Optimized expression of Peptidyl-prolyl cis/transisomerase cyclophilinB with prokaryotic toxicity from Sporothrix globosa.

Cyclophilin B (CypB), a significant member of immunophilins family with peptidyl-prolyl cis-trans isomerase (PPIase) activity, is crucial for the growth and metabolism of prokaryotes and eukaryotes. Sporothrix globosa (S. globosa), a principal pathogen in the Sporothrix complex, causes sporotrichosis. Transcriptomic analysis identified the cypB gene as highly expressed in S. globosa. Our previous study demonstrated that the recombinant Escherichia coli strain containing SgcypB gene failed to produce sufficient product when it was induced to express the protein, implying the potential toxicity of recombinant protein to the bacterial host. Bioinformatics analysis revealed that SgCypB contains transmembrane peptides within the 52 amino acid residues at the N-terminus and 21 amino acids near the C-terminus, and 18 amino acid residues within the cytoplasm. AlphaFold2 predicted a SgCypB 3D structure in which there is an independent PPIase domain consisting of a spherical extracellular part. Hence, we chose to express the extracellular domain to yield high-level recombinant protein with PPIase activity. Finally, we successfully produced high-yield, truncated recombinant CypB protein from S. globosa (SgtrCypB) that retained characteristic PPIase activity without host bacterium toxicity. This study presents an alternative expression strategy for proteins toxic to prokaryotes, such as SgCypB. ONE-SENTENCE SUMMARY: The recombinant cyclophilin B protein of Sporothrix globosa was expressed successfully by retaining extracellular domain with peptidyl-prolyl cis-trans isomerase activity to avoid toxicity to the host bacterium.

Chitin-rich heteroglycan from Sporothrix schenckii sensu stricto potentiates fungal clearance in a mouse model of sporotrichosis and promotes macrophages phagocytosis.

BACKGROUND: Fungal cell wall polysaccharides maintain the integrity of fungi and interact with host immune cells. The immunomodulation of fungal polysaccharides has been demonstrated in previous studies. However, the effect of chitin-rich heteroglycan extracted from Sporothrix schenckii sensu stricto on the immune response has not been investigated. RESULTS: In this study, chitin-rich heteroglycan was extracted from S. schenckii sensu stricto, and immunomodulation was investigated via histopathological analysis of skin lesions in a mouse model of sporotrichosis and evaluation of the phagocytic function and cytokine secretion of macrophages in vitro. The results showed that the skin lesions regressed and granulomatous inflammation was reduced in infected mice within 5 weeks. Moreover, heteroglycan promoted the fungal phagocytosis by macrophages and modulated the cytokine secretion. Heteroglycan upregulated TNF-α expression early at 24 h and IL-12 expression late at 72 h after incubation, which might result from moderate activation of macrophages and contribute to the subsequent adaptive immune response. CONCLUSIONS: Chitin-rich heteroglycan extracted from S. schenckii sensu stricto potentiated fungal clearance in a mouse model of sporotrichosis. Moreover, chitin-rich heteroglycan promoted fungus phagocytosis by macrophages and modulated cytokines secretion. These results might indicate that chitin-rich heteroglycan could be considered as an immunomodulator used in the treatment of sporotrichosis.

Antibodies against Schistosoma japonicum lactate dehydrogenase B enhance enzyme active.

Lactate dehydrogenase (LDH) is a key enzyme in glycolysis process. It catalyzes the interconversion between pyruvic acid and lactic acid. Schistosoma japonicum adult worms largely rely on glycolysis for energy production when they parasitize in human. S. japonicum may be killed if energy production is suppressed. So, we wonder whether antibody against S. japonicum LDH is a harmful factor for S. japonicum surviving. In this study, we cloned and characterized S. japonicum lactate dehydrogenase B (SjLDHB) to evaluate its role in parasite survival. We found SjLDHB was highly similar to S. japonicum lactate dehydrogenase A (SjLDHA) which is another LDH subtype in S. japonicum in amino acid sequence. The optimal temperature of SjLDHB catalytic activity was 37 °C, the optimal pH values for pyruvate reduction and lactate oxidation were 7.0 and 6.0 and Km values of pyruvate and lactate were 0.2752 and 0.2339 mM respectively. Then, we identified SjLDHB expression in male and female S. japonicum. Finally, we evaluated the influence of antibodies on SjLDHB enzymatic activity. Interestingly, we found anti-SjLDHA antibody suppressed SjLDHB enzymatic activity, while anti-SjLDHB antibody and mixed antibody enhanced SjLDHB enzymatic activity in vitro. Although further investigation is needed, we suggest that anti-SjLDHB antibody may be not a negative factor, but a valuable compensation for S. japonicum adult worm surviving and pathogenicity.

