that DW-3-15 showed potent anti-schistosomal activities in vivo. The results strongly support our conjugation design strategy of artemisinin analogs and further development of DW-3-15 as a new lead compound as anti-schistosomal agent.

[In vitro effect of photoactivated hypericin on anti-Schistosoma japonicum adult male worms].

OBJECTIVE: To investigate the in vitro effect of photoactivated hypericin on anti-Schistosoma japonicum adult male worms. METHODS: 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). RESULTS: 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. CONCLUSION: Photoactivated hypericin has anti-Schistosoma japonicum adult male worms effect in vitro.

Development-specific differences in the proteomics of Angiostrongylus cantonensis.

Angiostrongyliasis is an emerging communicable disease. Several different hosts are required to complete the life cycle of Angiostrongylus cantonensis. However, we lack a complete understanding of variability of proteins across different developmental stages and their contribution to parasite survival and progression. In this study, we extracted soluble proteins from various stages of the A. cantonensis life cycle [female adults, male adults, the fifth-stage female larvae (FL5), the fifth-stage male larvae (ML5) and third-stage larvae (L3)], separated those proteins using two-dimensional difference gel electrophoresis (2D-DIGE) at pH 4-7, and analyzed the gel images using DeCyder 7.0 software. This proteomic analysis produced a total of 183 different dominant protein spots. Thirty-seven protein spots were found to have high confidence scores (>95%) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Comparative proteomic analyses revealed that 29 spots represented cytoskeleton-associated proteins and functional proteins. Eight spots were unnamed proteins. Twelve protein spots that were matched to the EST of different-stage larvae of A. cantonensis were identified. Two genes and the internal control 18s were chosen for quantitative real-time PCR (qPCR) and the qPCR results were consistent with those of the DIGE studies. These findings will provide a new basis for understanding the characteristics of growth and development of A. cantonensis and the host-parasite relationship. They may also assist searches for candidate proteins suitable for use in diagnostic assays and as drug targets for the control of eosinophilic meningitis caused by A. cantonensis.

Development of a novel class of pyrrolo-[1,2,5]benzothiadiazepine derivatives as potential anti-schistosomal agents.

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.

[Effect of immune response mediated by ICOS signaling pathway on hepatic fibrosis in mice infected with Schistosoma japonicum].

OBJECTIVE: To investigate the effect of Th2 polarization mediated by ICOS signaling pathway on hepatic fibrosis in mice infected with Schistosoma japonicum. METHODS: 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. RESULTS: 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 (P < 0.05). However, Th1 cytokines IFN-gamma and IL-12 showed no significant difference between the two groups (P > 0.05). 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 (P < 0.05 or P < 0.01). 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 (P < 0.05 or P < 0.01, 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 (P < 0.05). The levels of HA on 7, 12, 16, and 20 weeks post-infection (P < 0.05 or P < 0.01) and HYP on 12, 16, and 20 weeks post-infection (P < 0.05) 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 (P < 0.05 or P < 0.01); 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 (P < 0.05). 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 (P > 0.05). CONCLUSION: 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.

The sources and metabolic dynamics of Schistosoma japonicum DNA in serum of the host.

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.

Sensitive and specific target sequences selected from retrotransposons of Schistosoma japonicum for the diagnosis of schistosomiasis.

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.

Praziquantel derivatives exhibit activity against both juvenile and adult Schistosoma japonicum.

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.

[Advances in research on antischistosomal drugs].

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.

Schistosoma japonicum: a PCR assay for the early detection and evaluation of treatment in a rabbit model.

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.