[Bioinformatics analysis of differentially expressed genes associated with chronic schistosomiasis japonica-induced hepatic fibrosis].

OBJECTIVE: To screen differentially expressed genes (DEGs) associated with chronic schistosomiasis japonica-induced hepatic fibrosis and analyze their functions. METHODS: The dataset of gene expression profiles of patients with chronic schistosomiasis japonica-induced hepatic fibrosis was downloaded from the Gene Expression Omnibus (GEO) database, and DEGs were screened using R package. The biological functions of DEGs were characterized using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In addition, the protein-protein interaction (PPI) network of DEGs was created to screen the hub genes. RESULTS: A total of 62 DEGs were identified, including 12 down-regulated genes and 50 up-regulated genes. GO enrichment analysis showed that DEGs were mainly enriched in 116 biological processes, including fatty acid, sulfur compound, acyl-coenzyme A and thioester metabolism; 19 cellular components, including mitochondrial matrix, outer mitochondrial membrane and organelle outer membrane; and 7 molecular functions, including insulin-like growth factor binding and oxidoreductase activity. KEGG pathway enrichment analysis that the DEGs were significantly enriched in phosphatidylinositol-3-kinase/serine/threonine protein kinase (PI3K/Akt), mitogen-activated protein kinase (MAPK), calcium metabolism and cyclic adenosine monophosphate (cAMP) signaling. PPI network analysis identified six hub genes involved in the development of chronic schistosomiasis japonica-induced hepatic fibrosis, including ACACA, ACSL1, GPAM, THRSP, PLIN1 and DGAT2, and ACSL1, ACACA and PLIN1 were the top 3 hub genes. CONCLUSIONS: ACSL1, ACACA and PLIN1 may be the hub genes associated with the development of chronic schistosomiasis japonica-induced hepatic fibrosis, and abnormal lipid metabolism mediated by these DEGs may play an important role in the development of chronic schistosomiasis japonica-induced hepatic fibrosis.

STAT3 Promotes Schistosome-Induced Liver Injury by Inflammation, Oxidative Stress, Proliferation, and Apoptosis Signal Pathway.

Schistosomiasis is a parasitic helminth disease that can cause organ lesions leading to health damage. During a schistosome infection, schistosome eggs can flow into the liver along the portal vein. Numerous inflammatory cells gather around the eggs, causing granulomas and fibrosis in the liver. In this process, many molecules are involved in the initiation and regulation of the fibrous scar formation. However, the precise molecular mechanisms responsible for the progression of granuloma formation and fibrosis initiation caused by schistosome infection have not been extensively studied. In this study, C57BL/6 wild-type mice and Stat3flox/flox Alb-Cre mice were infected with cercariae of Schistosoma japonicum Liver injury, effector molecule levels, and RNA transcriptome resequencing of liver tissue were detected at 4, 5, and 6 weeks postinfection. We investigated the role of STAT3 (signal transducer and activator of transcription 3) in Schistosoma-induced liver injury in mice. After 6 weeks postinfection, there was obvious liver fibrosis. A sustained pathological process (inflammation, oxidative stress, proliferation, and apoptosis) occurred in S. japonicum-induced liver fibrosis initiation. Meanwhile, we observed activation of the STAT3 pathway in hepatic injury during S. japonicum infection by RNA transcriptome resequencing. Liver deficiency of phospho-STAT3 alleviated infection-induced liver dysfunction, hepatic granuloma formation, and fibrosis initiation. It also promoted STAT3-dependent apoptosis and reduced liver inflammation, oxidative stress, and proliferation. Our results suggest that STAT3 signal pathway and its mediating inflammation, oxidative stress, proliferation, and apoptosis are involved in S. japonicum-induced liver injury and may be a new potential guideline for the treatment of schistosomiasis.

Association of CX3CL1 and CX3CR1 Expression with Liver Fibrosis in a Mouse Model of Schistosomiasis.

