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Common in fungal extracellular membrane (CFEM) domain is unique in fungal proteins and some of which contribute to iron acquisition in yeast. However, their roles in iron acquisition remain largely unknown in filamentous fungi. In this study, 12 CFEM-containing proteins were bioinformatically identified in the filamentous entomopathogenic fungus Beauveria bassiana, and the roles of 11 genes were genetically characterized. Transmembrane helices were critical for their association with intracellular membranes, and their number varied among proteins. Eleven CFEM genes significantly contribute to vegetative growth under iron starvation and virulence. Notably, the virulence of most disruptants could be significantly weakened by a decrease in iron availability, in which the virulence of ΔBbcfem7 and 8 strains was partially recovered by exogenous hemin. ΔBbcfem7 and 8 mutants displayed defective competitiveness against the sister entomopathogenic fungus Beauveria brongniartii. All 11 disruptants displayed impaired growth in the antagonistic assay with the saprotrophic fungus Aspergillus niger, which could be repressed by exogenous ferric ions. These findings not only reveal the systematic contributions of CFEM proteins to acquire two forms of iron (i.e. heme and ferric ion) in the entire lifecycle of entomopathogenic fungi but also help to better understand the mechanisms of fungus-host and inter-fungus interactions.

Yang Bai,1,* Fei Guan,1,* Feifan Zhu,1 Chunjie Jiang,1 XiaoXiao Xu,1 Fang Zheng,2 Wenqi Liu,1 Jiahui Lei1 1Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China; 2Department of Immunology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jiahui LeiDepartment of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of ChinaTel +86-13329722645Fax +86-27-83691246Email leijiahui@hotmail.comPurpose: Schistosoma japonicum-infected IL-33 and ST2 gene deficiency (IL-33−/− and ST2−/−, respectively) mice were used to explore the role of the IL-33/ST2 axis in liver pathology targeting regulatory T cells (Treg)/T helper 17 cells (Th17).Materials and Methods: Each mouse was infected percutaneously with 20 S. japonicum cercariae. Hepatic mass index (HMI), liver egg granulomas, hepatic fibrosis biomarkers and serum levels of alanine aminotransferase (ALT) were investigated. Treg and Th17 frequency was determined by flow cytometry. Expressions of Foxp3, ST2, TGF-β 1, IL-10, RORγt, and IL-17A were measured via quantitative real-time polymerase chain reaction (qRT-PCR). Concentrations of TGF-β 1, IL-10 and IL-17A were tested with ELISA. In vitro experiments, mRNA expressions of Foxp3, TGF-β 1, IL-10, Atg5, Beclin-1 and p62 associated with polarization of Treg by recombinant mouse IL-33 (rmIL-33) were detected by qRT-PCR.Results: An increased expression of IL-33/ST2 was shown in S. japonicum-infected mice. Deficiency of IL-33 or ST2 gene led to an aggravated liver pathology, which was evidenced by elevated hepatic granuloma volume, HMI and ALT levels and fibrosis, which was demonstrated by increased hepatic collagen deposition in the infected mice. Injection of rmIL-33 into the infected IL-33−/− mice strongly abrogated the liver pathology and fibrosis, whereas no detectable effect with injecting rmIL-33 into the infected ST2−/− mice. Furthermore, depletion of the IL-33/ST2 axis inhibited Treg, accompanied by increased Th17. rmIL-33 treatment upregulated Treg and downregulated Th17 in the infected IL-33−/− mice, while no effect in the infected ST2−/− mice. rmIL-33 led to elevated expressions of Atg5, Beclin-1 and inhibited expression of p62 in expansion of Treg.Conclusion: The IL-33/ST2 axis plays a protective role in S. japonicum infected mice, which is closely related to increasing Treg responses as well as suppressing Th17 responses. Expansion of Treg by IL-33 may be associated with its regulation of autophagy.Keywords: IL-33, ST2, liver pathology, fibrosis, Treg, Th17, autophagy

Acetyl-coenzyme A (CoA) synthetase (Acs) links cellular metabolism and physiology by catalyzing acetate and CoA into acetyl-CoA. However, the biological roles of Acs are not well studied in entomopathogenic fungi. In this study, two Acs proteins (BbAcs1 and BbAcs2) was functionally characterized in the filamentous insect pathogenic fungus Beauveria bassiana. BbAcs1 and BbAcs2 localize in cytoplasm and peroxisome, respectively. BbAcs1 contributes to vegetative growth on fatty acids as carbon source, and BbAcs2 did not. Both genes did not contribute to fungal response to stresses. The BbAcs1 loss conferred a slight influence on conidiation, and did not result in the defects in blastospore formation. On the contrary, BbAcs2 significantly contributes to lipid metabolism in germlings, blastospore formation, and virulence. The results indicated that Acs2 played a more predominant role than Acs1 in B. bassiana, which links the acetyl-CoA metabolism with the lifestyle of entomopathogenic fungi.