Clonorchis sinensis cyclophilin A immunization protected mice from CLP-induced sepsis.

Cyclophilin A (CyPA), ubiquitously existing in cytoplasm of all eukaryotes, can be secreted in response to inflammatory stimuli. Extracellular CyPA plays a prominent role in the pathological processes of inflammatory diseases, acting as a proinflammatory mediator, exerting chemotactic effects, promoting apoptosis of endothelial cells and amplifying ROS generation, thus being considered as a potential treatment target of sepsis, a systemic inflammatory response syndrome. Our previous study found that antibodies against cyclophilin A of Clonorchis sinensis (CsCyPA) could neutralize mouse cyclophilin A (MuCyPA). In this study, we explored whether CsCyPA immunization could prevent or ameliorate mice sepsis induced by cecum ligation puncture (CLP). The results showed that CsCyPA immunization could improve the 72 h survival rate of mice after CLP. Moreover, the protective effect presented in a titer-dependent manner. The levels of cytokine IL-1ß, IL-6, TNF-α, MCP-1 and AST in serum were remarkably decreased compared to CLP control group mice. Pathological damages of liver, lung and kidney were ameliorated accompanied by less inflammatory cell infiltration. CFU per whole peripheral blood at 12 h and 24 h after CLP surgery was significantly lower than that of CLP control group. In vitro, intracellular ROS generation and cytokine mRNA expression in peritoneal macrophages stimulated by LPS were reduced obviously with anti-CsCyPA antibodies (anti-CsCyPAs) preincubation. All these results demonstrated that CsCyPA immunization protected mice from CLP induced sepsis.

Survival advantage depends on cecal volume rather than cecal length in a mouse model of cecal ligation and puncture.

BACKGROUND: Cecal ligation and puncture (CLP) is the most commonly used model to simulate human polymicrobial sepsis. However, the severity of CLP is difficult to be standardized across different laboratories. The aim of the present study was to evaluate the influence of ligated cecal volume and length on mortality in mouse CLP model. METHODS: Cecal length and volume were measured from 120 Kunming mice subjected to CLP or sham operation. According to cecal volume, mice were divided into three groups, volume0.0∼0.2 (0.0 cm(3)-0.2 cm(3)), volume0.2∼0.4 (0.2 cm(3)-0.4 cm(3)), and volume>0.4 (larger than 0.4 cm(3)). The contents of cytokines, including interleukin-1ß, interleukin-6, and TNF-α, were measured at 3 h after surgery. The blood bacterial load and oxidative stress indicators (including malondialdehyde and superoxide dismutase) were measured at 12 h after surgery. RESULTS: There was no significant difference on 72-h survival rate between the mice with cecum longer than 2 cm and shorter than 2 cm. Compared to the other volume groups, volume>0.4 group showed significantly increased blood bacterial load, malondialdehyde levels in lung and liver, and pro-inflammatory cytokines in serum. Surprisingly, the survival rate in volume>0.4 (0%) group showed significant difference from those of volume0.0∼0.2 group (40%) and volume0.2∼0.4 group (40%). CONCLUSIONS: The mice in volume>0.4 group have much serious inflammatory reaction and are easier to die. As the proportion of volume>0.4 mice is near 20%, it can have large influence on most of the related studies using this CLP model.

Recombinant Trichinella spiralis 53-kDa protein activates M2 macrophages and attenuates the LPS-induced damage of endotoxemia.