Immunopathological mechanisms of schistosomiasis, a debilitating parasitic disease, are still unclear. In this study, we investigated the involvement of CX3C chemokine ligand 1 (CX3CL1) and its sole receptor CX3CR1 in the development of liver fibrosis in schistosomiasis. The animal model of schistosomiasis was established by infection of C57BL/6 mice with Schistosoma japonicum cercariae; mice injected with carbon tetrachloride (CCl4) were used as positive control of liver injury. After 4 and 8 weeks, the degree of liver lesions was assessed by hematoxylin and eosin staining, serum levels of hyaluronic acid (HA) were analyzed by a chemiluminescence immunoassay, liver fibrosis was evaluated by immunohistochemistry analysis of α-smooth muscle actin (α-SMA) expression, and CX3CL1 and CX3CR1 expression in the liver was measured by immunohistochemistry and real-time PCR. The results showed that at 8 weeks after Schistosoma infection, serum HA levels were increased and α-SMA-expressing cells appeared in the liver, indicating fibrogenesis. CX3CL1- and CX3CR1-positive cells were observed in the outer layer of granulomas formed around Schistosoma eggs in liver tissues, which was consistent with the significant upregulation of hepatic CX3CL1 and CX3CR1 mRNA expression at 4 and 8 weeks post-infection. Furthermore, correlation analysis revealed positive association between CX3CL1 and CX3CR1 expression and serum HA levels at 8 weeks post-infection, indicating a link between fibrogenesis and the CX3CL1/CX3CR1 axis in schistosomiasis. In conclusion, our data suggest the involvement of CX3CL1 and CX3CR1 in the progression of liver fibrosis caused by Schistosoma infection.

Schistosoma japonicum infection causes a reprogramming of glycolipid metabolism in the liver.

BACKGROUND: Recent investigations indicate that schistosome infection is closely associated with aberrant glycolipid metabolism. However, the actual glycolipid metabolism gene expression, as well as the possible pathways that regulate glycolipid metabolism in the schistosome-infected liver, has not been extensively explored. METHODS: In this study, we evaluated the dynamic expression of glycolipid metabolism-associated genes and proteins in the livers from mice infected with Schistosoma japonicum at the indicated time points using real-time PCR and immunofluorescence. Then, cultures of macrophages were treated with schistosome soluble egg antigen (SEA) to detect the expression levels of genes associated with glucose and lipid metabolism in order to identify macrophages metabolic characteristics in response to these antigens. Furthermore, SEA-stimulated macrophages were co-cultures with hepatocytes and detected the effects of macrophages on the gene expression of hepatocytes metabolism. RESULTS: The expression of glycolysis-related genes (Ldha, Glut4, Pkm2, Glut1, Pfkfb3, Aldoc, HK2, Pfk) in the liver were upregulated but the gluconeogenesis gene (G6pc) was downregulated during S. japonicum infection. In addition, the mRNA levels of fatty acid (FA) oxidation-related genes (Ucp2, Atp5b, Pparg) in the liver were significantly upregulated; however, the FA synthesis genes (Fas, Acc, Scd1, Srebp1c) and lipid uptake gene (Cd36) were downregulated post-S. japonicum-infection. In consistence with these data, stimulation with SEA in vitro significantly enhanced the gene expression that involved in glycolysis and FA oxidation, but decreased genes related to gluconeogenesis, FA synthesis and lipid uptake in macrophages. The levels of phosphorylated AMPK, AKT and mTORC1 were increased in macrophages after SEA stimulation. Inhibition of phosphorylated AMPK, AKT and mTORC1 promoted SEA-treated macrophages to produce glucose. In addition, suppression of phosphorylated-AMPK, but not phosphorylated-AKT and phosphorylated-mTOR, induced the lipid accumulation in SEA-stimulated macrophages. Furthermore, SEA-treated macrophages significantly reduced the expression of Acc mRNA in hepatocytes in vitro. CONCLUSIONS: These findings reveal S. japonicum infection induces dynamic changes in the expression levels of genes involved in catabolism (glucose uptake, glycolysis and fatty acid oxidation) and suppressing anabolism (glycogen synthesis) in the liver, which could occur via macrophages' metabolic states, particularly those involved in the AMPK, AKT and mTORC1 pathways.

Diagnostic Value of SjR2 Gene in Colonic Tissue from Schistosoma Japonicum Infected Hosts.