PurposeThis study aims to determine the prevalence of malaria and HIV seropositivity among children with undernutrition in the Democratic Republic of the Congo.MethodsA cross-sectional study of undernourished children aged between 12 and 60 months in Kalembe-Lembe hospital was carried out. Blood samples were collected for the analyses of malaria parasite, haemoglobin and haematocrit levels. HIV serostatus was determined with rapid HIV antibody tests and enzyme-linked immunosorbent assay. Logistic regression analyses were used to identify clinical predictors of HIV seropositivity.ResultsOf 225 children, 88.9% had malaria; the parasite loads were 16 000 para per μL (38.0%); 24 400 para per μL (56.8%), P < 0.001 and malaria and associated HIV infection accounted for 29.2%. In children aged >12 months, HIV seroprevalence was 29.3%; 86.0% had undernutrition and malaria, 6.8% had undernutrition and HIV and 4.3% had undernutrition, HIV and malaria (P < 0.001). The occurrence of at least three or more symptoms was highly specific (96.4–100.0%) for HIV seropositivity (P < 0.05). The overall mortality rate was 18.4%, higher in children with malaria and HIV (39.6% vs 12.2%, P < 0.001) and those with lower weight gain (4.3 vs 7.5 g kg−1 day−1, P < 0.001).ConclusionsThere was high prevalence of malaria and HIV and mortality among severely undernourished children with malaria and HIV.

Trichinellosis is one of most neglected foodborne zoonoses worldwide. During Trichinella spiralis infection, the intestinal immune response is the first line of defense and plays a vital role in the host’s resistance., Trichinellosis is one of most neglected foodborne zoonoses worldwide. During Trichinella spiralis infection, the intestinal immune response is the first line of defense and plays a vital role in the host’s resistance. Previous studies indicate that purinergic P2X7 receptor (P2X7R) and pyrin domain-containing protein 3 (NLRP3) inflammasome are involved in the intestinal immune response in T. spiralis infection. However, the precise role of P2X7R and its effect on NLRP3 remains largely underdetermined. In this study, we aimed to investigate the role of P2X7R in the activation of NLRP3 in macrophages during the intestinal immune response against T. spiralis. We found that T. spiralis infection upregulated expression of P2X7R and activation of NLRP3 in macrophages in mice. In vivo, P2X7R deficiency resulted in increased intestinal adult and muscle larval burdens, along with decreased expression of NLRP3/interleukin-1β (IL-1β) in macrophages from the infected mice with T. spiralis. In In vitro experiments, P2X7R blockade inhibited activation of NLRP3/IL-1β via NF-κB and thus reduced the capacity of macrophages to kill newborn larvae of T. spiralis. These results indicate that P2X7R mediates the elimination of T. spiralis by activating the NF-κB/NLRP3/IL-1β pathway in macrophages. Our findings contribute to the understanding of the intestinal immune mechanism of T. spiralis infection.

Aims The Schistosoma japonicum (S japonicum)-infected ApoE gene deficiency (ApoE-/- ) mice were used to determine effect of ApoE on hepatic immunopathology. Methods Murine activities and appetite, body weight, and ratio of liver weight to its body weight (Hepatic mass index, HMI) were observed. Worm load and liver egg burden were evaluated as the infection intensity. Number and size of liver egg granulomas and serum levels of alanine aminotransferase (ALT) were investigated. We analysed hepatic fibrosis by markers of fibrosis in tissue, detected hepatic Th17 and Treg frequency by flow cytometry, and measured hepatic expressions of RORγt, Foxp3, IL-17A and TGF-β1 via qPCR. Lipid metabolism was determined by serum levels of cholesterol (TC) and triglyceride (TG) as well as hepatic Oil red O staining. Results In the infected ApoE-/- mice, the increased infection intensity aggravated the hepatic immunopathology (evidenced by increased HMI, elevated egg granulomas and increased ALT levels) and fibrosis (increased hepatic collagen deposition). ApoE deficiency resulted in significantly elevated ratio of hepatic Th17/Treg and higher serum levels of TC and TG, along with higher level of hepatic Oil red O staining. Conclusions ApoE deficiency promotes hepatic pathology and fibrosis by exacerbating Th17/Treg imbalance and altering lipid metabolism in murine schistosomiasis japonica.