Sepsis is a serious clinical condition of excessive systemic immune response to microbial infection. The pro-inflammatory stage of sepsis is generally launched by innate cells such as macrophages. They release inflammatory cytokines, activate other immune cells and cause severe tissue/organ damage. In this study, we have revealed that recombinant Trichinella spiralis (TS) excretory-secretory protein (rTsP53) exhibited anti-inflammatory properties and rescued mice from LPS-induced endotoxemia, which is a common model for sepsis study, potentially through the induction of M2 macrophages. rTsP53 treatment significantly decreased inflammatory cytokines (IL-6, IFN-γ and TNF-α) and increased IL-4, IL-10, IL-13 and TGF-ß secretion, both in circulation and in tissues. rTsP53 also induced the activation and infiltration of F4/80(+)CD163(+) macrophages to inflammatory tissues, increased M2 macrophage-related Arg1 and Fizz1 expression, and decreased M1 macrophage-related iNOS expression. PCR array showed that rTsP53 activated several genes that involve the survival of macrophages and also anti-inflammatory genes such as SOCS3. Together, our results show that rTsP53 activates M2 macrophages, which has strong anti-inflammatory potential to prevent LPS-induced lethal sepsis.

Antifungal curcumin promotes chitin accumulation associated with decreased virulence of Sporothrix schenckii.

Curcumin, a yellow polyphenol compound, is known to possess antifungal activity for a range of pathogenic fungi. However, the fungicidal mechanism of curcumin (CUR) has not been identified. We have occasionally found that chitin redistributes to the cell wall outer layer of Sporothrix schenckii (S. schenckii) upon sublethal CUR treatment. Whether CUR can affect chitin synthesis via the protein kinase C (PKC) signaling pathway has not been investigated. This study describes a direct fungicidal activity of CUR against S. schenckii demonstrated by the results of a checkerboard microdilution assay and, for the first time, a synergistic effect of CUR with terbinafine (TRB). Furthermore, the results of real-time PCR showed that sublethal CUR upregulated the transcription of PKC, chitin synthase1 (CHS1), and chitin synthase3 (CHS3) in S. schenckii. The fluorescence staining results using wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC) and calcofluor white (CFW) consistently showed that chitin exposure and total chitin content were increased on the conidial cell wall of S. schenckii by sublethal CUR treatment. A histopathological analysis of mice infected with CUR-treated conidia showed dampened inflammation in the local lesion and a reduced fungal burden. The ELISA results showed proinflammatory cytokine secretion at an early stage from macrophages stimulated by the CUR-treated conidia. The present data led to the conclusion that CUR is a potential antifungal agent and that its fungicidal mechanism may involve chitin accumulation on the cell wall of S. schenckii, which is associated with decreased virulence in infected mice.

Antibodies against Clonorchis sinensis LDH could cross-react with LDHB localizing on the plasma membrane of human hepatocarcinoma cell SMMC-7721 and induce apoptosis.

Lactate dehydrogenase (LDH) is a terminal enzyme in anaerobic glycolytic pathway. It widely exists in various organisms and is in charge of converting the glycolysis product pyruvic acid to lactic acid. Most parasites, including Clonorchis sinensis, predominantly depend on glycolysis to provide energy. Bioinformatic analysis predicts that the LDHs from many species have more than one transmembrane region, suggesting that it may be a membrane protein. C. sinensis LDH (CsLDH) has been confirmed as a transmembrane protein mainly located in the tegument. The antibodies against CsLDH can inhibit the worm's energy metabolism, kill the worm, and may have the same effects on human cancer cells. In this study, we cloned and characterized human LDHA (HsLDHA), HsLDHB, and CsLDH. Semi-quantitative real-time RCP showed that HsLDHB only existed in hepatocarcinoma cell SMMC-7721. Confocal microscopy and Western blot experiments revealed that HsLDHB was localized in the plasma membrane of SMMC-7721 cells, and the antibodies against CsLDH could cross-react with it. This cross-reaction could inhibit the enzymatic activity of HsLDHB. The cancer cells co-cultured with anti-CsLDH sera showed a significant decrease in cell proliferation rate and increases in caspase 9 and reactive oxygen species (ROS) levels. Therefore, anti-CsLDH antibodies can induce the apoptosis of cancer cells SMMC-7721 and may serve as a new tool to inhibit tumor.

Should we abandon quinine plus antibiotic for treating uncomplicated falciparum malaria? A systematic review and meta-analysis of randomized controlled trials.