BACKGROUND The prevalence and intensity of schistosomiasis infection in China has decreased markedly in recent years. Therefore, more accurate methods are critically needed to ensure further control of low-intensity schistosomiasis infection. For chronic schistosomiasis patients, the detection of schistosome eggs in colorectal mucosa tissues is commonly used. This work aimed to explore differences in sensitivity of the Schistosoma japonicum (S. japonicum) retrotransposon (SjR2) gene in colon tissue from S. japonicum infected hosts and to develop an ideal method for genetic diagnosis of low-intensity schistosomiasis. MATERIAL AND METHODS Serum and colon samples were collected from mice at different time points, either post-infection (PI) or post-treatment (PT). Colorectal biopsy specimens from outpatients with schistosomiasis were collected. All samples from mice and patients, including serum as well as colon tissue containing eggs and tissue containing no eggs, were examined using the polymerase chain reaction technique. RESULTS The results showed that the SjR2 gene could be detected in all colon tissue containing at least one egg, except for when the egg was completely degraded. The positive rate of gene detection in serum was low. The results from egg-free colon tissue from around the eggs were more consistent with the actual parasitism in vivo. CONCLUSIONS The results indicate that detection of the gene in colon tissue located within a 0.5 cm distance from the eggs would be a practical and ideal method for genetic diagnosis of schistosomiasis. After the colorectal biopsy, this method can be a sensitive assisted examination to the clinical diagnosis of low-intensity schistosomiasis infection.

MicroRNA-29b-3p prevents Schistosoma japonicum-induced liver fibrosis by targeting COL1A1 and COL3A1.

Schistosomiasis is one of the world's major public health problems in terms of morbidity and mortality, causing granulomatous inflammation and cumulative fibrosis. This study explored in vivo and vitro effects of miR-29b-3p in granulomatous liver fibrosis by targeting COL1A1 and COL3A1 in Schistosoma japonicum infection. Thirty male Balb/c mice were assigned to normal control and model (percutaneous infection of cercariae of S. japonicum) groups. NIH-3T3 mouse embryonic fibroblasts were designated into blank, NC, miR-29b-3p mimic, TGF-ß1, TGF-ß1 + NC, and TGF-ß1 + miR-29b-3p mimic groups. HE and Masson staining were employed to observe the pathological changes and collagenous fibrosis. The expression of α-SMA, COL1A1, COL3A1, TIMP-1 was determined by immunohistochemistry. The RT-qPCR, Western blotting and immunofluorescence staining were conducted to determine expression of miR-29b-3p, COL1A1, and COL3A1. CCK-8 assay and flow cytometry were performed to evaluate viability and apoptosis. The relative expression of miR-29b-3p decreased in the model group. The model group showed marked fibrosis in liver tissues. The expression of α-SMA, COL1A1, COL3A1, TIMP-1 was higher in the model group than that in the normal control group. Dual luciferase reporter gene assay revealed that miR-29b-3p directly targeted COL1A1 and COL3A1. Compared with the blank, NC, TGF-ß1 and TGF-ß1 + NC groups, the miR-29b-3p mimic group exhibited up-regulated expression of miR-29b-3p and MMP-9 but down-regulated expression of TIMP-1, HSP47, α-SMA, COL1A1, and COL3A1; while lower cell viability but higher apoptosis rate showed. It indicated that miR-29b-3p prevents S. japonicum-induced liver fibrosis by inhibiting COL1A1 and COL3A1.

Protective effects of hydrogen sulfide on portal hypertensive vasculopathy in rabbits by activating AKT-NF-κB pathway.

The role of hydrogen sulfide (H2S) in portal hypertension (PH)-induced esophagus-gastric junction vascular lesions in rabbits was observed. The rabbit PH models were established. The animals were randomly divided into the following groups: normal, PH, PH+sodium hydrosulfide (PH+S), PH+propargylglycine (PH+PPG). The plasma H2S levels, apoptosis of esophageal-gastric junction vascular smooth muscle cells, and the expression of nuclear transcription factor-κB (NF-κB), p-AKT, IκBa and Bcl-2 were detected. The cystathionine γ lyase (cystathionine-gamma-splitting enzyme, CSE) in the junction vascular tissue was measured. The results showed that the plasma H2S levels and the CSE expression levels had statistically significant difference among different groups (P<0.05). As compared with PH group, plasma H2S levels were declined obviously (11.9±4.2 vs. 20.6±4.5, P<0.05), and CSE expression levels in the junction vascular tissue were notably reduced (1.7±0.6 vs. 2.8±0.8, P<0.05), apoptosis rate of vascular smooth muscle cells per unit area was significantly decreased (0.10±0.15 vs. 0.24±0.07, P<0.05), and the expression levels of p-AKT and NF-κB were significantly decreased (2.31±0.33 vs. 3.04±0.38, P<0.05; 0.33±0.17 vs. 0.51±0.23, P<0.05), however, IκBa and Bcl-2 expression increased obviously (5.57±0.17 vs. 3.67±0.13, P<0.05; 0.79±0.29 vs. 0.44±0.36, P<0.05) in PH+PPG group. As compared with PH group, H2S levels were notably increased (32.7±7.3 vs. 20.6±4.5, P<0.05), the CSE levels in the junction vascular tissue were significantly increased (6.3±0.7 vs. 2.8±0.8, P<0.05), apoptosis rate of vascular smooth muscle cells per unit area was significantly increased (0.35±0.14 vs. 0.24±0.07, P<0.05), and the expression levels of p-AKT and NF-κB were significantly increased (4.29±0.49 vs. 3.04±0.38, P<0.05; 0.77±0.27 vs. 0.51±0.23, P<0.05), yet IκBa and Bcl-2 expression decreased significantly (3.23±0.24 vs. 3.67±0.13, P<0.05; 0.31±0.23 vs. 0.48±0.34, P<0.05) in PH+S group. It is concluded that esophagus-gastric junction vascular lesions happen under PH, and apoptosis of smooth muscle cells is declined. H2S can activate NF-κB by the p-AKT pathway, leading to the down-regulation of Bcl-2, eventually stimulating apoptosis of vascular smooth muscle cells, easing PH. H2S/CSE system may play an important role in remission of PH via the AKT-NF-κB pathway.