Background: Hypoglycemia is an important complication of severe malaria, especially in children and pregnant women. In 2017 Kikimi hospital changed quinine treatment policy from 15 mg/kg loading (plus 10 mg/kg 12-hourly) to 20 mg/kg loading (plus 10 mg/kg 8-hourly) to comply with new WHO guidelines. This presented us with the opportunity to examine whether there was any dose relationship of quinine and hypoglycemia occurrence, in particular, in severe malaria in children from 5 to 144 months. Methods: The study was retrospective of children aged 5 to 144 months admitted to KIkimi hospital with severe malaria between April 2017 and July 2018, before and after the introduction of the new quinine treatment regimen by the WHO. The patient's blood glucose at four hours was measured until intravenous quinine was stopped. Clinical events immediately before or simultaneously with each episode of hypoglycemia (glucose

Crohn’s disease and ulcerative colitis are two forms of inflammatory bowel diseases. They are autoimmune disorders caused by excessive inflammatory response to antigens in the intestine. In addition to the hygiene hypothesis which suggests the potential application of helminthic infection in the treatment of inflammatory bowel diseases, helminthic infection has shown preventive and treatment effects in animal models of inflammatory bowel disease. Clinical trials have been initiated. For example, Trichuris suis ova infection at a certain dose has a promising efficacy in the treatment of inflammatory bowel diseases. Helminthic infection may also have adverse effects. Therefore, helminth-derived immunomodulatory molecules are needed to overcome these problems. It is traditionally considered that the Th1/Th2 axis is involved in the mechnisms of the efficacy of helminthic infection. More recent research has pointed out the much more participation of the Tregs/Th17 axis. The mechanisms may also involve other palyers such as mucosal barrier, Toll like receptor and macrophages. This paper reviews the effect and mechanism of helminthic infection on the prevention and treatment of inflammatory bowel disease.

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

Allograft inflammatory factor-1 (AIF-1) plays an important role in various inflammatory conditions. Our previous study demonstrated that AIF-1 was over-expressed in the liver of BALB/c mice infected with Schistosoma japonicum and played significant role in the pathogenesis of schistosomiasis. The aim of this study was to focus on the effect of AIF-1 treatment on liver fibrosis and necrosis of BALB/c mice infected with S. japonicum. Seventy-two BALB/c mice were infected with cercariae of S. japonicum and then divided into three groups: AIF-1-treated group, saline-treated group, and control group. The vital signs, liver function, egg load, and hepatic pathological changes of the mice were assessed, and the levels of AIF-1 and TNF-α in the liver and spleen were measured at 5, 8, and 14 weeks postinfection. The treatment of AIF-1 on the mice infected with S. japonicum suppressed the expression of TNF-α and increased the effectiveness of AIF-1 in the liver and spleen at 14 weeks postinfection. Histopathological analysis and Masson trichrome staining for the liver tissues showed that the liver fibrosis and necrosis were alleviated previously compared with other infected mice at 14 weeks postinfection. The treatment of AIF-1 on the mice infected with S. japonicum can alleviate hepatic fibrosis and necrosis which indicate that AIF-1 use may prevent and cure the liver fibrosis.

In a previous study we demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) contributed to the escape of Schistosoma japonicum (S. japonicum) from the host's immune responses. In this paper, we studied the effect of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on CD4(+)CD25(+) Tregs in murine Schistosomiasis japonica and its corresponding role in the immune evasion of S. japonicum in mice. The results showed substantial reductions of worm burden and egg production in worm groups treated with anti-CD25 or anti-CTLA-4 monoclonal antibodies (mAb) compared to an infected but untreated control. The reduction effect was even enhanced in an experimental group co-treated with both mAbs. Compared to the control group, the percentage of CD4(+)CD25(+) Tregs was very much lower in the anti-CD25 mAb group as determined by FACS analyses and higher in the anti-CTLA-4 mAb group. ELISA analyses showed that both the anti-CTLA-4 mAb and the co-treated groups had higher levels of cytokines compared to the control group as well as larger egg granuloma sizes as determined by microscopical analyses of liver sections of infected mice. These results suggest that treatment with an anti-CTLA-4 mAb allows the host to clear S. japonicum, but at the cost of elevated pathological damage. The latter indicated a role of CTLA-4 in granuloma formation. Moreover, CD4(+)CD25(+) Tregs and CTLA-4 may exert synergistic effects during immune evasion processes by enhancing Th1-type immune response.

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

SUMMARYA traditional assumption is that schistosome cercariae lose their tails at the onset of penetration. It has, however, recently been demonstrated that, for Schistosoma mansoni, cercarial tails were not invariably being shed as penetration took place and a high proportion of tails entered human skin under experimental conditions. This phenomenon was termed delayed tail loss (DTL). In this paper, we report that DTL also happens with S. japonicum cercariae during penetration of mouse skin. It occurred at all cercarial densities tested, from as few as 10 cercariae/2·25 cm2 of mouse skin up to 200 cercariae. Furthermore, it was demonstrated that there was a density-dependent increase in DTL as cercarial densities increased. No such density-dependent enhancement was shown for percentage attachment over the same cercarial density range.

Cimetidine (CIM), a histamine-2-receptor antagonist, has a long history of safe use in gastric acid-mediated gastrointestinal disorders. In this study, we used CIM, as an adjuvant, with pEGFP-Sj26 GST (the recombinant plasmid containing enhanced green fluorescent protein gene and the gene encoding 26kDa glutathione S-transferase of Schistosoma japonicum) DNA vaccine to immunized mice and attempted to enhance the protective effect against S. japonicum. The results showed that the reduction rate of worm and egg burdens in the pEGFP-Sj26GST plus CIM group were 79.0% and 68.4%, respectively, significantly higher than that in pEGFP-Sj26GST alone group (27.0% and 22.5%, P

CD4(+) T-helper (Th) cell is widely recognized to be capable of influencing worm development and egg granuloma formation after schistosome infection. Interleukin (IL)-12 and IL-4 play key roles in regulation of Th cell differentiation. In the present study, we subcutaneously inoculated mice with hybridoma cells secreting monoclonal antibodies to neutralize IL-12 and IL-4 and explored the effects of IL-12 and IL-4 deficiency on the worm development and granuloma formation in mice infected with cercariae of Schistosoma japonicum. It was found that deficiency of host IL-12 and IL-4 supported normal parasite survival and fecundity. However, worm development (length and female fecundity) was significantly enhanced in anti-IL-12-treated mice. Mean length of worms in anti-IL-12-treated group was significantly greater than that of intact controls on day 28 after infection (females, 11.84 ± 1.20 mm vs. 9.45 ± 1.34; males, 9.35 ± 1.21 mm vs. 8.10 ± 0.85 mm, p 0.05). Liver egg load per pair of worms (1,770.12 ± 470.67 vs. 806.08 ± 232.37, p 0.05) and uterine egg load of ovigerous females (93.08 ± 27.85 vs. 46.05 ± 34.24, p 0.05) in anti-IL-12-treated mice were significantly higher than those in intact control 28 days postinfection. But these effects diminished 42 days postinfection (p 0.05). Granuloma size in anti-IL-12-treated mice was significantly larger than that in intact mice 42 days postinfection (398.3 ± 80.7 μm vs. 294.4 ± 72.2 μm, p 0.05). Granuloma fibrosis dramatically intensified in anti-IL-12-treated mice but diminished in anti-IL-4-treated mice. The results suggest that IL-12 may play an impeditive role in the development of S. japonicum and in granuloma formation as well as fibrosis. IL-4 may promote granuloma formation but have no effect on worm development.

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

Asthma, a chronic inflammatory disorder of the airways, is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. It has been shown that helminth infections including Schistosoma mansoni may modulate atopic diseases including asthma. In the present study, BALB/c mice were infected with bisexual and unisexual (male) S. japonicum, respectively, prior to ovalbumin (OVA) sensitization and challenge. Compared to mice with OVA sensitization/challenge alone, S. japonicum infection led to a significant decrease of eosinophil accumulation in bronchoalveolar lavage fluid (BALF) collected 48 h postchallenge, as well as to a marked reduction in inflammatory cell infiltration around the airways and pulmonary blood vessels. Compared to OVA-immunized uninfected mice, the level of OVA-specific serum IgE as well as interleukin (IL)-4 and IL-5 in BALF were reduced, but IL-10 was strongly elevated in mice with preexisting S. japonicum infection prior to OVA immunization. These results suggest that both bisexual and male S. japonicum infections may modulate the development of allergic asthma.

It is widely accepted that the immune response of the host attacks the parasite and the parasite appears to develop strategies to evade the assault. However, there is increasing evidence that the development of a parasite may be also positively influenced by the immune response of host. In this paper, we explore the effects of T cell deficiency on the development of the worms and granuloma formation in mice infected with cercariae of Schistosoma japonicum. T cell-deficient (nude) mice supported normal parasite survival and fecundity, but compared to normal mice delayed the worms' development (length and female fecundity) until 28 days after infection. However, these differences equaled out at 35 and 42 days. The nude mice apparently suppressed the size of granuloma in the livers around the eggs of S. japonicum. The granulomas were composed predominantly of neutrophils but with significantly fewer eosinophils in nude compared to normal mice. In addition, hepatocyte necrosis occurred in the vicinity of granulomas in nude but not normal mice. This is consistent with egg-granuloma formation in the host being dependent on T-lymphocyte functions and shows that the effect of T cell deficiency on the development of the worms is transitory in S. japonicum.