Characteristics of IL-9 induced by Schistosoma japonicum infection in C57BL/6 mouse liver.

Liver granulomatous inflammation and fibrosis were the primary pathological changes observed during Schistosoma japonicum (S. japonicum) infection. In the present study, the characteristics of IL-9 were investigated in the liver of S. japonicum infection C57BL/6 mice. Immunofluorescence, qRT-PCR, and ELISA results demonstrated that the expression of IL-9 significantly increased after infection (P < 0.01). FACS results indicated that the peak of IL-9+ Th9 cells in the liver mononuclear cells appeared at the early phase of infection (week 5), except that Th9 cells, CD8+ Tc cells, NKT and γδT cells could secrete IL-9 in this model. Although IL-9 neutralization has a limited effect on liver granulomatous inflammation, it could decrease the level of fibrosis-associated factor, PC-III, in the serum of infected mice (P < 0.05). Taken together, our results indicated that IL-9 was an important type of cytokine involved in the progression of S. japonicum infection-induced hepatic damage.

Cysteine protease inhibitor of Schistosoma japonicum - A parasite-derived negative immunoregulatory factor.

Studies have shown that cysteine protease inhibitors from some parasites have immunosuppressive effects on the host. We previously have cloned a novel cysteine protease inhibitor from Schistosoma japonicum and purified its recombinant version (protein named rSj-C). Its possible inhibitory effect on the host immune response has not been described.This study shows that rSj-C inhibits lysosomal cysteine protease of murine dendritic cells (DCs). After DCs were incubated with rSj-C and then with soluble adult worm antigen (AWA) of S. japonicum, the mean fluorescence intensity of MHC class II antigens on the surface of DCs decreased significantly by flow cytometry. These results indirectly proved that rSj-C can suppress exogenous-antigen presentation by DCs. The flow cytometric assay revealed that in comparison with control groups, the proportion of CD4+CD25+Foxp3+ T cells among CD4+CD25+ T cells of Schistosom-infected mice increased significantly 8 weeks after the infected mice were injected with rSj-C (p Ë‚ 0.05). Additionally, the expression levels of cytokines IL-4 and TGF-ß produced by T cells increased significantly as compared with these levels in the normal group (p Ë‚ 0.05). These results clearly show that the cysteine protease inhibitor from S. japonicum is a new parasite-derived immunosuppressive factor.

Differential pulmonic NK and NKT cell responses in Schistosoma japonicum-infected mice.

Natural killer cells (NK cells) and natural killer T cells (NKT cells) play a role in anti-infection, anti-tumor, transplantation immunity, and autoimmune regulation. However, the role of NK and NKT cells during Schistosoma japonicum (S. japonicum) infection has not been widely reported, especially regarding lung infections. The aim of this study was to research the NK and NKT cell response to S. japonicum infection in the lungs of mice. Using immunofluorescent histological analysis, NK and NKT cells were found near pulmonary granulomas. Moreover, flow cytometry revealed that the percentage and number of pulmonic NK cells in S. japonicum-infected mice were significantly increased (P < 0.05). However, the percentage and cell number of NKT cells were decreased compared to those of normal mice (P < 0.05). The expression of CD69 on pulmonic NK and NKT cells was increased after infection (P < 0.05), and CD25 expression increased only on NKT cells (P < 0.05). Intracellular cytokine staining showed a higher percentage of IFN-γ+ and lower percentage of IL-5+ pulmonic NK cells (P < 0.05) compared to controls. However, the percentage of IL-17+, IL-10+, and IL-5+ pulmonic NKT cells significantly increased (P < 0.05). Additionally, there was a significant decrease in NKG2A/C/E (CD94) expression and an increase of NKG2D (CD314) expression on pulmonic NKT cells (P < 0.05), which might serve as a mechanism for NKT cell activation during S. japonicum infection.

Blockade of PD-1 Signaling Enhances Th2 Cell Responses and Aggravates Liver Immunopathology in Mice with Schistosomiasis japonica.

BACKGROUND: More than 220 million people worldwide are chronically infected with schistosomes, causing severe disease or even death. The major pathological damage occurring in schistosomiasis is attributable to the granulomatous inflammatory response and liver fibrosis induced by schistosome eggs. The inflammatory response is tightly controlled and parallels immunosuppressive regulation, constantly maintaining immune homeostasis and limiting excessive immunopathologic damage in important host organs. It is well known that the activation of programmed death 1 (PD-1) signaling causes a significant suppression of T cell function. However, the roles of PD-1 signaling in modulating CD4+ T cell responses and immunopathology during schistosome infection, have yet to be defined. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that PD-1 is upregulated in CD4+ T cells in Schistosoma japonicum (S. japonicum)-infected patients. We also show the upregulation of PD-1 expression in CD4+ T cells in the spleens, mesenteric lymph nodes, and livers of mice with S. japonicum infection. Finally, we found that the blockade of PD-1 signaling enhanced CD4+ T helper 2 (Th2) cell responses and led to more severe liver immunopathology in mice with S. japonicum infection, without a reduction of egg production or deposition in the host liver. CONCLUSIONS/SIGNIFICANCE: Overall, our study suggests that PD-1 signaling is specifically induced to control Th2-associated inflammatory responses during schistosome infection and is beneficial to the development of PD-1-based control of liver immunopathology.

Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum.

Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42-44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica.

Tim-3 induces Th2-biased immunity and alternative macrophage activation during Schistosoma japonicum infection.

T cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) has been regarded as an important regulatory factor in both adaptive and innate immunity. Recently, Tim-3 was reported to be involved in Th2-biased immune responses in mice infected with Schistosoma japonicum, but the exact mechanism behind the involvement of Tim-3 remains unknown. The present study aims to understand the role of Tim-3 in the immune response against S. japonicum infection. Tim-3 expression was determined by flow cytometry, and increased Tim-3 expression was observed on CD4(+) and CD8(+) T cells, NK1.1(+) cells, and CD11b(+) cells from the livers of S. japonicum-infected mice. However, the increased level of Tim-3 was lower in the spleen than in the liver, and no increase in Tim-3 expression was observed on splenic CD8(+) T cells or CD11b(+) cells. The schistosome-induced upregulation of Tim-3 on natural killer (NK) cells was accompanied by reduced NK cell numbers in vitro and in vivo. Tim-3 antibody blockade led to upregulation of inducible nitric oxide synthase and interleukin-12 (IL-12) mRNA in CD11b(+) cells cocultured with soluble egg antigen and downregulation of Arg1 and IL-10, which are markers of M2 macrophages. In summary, we observed schistosome-induced expression of Tim-3 on critical immune cell populations, which may be involved in the Th2-biased immune response and alternative activation of macrophages during infection.

A boswellic acid-containing extract ameliorates schistosomiasis liver granuloma and fibrosis through regulating NF-κB signaling in mice.