Crohn's disease (CD) is considered to be caused by a disorder of the immune system and helminth infections may interact with development of the disease. We induced colitis in mice by trinitrobenzenesulfonic acid (TNBS) and observed the effects of intraperitoneally injected eggs of Schistosoma japonicum on the course of the disease. The inflammation in the colon was reduced in egg-treated mice and secretion of IFN-gamma (a Th1 cytokine) by cultured spleen cells in vitro was greatly suppressed, and of IL-4, IL-5 and IL-10 (Th2 cytokines) significantly elevated after egg injection. Also, the percentage of regulatory T-cells (Tregs) was increased in the spleens of egg-exposed mice with TNBS-induced colitis compared to non-egg exposed animals. The data suggest that Tregs may be activated by S. japonicum eggs and play a role in restoring immune disorders in TNBS-induced colitis of mice.

In 1975, an ancient corpse buried in 167 BC was found at Jiangling County, Hubei Province of China. The eggs of Clonorchis sinensis found in the gall bladder of the corpse were preserved well. In the present paper, we extracted the genomic DNA from the ancient eggs and modern eggs, respectively, and the internal transcribed spacer 1 and 2 (ITS1 and ITS2) at ribosomal RNA genes were studied. The results show that ITS2 sequences from the ancient sample were identical with those from modern samples, but in ITS1 differences in 15 nucleotide positions were found between the ancient and modern samples. The results demonstrated that it is possible to extract and sequence DNA from ancient parasite eggs. The ITS1 sequence obtained differed from all modern ones available to date. This might indicate sequence divergence through time, or might reflect a sequence polymorphism that may eventually be found also in modern samples.

Background Clonorchiasis, caused by Clonorchis sinensis, is widely distributed in Southeast Asia including China. Clonorchiasis is included in control programs of neglected tropical diseases by World Health Organization (WHO) because it is one of the major health problems in most endemic areas. Diagnosis of clonorchiasis plays a key role in the control programs. However, so far, there is no satisfactory method for clonorchiasis because of low sensitivity, poor practicality and high false positivity of available diagnostic tools. Methodology/Principal Findings We developed an immunomagnetic bead enzyme-linked immunosorbent assay (ELISA) based on IgY (egg yolk immunoglobulin) against cysteine proteinase of C. sinensis for detection of circulating antigen in serum samples of patients infected with C. sinensis. The polyclonal IgY, coated with magnetic beads, was used as a capture antibody and a monoclonal IgG labeled with horseradish peroxidase as a detection antibody in the IgY-based immunomagnetic bead ELISA system (IgY-IMB-ELISA). The results showed that the sensitivity of IgY-IMB-ELISA was 93.3% (14 of 15) in cases of heavy infection (5000 to 9999 eggs per gram feces, i.e, EPG 5000–9999), 86.7% (13 of 15) in cases of moderate infection (EPG 1000–4999) and 75.0% (9 of 12) in cases of light infection (EPG

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine kurarinone in rat plasma using chlorzoxazone as the internal standard (IS). Sample preparation was accomplished through a liquid-liquid extraction procedure with ethyl acetate to 0.2 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 437.0→301.2 for kurarinone and m/z 168.1→132.1 for IS. The linearity of this method was found to be within the concentration range of 20-2000 ng/mL with a lower limit of quantification of 20 ng/mL. Only 3.0 min was needed for an analytical run. The matrix effect was 94.7-107.2% for kurarinone. The intra- and inter-day precision (RSD%) were less than 8.2% and accuracy (RE%) was within ±9.0%. The recovery ranged from 77.3% to 85.6%. Kurarinone was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of kurarinone in rats.