Boswellic acid (BA)-containing extracts such as BSE have anti-inflammatory and immunomodulatory activity. In chronic schistosomiasis, the hepatic granuloma and fibrosis induced by egg deposition in the liver is the most serious pathological manifestations. However, little is known regarding the role of BAs in Schistosoma japonicum (S. japonicum) egg-induced liver granuloma and fibrosis. In order to investigate the effect of a water-soluble complex preparation of BSE, BSE-CD, on S. japonicum egg-induced liver pathology, liver granuloma and fibrosis were induced by infecting C57BL/6 mice with 18-22 cercariae of S. japonicum. S. japonicum cercariae infected mice were injected with BSE-CD at the onset of egg granuloma formation (early phase BSE-CD treatment after 4 weeks infection) or after the formation of liver fibrosis (late phase BSE-CD treatment after 7 weeks infection). Our data show that treatment of infected mice with BSE-CD significantly reduced both the extent of hepatic granuloma and fibrosis. Consistent with an inhibition of NF-κB signaling as evidenced by reduced IκB kinase (IKK) activation, the mRNA expression of VEGF (vascular endothelial growth factor, VEGF), TNF-α (tumor necrosis factor-alpha TNF-α) and MCP-1 (monocyte chemotactic protein 1, MCP-1) was decreased. Moreover, immunohistochemical analysis (IHC) revealed that the content of α-SMA in liver tissue of BSE-CD treated mice was dramatically decreased. Our findings suggest that BSE-CD treatment attenuates S. japonicum egg-induced hepatic granulomas and fibrosis, at least partly due to reduced NF-κB signaling and the subsequently decreased expression of VEGF, TNF-α, and MCP-1. Suppression of the activation of hepatic stellate cells (HSC) may also be involved in the therapeutic efficacy of BSE-CD.

Protective effects of membrane-anchored and secreted DNA vaccines encoding fatty acid-binding protein and glutathione S-transferase against Schistosoma japonicum.

In order to explore the high performance bivalent DNA-based vaccine against schistosomes, SjFABP and Sj26GST were selected and used to construct a vaccine. Two strategies were used to construct the bivalent DNA vaccine. In the first strategy, a plasmid encoding antigen in the secreted form was used, while in the other, a plasmid encoding a truncated form of SjFABP and Sj26GST targeted to the cell surface was used. Various parameters, including antibody and cytokine response, proliferation, histopathological examination, and characterization of T cell subsets were used to evaluate the type of immune response and the level of protection against challenge infection. Injection with secreted pIRES-sjFABP-sj26GST significantly increased the levels of antibody, splenocyte proliferation, and production of IFN-γ, compared with membrane-anchored groups. Analysis of splenic T cell subsets showed that the secreted vaccine significantly increased the percentage of CD3(+)CD4(+) and CD3(+)CD8(+) T cells. Liver immunopathology (size of liver granulomas) was significantly reduced in the secreted group compared with the membrane-anchored groups. Moreover, challenge experiments showed that the worm and egg burdens were significantly reduced in animals immunized with recombinant vaccines. Most importantly, secreted Sj26GST-SjFABP markedly enhanced protection, by reducing worm and egg burdens by 31.8% and 24.78%, respectively, while the membrane-anchored group decreased worm and egg burdens by 24.80% and 18.80%, respectively. Taken together, these findings suggest that the secretory vaccine is more promising than the membrane-anchored vaccine, and provides support for the development and application of this vaccine.

A potential screening factor for accumulation of cholesteyl ester transfer protein deficiency in East Asia: Schistosoma japonicum.

Cholesteryl ester transfer protein (CETP)-deficiency manifests a unique plasma lipoprotein profile without other apparent symptoms. It is highly common in East Asia while rather rare anywhere else. A potential environmental screening factor(s) may therefore contribute to this eccentric distribution, such as its selective advantage against a regional illness, most likely an infectious disease, in relation to plasma lipoproteins. Blood flukes use the host plasma lipoproteins as nutrient sources through the lipoprotein receptor-like systems. Its Asian-specific species, Schistosoma (S) japonicum, which has been endemic in East Asia, takes up cholesteryl ester (CE) from high-density lipoprotein (HDL) for the embryonation of their eggs to miracidia, a critical step of the hepatic pathogenesis of this parasite, but poorly from HDL of CETP-deficiency. CD36-related protein (CD36RP) was cloned from the adults and the eggs of S. japonicum, with 1880-bp encoding 506 amino-acid residues exhibiting the CD36 domains and two transmembrane regions. Its extracellular domain selectively bound human HDL but neither LDL nor CETP-deficiency HDL, and the antibody against the extracellular domain suppressed the selective HDL-CE uptake and embryonation of the eggs. When infected with S. japonicum, wild-type mice developed less hepatic granulomatosis than CETP-transgenic mice by the ectopic egg embryonation. CD36RP is thus a candidate receptor of S. japonicum to facilitate uptake of HDL-CE necessary for egg embryonation. Abnormal HDL caused by CETP-deficiency retards this process and thereby protects the patients from development of hepatic lesions. S. japonicum infection is a potential screening factor for high prevalence of CETP deficiency in East Asia.

[Expression of vascular growth factors in intestinal tissues in colorectal carcinoma patients with schistosomiasis japonica].

OBJECTIVE: To investigate the differences of mRNA quantitation and protein expression of vascular growth factors including platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) in intestinal tissues in colorectal carcinoma patients with and without schistosomiasis. METHODS: Thirty colorectal carcinoma patients with schistosomiasis and 30 colorectal carcinoma patients without schistosomiasis were included in this study. The mRNA quantitation and protein expression of PD-ECGF and VEGF in the normal tissue, peri-carcinoma tissue as well as carcinoma tissue obtained from surgical specimens were detected by qRT-PCR and Western blot. RESULTS: The mRNA relative quantitations of PD-ECGF in normal tissue, peri-carcinoma tissue and carcinoma tissue in the colorectal carcinoma patients with schistosomiasis were 1.726, 1.766 and 2.729 times to those in the colorectal carcinoma patients without schistosomiasis, respectively. The corresponding ones of VEGF were 2.138, 1.831 and 3.376 times, respectively. The protein expression levels of PD-ECGF and VEGF in normal tissue, peri-carcinoma tissue and carcinoma tissue were higher in the colorectal carcinoma patients with schistosomiasis than in the colorectal carcinoma patients without schistosomiasis. CONCLUSIONS: The expressions of vascular growth factors including PD-ECGF and VEGF are higher in the colorectal carcinoma patients with schistosomiasis than in the colorectal carcinoma patients without schistosomiasis. Therefore, schistosomiasis may be one of the risk factors of colorectal cancer.

MicroRNA-gene expression network in murine liver during Schistosoma japonicum infection.

BACKGROUND: Schistosomiasis japonica remains a significant public health problem in China and Southeast Asian countries. The most typical and serious outcome of the chronic oriental schistosomiasis is the progressive granuloma and fibrosis in the host liver, which has been a major medical challenge. However, the molecular mechanism underling the hepatic pathogenesis is still not clear. METHODOLOGY AND PRINCIPAL FINDINGS: Using microarrays, we quantified the temporal gene expression profiles in the liver of Schistosoma japonicum-infected BALB/c mice at 15, 30, and 45 day post infection (dpi) with that from uninfected mice as controls. Gene expression alternation associated with liver damage was observed in the initial phase of infection (dpi 15), which became more magnificent with the onset of egg-laying. Up-regulated genes were dominantly associated with inflammatory infiltration, whereas down-regulated genes primarily led to the hepatic functional disorders. Simultaneously, microRNA profiles from the same samples were decoded by Solexa sequencing. More than 130 miRNAs were differentially expressed in murine liver during S. japonicum infection. MiRNAs significantly dysregulated in the mid-phase of infection (dpi 30), such as mmu-miR-146b and mmu-miR-155, may relate to the regulation of hepatic inflammatory responses, whereas miRNAs exhibiting a peak expression in the late phase of infection (dpi 45), such as mmu-miR-223, mmu-miR-146a/b, mmu-miR-155, mmu-miR-34c, mmu-miR-199, and mmu-miR-134, may represent a molecular signature of the development of schistosomal hepatopathy. Further, a dynamic miRNA-gene co-expression network in the progression of infection was constructed. CONCLUSIONS AND SIGNIFICANCE: This study presents a global view of dynamic expression of both mRNA and miRNA transcripts in murine liver during S. japonicum infection, and highlights that miRNAs may play a variety of regulatory roles in balancing the immune responses during the development of hepatic pathology. The data provide robust information for further researches on the pathogenesis and molecular events of hepatopathy induced by schistosome eggs.

Spatial and temporal transcriptomics of Schistosoma japonicum-induced hepatic granuloma formation reveals novel roles for neutrophils.