Vaccination is the most effective and cost-effective way to treat hepatitis B virus (HBV) infection. Collective data suggest that helminth infections affect immune responses to some vaccines. Therefore, it is important to reveal the effects of helminth infections on the efficacy of protective vaccines in countries with highly prevalent helminth infections. In the present work, effects of Trichinella spiralis infection on the protective efficacy of HBV vaccine in a mouse model were investigated. This study demonstrated that the enteric stage of T. spiralis infection could inhibit the proliferative response of spleen lymphocytes to hepatitis B surface antigen (HBsAg) and lead to lower levels of anti-HBsAg antibodies, interferon-γ, and interleukin (IL)-2, along with higher levels of IL-4 and IL-5. However, these immunological differences are absent in the muscle stage of T. spiralis infection. The results suggest that the muscle stage of T. spiralis infection does not affect the immune response to HBV vaccination, while the enteric-stage infection results in a reduced immune response to HBsAg.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease with rising prevalence. Increasing evidence has demonstrated that resveratrol, a dietary phytochemical, is capable of attenuating NAFLD development and progression; however, results from clinical studies are inconsistent and inconclusive. Here, we conducted a meta-analysis to evaluate the efficacy of resveratrol on NAFLD, using several parameters to provide new insights for clinical application. We systematically searched EMBASE, PubMed, Science Citation Index, Elsevier, and Cochrane Library databases for studies published up to date (July 2016), in English, to identify and screen eligible, relevant studies. Either a fixed-effect model or random model was used to estimate mean difference (MD) and 95% confidence intervals (CIs) for the effect of resveratrol on NAFLD. Four randomized, double-blinded, placebo-controlled trials involving 156 patients were included in the meta-analysis. Levels of low-density lipoprotein (MD = 0.47, 95% CI: 0.21, 0.74, P < 0.05) and total cholesterol (MD = 0.49, 95% CI: 0.18, 0.80, P < 0.05) were higher in the resveratrol treatment groups than in placebo control groups, whereas other parameters were not altered. Overall, this study indicates that resveratrol treatment has negligible effects on attenuating NAFLD, given the small improvement in NAFLD features. More high-quality clinical trials of resveratrol for NAFLD are required to confirm these results.

Liver fibrosis is a reversible wound-healing process aimed at maintaining organ integrity, and presents as the critical pre-stage of liver cirrhosis, which will eventually progress to hepatocellular carcinoma in the absence of liver transplantation. Fibrosis generally results from chronic hepatic injury caused by various factors, mainly viral infection, schistosomiasis, and alcoholism; however, the exact pathological mechanisms are still unknown. Although numerous drugs have been shown to have antifibrotic activity in vitro and in animal models, none of these drugs have been shown to be efficacious in the clinic. Importantly, hepatic stellate cells (HSCs) play a key role in the initiation, progression, and regression of liver fibrosis by secreting fibrogenic factors that encourage portal fibrocytes, fibroblasts, and bone marrow-derived myofibroblasts to produce collagen and thereby propagate fibrosis. These cells are subject to intricate cross-talk with adjacent cells, resulting in scarring and subsequent liver damage. Thus, an understanding of the molecular mechanisms of liver fibrosis and their relationships with HSCs is essential for the discovery of new therapeutic targets. This comprehensive review outlines the role of HSCs in liver fibrosis and details novel strategies to suppress HSC activity, thereby providing new insights into potential treatments for liver fibrosis.

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

To investigate the dynamic expressions of IL-17 and IL-23 in mice with Schistosoma japonicum infection.A murine model of the infection of S. japonicum was established. The suspension of spleen cells incubated with ConA was collected at 0, 1, 2, 4, 6, 8 and 10 weeks post-infection. Sandwich ELISA and RT-PCR were used to measure the expressions of IL-17A and IL-23p19 on protein and mRNA level.The dynamic changes of IL-17A and IL-23p19 showed a positive correlation. The level of IL-17A increased and reached the peak at 1 week after the infection, reduced at 4 weeks after the infection, and was even lower at 8 weeks post-infection. The level of IL-17 mRNA increased at 1 week post-infection, and then decreased gradually at 2 weeks post-infection. Being consistent with the dynamic expression of IL-17A, the IL-23p19 expression increased at 1 week post-infection, went up to the peak at 2 weeks post-infection, and gradually reduced into the normal level at 4 weeks. However, the expression of IL-23p19 mRNA fluctuated in the normal range, increased at 4 weeks post-infection, and reached the peak at 6 weeks post-infection.IL-17 and IL-23 are of co-expression in the mice after schistosome infection. There is a significant increase in the early stage of the infection. IL-17 and IL-23 may play important roles during the immune process in the very early stage of Schistosoma infection.