The severity of schistosome egg-induced hepatic granulomatous pathology depends markedly on the nature of the host immune responses. In this study, we used LMM and microarray analysis to compare gene expression profiles of histologically distinct zones within, and directly proximal to, hepatic granulomas that developed in C57BL/6 mice infected with Schistosoma japonicum. There was significant up-regulation of type-1, type-2, and type-17 immune-associated genes within the granuloma core (adjacent to eggs), followed by increased expression of type-2 and fibrotic genes at the outer zones of granulomas. Neutrophil-associated genes were also found to be expressed differentially in the core and at the peripheral zone of granulomas, present at 7 weeks p.i., demonstrating a significant role of neutrophils in S. japonicum granulomatous pathology. The release of NETs was observed microscopically in granulomas obtained from the livers of infected mice and when human neutrophils were incubated in vitro in the presence of S. japonicum eggs. These finding are the first to suggest a novel, dual role for neutrophils in the mediation of tissue damage and repair in S. japonicum egg-induced hepatic granulomatous lesions. Together, these results provide an overview of the local events occurring within the granuloma microenvironment.

Exogenous bone morphogenetic protein-7 reduces hepatic fibrosis in Schistosoma japonicum-infected mice via transforming growth factor-ß/Smad signaling.

AIM: To investigate the antifibrotic effects of bone morphogenetic protein-7 (BMP-7) on Schistosoma japonicum (S. japonicum)-induced hepatic fibrosis in BALB/C mice. METHODS: Sixty BALB/C mice were randomly divided into three groups, including a control group (group A, n = 20), model group (group B, n = 20) and BMP-7 treated group (group C, n = 20). The mice in group B and group C were abdominally infected with S. japonicum cercariae to induce a schistosomal hepatic fibrosis model. The mice in group C were administered human recombinant BMP-7. Liver samples were extracted from mice sacrificed at 9 and 15 wk after modeling. Hepatic histopathological changes were assessed using Masson's staining. Transforming growth factor-beta 1 (TGF-ß1), alpha-smooth muscle actin (α-SMA), phosphorylated Smad2/3 (pSmad2/3) and Smad7 protein levels and localization were measured by Western blotting and immunohistochemistry, respectively, and their mRNA expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The schistosomal hepatic fibrosis mouse model was successfully established, as the livers of mice in group B and group C showed varying degrees of typical schistosomal hepatopathologic changes such as egg granuloma and collagen deposition. The degree of collagen deposition in group C was higher than that in group A (week 9: 22.95 ± 6.66 vs 2.02 ± 0.76; week 15: 12.84 ± 4.36 vs 1.74 ± 0.80; P < 0.05), but significantly lower than that in group B (week 9: 22.95 ± 6.66 vs 34.43 ± 6.96; week 15: 12.84 ± 4.36 vs 18.90 ± 5.07; P < 0.05) at both time points. According to immunohistochemistry data, the expressions of α-SMA, TGF-ß1 and pSmad2/3 protein in group C were higher than those in group A (α-SMA: week 9: 21.24 ± 5.73 vs 0.33 ± 0.20; week 15: 12.42 ± 4.88 vs 0.34 ± 0.27; TGF-ß1: week 9: 37.00 ± 13.74 vs 3.73 ± 2.14; week 15: 16.71 ± 9.80 vs 3.08 ± 2.35; pSmad2/3: week 9: 12.92 ± 4.81 vs 0.83 ± 0.48; week 15: 7.87 ± 4.09 vs 0.90 ± 0.45; P < 0.05), but significantly lower than those in group B (α-SMA: week 9: 21.24 ± 5.73 vs 34.39 ± 5.74; week 15: 12.42 ± 4.88 vs 25.90 ± 7.01; TGF-ß1: week 9: 37.00 ± 13.74 vs 55.66 ± 14.88; week 15: 16.71 ± 9.80 vs 37.10 ± 12.51; pSmad2/3: week 9: 12.92 ± 4.81 vs 19.41 ± 6.87; week 15: 7.87 ± 4.09 vs 13.00 ± 4.98; P < 0.05) at both time points; the expression of Smad7 protein in group B was higher than that in group A and group C at week 9 (8.46 ± 3.95 vs 1.00 ± 0.40 and 8.46 ± 3.95 vs 0.77 ± 0.42; P < 0.05), while there were no differences in Smad7 expression between the three groups at week 15 (1.09 ± 0.38 vs 0.97 ± 0.42 vs 0.89 ± 0.39; P > 0.05). Although minor discrepancies were observed, the results of RT-PCR and Western blotting were mainly consistent with the immunohistochemical results. CONCLUSION: Exogenous BMP-7 significantly decreased the degree of hepatic fibrosis in both the acute and chronic stages of hepato-schistosomiasis, and the regulatory mechanism may involve the TGF-ß/Smad signaling pathway.