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

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

To investigate the effect of ageing on the immune responses against Schistosoma japonicum infection in mice.Female BALB/c mice were divided into young group (2 months) and old group (18 months), each composed of 8 mice. Each mouse was percutaneously infected with 40 +/- 1 S. japonicum cercariae. At 6 weeks post-infection, the mice were sacrificed, and the spleens were removed and single-cell suspensions of splenocytes were prepared. Worms were perfused from hepatic portal system and counted. The number of eggs in the liver was determined after KOH digestion. Mean single-egg granulomas sizes were determined in stained histological sections. Splenocyte proliferation responses were analyzed by MTT colorimetry. Level of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the splenocyte culture supernatants was determined by ELISA.The worm burden and egg per gram of liver in old mice [19.75 +/- 1.95, (1.59 +/- 1.05) x 10(4)] were significantly lower than that of young mice [26.00 +/- 2.42, (208 +/- 0.87) x 10(4)] (P0.05). The mean volume of single-egg granulomas of the livers in old mice [(30.13 +/- 10.97) x 10(3) mm3] was significantly lower than that of the young mice [(47.02 +/- 24.13) x l0(3) mm3] (P0.05).of T cell proliferation showed that the splenocytes had poorer immune reactivity to ConA in old mice (SI: 1.08 +/- 0.12) than that in young mice (SI: 131 +/- 0.14) (P0.05). Levels of IFN-gamma and IL-4 in the splenocyte culture supernatants [(24.05 +/- 6.24), (4.15 +/- 0.68) pg/ml] from old mice were lower than that of young mice [(34.25 +/- 869), (7125 +/- 0.83) pg/ml](P0.05).Ageing down-modulates the immune responses and the poorer immune reactivity might decrease pathological alterations in mice infected with Schistosoma japonicum.

To investigate the effect of antigens of different stage Schistosoma japonicum on airway inflammation in a murine model of asthma.48 female BALB/c mice were randomly divided into eight groups. Mice in group A were given normal saline of equal volume as control. Group B was asthma model which was established by intraperitoneal and intranasal challenge with OVA. Mice in groups C, D and E were immunized with soluble egg antigen (SEA), soluble male worm antigen (SWA), and schistosomulum antigen (SSA) respectively 4 times in a week interval, followed by OVA sensitization as in group B 1 week after the final immunization. Mice in groups F, G, and H were immunized with SEA, SWA, and SSA respectively but sensitized and challenged with saline instead of OVA. 48 hours after asthma was induced, the mice were sacrificed. Leukocytes and eosinophils were counted in bronchoalveolar lavage fluid (BALF). The level of IL-5, IL-10 and IFN-gamma in BALF was detected. Pathologic changes in lung tissues were observed.Inflammation cells, especially eosinophils, appeared in airways of mice in groups B, C, D and E, but with much less number in groups C, D and E. No inflammation cells were seen in airways of group A mice. The number of leukocytes, eosinophils and level of IL-5 in BALF of group B [(98.4 +/- 16.1) x 10(4)/ml, (17.6 +/- 4.3) x 10(4)/ml, (197.9 +/- 36.5) pg/ml respectively] were significantly higher than those of group A [(8.2 +/- 1.1) x 10(4)/ml, (0.02 +/- 0.01) x 10(4)/ ml, (12.3 +/- 7.4) pg/ml], however the levels of IL-10 and IFN-gamma were significantly lower than that of group A (P0.05). The number of leukocytes, especially eosinophils, in BALF of groups C, D and E was significantly lower than that of group B. The level of IL-5 in BALF of groups C, D and E was significantly reduced, while that of IL-10 and IFN-gamma in BALF of the 3 groups was significantly higher than group B (P0.05).The immunization with S. japonicum antigens can effectively modulate the level of cytokines and inhibit the eosinophil infiltration and airway inflammation in asthmatic mice.

We developed a novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) for detection of circulating antigen (CA) in sera of mice infected with Schistosoma japonicum. The assay involved the use of chicken polyclonal antibodies IgY against soluble egg antigens (SEA) of S. japonicum as a capture antibody and anti-SEA mouse monoclonal antibody NP28-5B labeled horseradish peroxidase (HRP) as a detecting antibody. Two groups of BALB/c mice infected with S. japonicum cercariae were used: lightly infected mice (infected with 10 S. japonicum cercariae) and heavily infected mice (infected with 30 S. japonicum cercariae). The CA was detectable as early as 4 and 5 weeks after infection in the sera of heavily and lightly infected mice, respectively. The CA levels rose rapidly and reached a peak in 8 weeks after infection and then remained a plateau for at least another 6 weeks in both groups. Moreover, the effect of praziquantel on the CA levels was also investigated. The heavily infected mice were treated with praziquantel and the CA levels in sera increased dramatically in the first week post-treatment and then decreased to the control level by 6 weeks after treatment. The novel assay appears to be sensitive for detection of schistosomal antigenemia and valuable to judge the efficacy of chemotherapy in murine schistosomiasis. © 2009 Elsevier B.V. All rights reserved. Refereed/Peer-reviewed

To study the suppression of Schistosoma japonicum eggs against the trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice.50 female BALB/c mice (6-8 week-old) were randomly divided into normal control group, ethanol control group, schistosome egg immunized control group, TNBS-induced colitis group and TNBS-induced colitis with egg immunization group. In TNBS-induced colitis with egg immunization group, mice were immunized 4 times with 10,000 schistosome eggs by intraperitoneal injection with one-week interval. On day 6 after the last immunization the mice were induced by TNBS and the body weight, histological change on colon and level of cytokines of mice were observed in egg-immunized and -unimmunized colitis mice.The unimmunized mice developed significant inflammation in colon with bloody mucus feces and decreased body weight after TNBS induction. Distinct hyperemia, edema and transmural inflammatory infiltration accompanied with ulceration were shown in colon. The level of IFN-gamma was (3.47 +/- 0.87) ng/ml and IL-4 was (146.06 +/- 45.76) pg/ml. However, in egg-immunized mice, the inflammation was suppressed greatly and the body weight recovered soon after TNBS induction. The production of IL-4 was enhanced to (598.50 +/- 135.90) pg/ml, and IFN-gamma was significantly diminished to (1.53 +/- 0.51) ng/ml.S.japonicum eggs protect mice from colitis induced by TNBS.

To study the protective efficacy of co-immunization with Schistosoma japonicum glutathione S-transferase (Sj26) DNA and recombinant Sj26 protein (rSj26 GST) vaccine against Schistosoma japonicum in BALB/c mice.The Sj26 gene was cloned into eukaryotic expression vector pEGFP-N3 with enhanced green fluorescence protein. The recombinant plasmid pEGFP-Sj26 was transfected into baby hamster kidney (BHK) cells, fluorescent microscope and Western blotting were employed to identify the expressed products. Each mouse in co-immunization group was primed with plasmid pEGFP-Sj26, boosted 2 weeks later and immunized with rSj26 GST 4 weeks later. While each mouse in pEGFP-Sj26 group and rSj26 GST group was primed and boosted with pEGFP-Sj26 or rSj26 GST independently. Two weeks after last immunization, each mouse was challenged with 40 +/- 1 cercariae of S. japonicum Chinese strain. At the 45th day post-infection, mice were sacrificed and the worms were perfused from portal vein and the number of worms and eggs in liver tissue were counted.In BHK cells transfected with the recombinant plasmid pEGFP-Sj26, the expression of Sj26-EGFP fusion protein was confirmed by fluorescent microcopy and Western blotting. The worm reduction rate in co-immunized group was 50.8%, significantly higher than that in pEGFP-Sj26 group (28.0%, P0.01) and rSj26 GST group (25.5%, P0.01). Liver egg reduction rate in co-immunized group, pEGFP-Sj26 group and rSj26 GST group were 32.7%, 20.6% and 33.0% respectively. The number of eggs per female in liver of co-immunized mice and rSj26 GST group were significantly higher than that in control group (P0.01).Compared to the immunization with pEGFP-Sj26 or rSj26 GST alone, the co-immunization with pEGFP-Sj26 and rSj26 GST can enhance protective efficacy in BALB/c mice.

To screen special mimic epitopes of Trichinella spiralis antigen from peptide library for exploring new diagnostic antigens.Ts-IgG purified from serum of trichinosis patients was used to screen the phage 12-mer peptide library for 5 rounds. 24 clones were picked out randomly to detect the immunoactivity. The sensitivity and specificity of the 6 clones (T1 - T6) whose A values were higher than others were tested by ELISA.The sensitivity of the clones T1 - T6 was the same with larval antigen of Trichinella spiralis (TsA) (positive rate: 100%, P0.05), and there was no difference in specificity between T1 - T6 and TsA (negative rate: 0 - 40%, P0.05); T3 and T6 did not react with sera from patients of paragonimiasis, showing higher specificity than TsA (P0.05); T6 did not react with sera from patients of schistosomiasis, also showing higher specificity than TsA (P0.05).The mimic antigenic epitopes of Trichinella spiralis have been successfully obtained by screening phage 12-mer peptide library.

To obtain the mimic epitope of specific and sensitive diagnostic antigen in schistosomiasis japonica from phage 12-mer peptide library.Specific Ig was purified from sera of patients with acute schistosomiasis and used to immunoscreen the phage peptide library (PH. D.-12). After 3 rounds of panning, 10 positive plaques were selected and amplified. The immunoactivity of each clone was examined by ELISA. The sensitivity and specificity of immunoactive clones were confirmed by detecting the sera of patients with different parasitosis.Six clones could bind to the specific Ig purified from sera of patients with acute schistosomiasis. One clone with the highest A492 value showed a high sensitivity and specificity.The clone (SjA1) identified by the specific Ig from the library played a better part in the immunodiagnosis of schistosomiasis.