In this study, we compared the efficacies and adverse effects of quinine plus antibiotics and other anti-malaria drugs on treating uncomplicated falciparum malaria. By systematically searching the major databases PubMed, Embase, and the Cochrane Library, 14 randomized controlled trials (RCTs) including 1996 cases were identified. Then, we performed a systematic review and cumulative meta-analysis on these data. The primary outcome of these treatments was parasite failure at day 28. There was no significant difference between quinine plus antibiotic therapy (QACT) and artemisinin-based therapies (odds ratio (OR) 0.69, 95 % confidence interval (CI) 0.28 to 1.71) or non-artemisinin-based therapies except quinine monotherapy and chloroquine monotherapy (OR 0.56, 95 % CI 0.18 to 1.74). The secondary outcome was the adverse effects within 28 days, including nausea, dizziness, vomiting, diarrhea, abdominal pain, headache, and tinnitus. QACT significantly increased the risk of tinnitus compared with artemisinin-based therapies (OR 111.65, 95 % CI 12.63 to 986.87) and non-artemisinin-based therapies (OR 48.16, 95 % CI 16.23 to 142.92). Vomiting was more frequently reported in QACT compared with non-artemisinin-based therapies (OR 2.02, 95 % CI 1.14 to 3.56). This meta-analysis suggests that almost all regimens have equivalent treatment effect at the 28th day. However, the patients with QACT had a higher chance to suffer from vomiting and tinnitus. Therefore, QACT does not have significant advantage on treating uncomplicated falciparum malaria.

[Protective effects of recombinant trichinella spiralis-53 000 protein combining with imipenem on polymicrobial septic mice].

Objective: To observe the protective effects of recombinant trichinella spiralis-53 000 protein (rTsP53 protein) combining with imipenem (IMP) on septic mice and their underlying mechanisms. . Methods: Male BALB/c mice were divided into five groups randomly. Cecal ligature and puncture (CLP) operation was used for building polymicrobial septic model (CLP group).Mice in sham group were only subjected to laparoromy and abdominal closure without cecum ligation. At 6 hours post CLP, mice in CLP+IMP,CLP+rTsP53,and CLP+IMP+rTsP53 groups were injected intraperitoneally with IMP (20 mg/kg) + 0.1 mL albumin,rTsP53 protein (6 mg/kg) + 0.1 mL normal saline (NS),IMP (20 mg/kg) + rTsP53 protein (6 mg/kg) respectively, mice in sham group and CLP group were injected intraperitoneally with 0.1 mL albumin + 0.1 mL NS, then these therapies were repeated every 12 hours until the experiment ended. Twenty mice were extracted randomly from each group for surveying 72-hour survival rate.At 0,6,12,24,36,48,72 hours post CLP,3 mice in each group were collected and cytokines in serum were tested by enzyme-linked immunosorbent assay (ELISA).Whole blood was cultured, then the numbers of bacteria colony-forming units (CFU) were counted. Mice were executed at 24 hours, then the epithelial cells ultrastructures of the mice small intestinal mucosa were observed by transmission electron microscope (TEM). Results: ① Compared with CLP,CLP+IMP or CLP+rTsP53 group,72-hour survival rate of the mice in CLP+IMP+rTsP53 group was significantly elevated (85％ vs.20％,55％,25％,all P ＜ 0.05).② No bacteria was found in cultured whole blood of mice in sham group at all time-points. At 6 hours post CLP operation, the number of bacterial clone of all experimental groups was rose significantly. The changed trend of bacterial number in CLP group was rising at the beginning, then declining, and the bacterial number reached the peak at 24 hours (× 106 cfu/L:12.74± 2.33).From 12 hours, the bacterial numbers of CLP+rTsP53 group were higher than those of CLP group, and reached the peak at 36 hours (× 106 cfu/L:22.13 ± 4.28),then declined gradually. The bacterial numbers of CLP+IMP and CLP+IMP+rTsP53 groups reached the peak at 6 hours (× 106 cfu/L:5.72 ± 0.50,5.49 ± 0.59),then declined. They were significantly less than those of CLP group from 12 hours.③ From 6 hours after CLP,the cytokines levels of mice in all experimental groups were higher than those in sham group. The tumor necrosis factor-α (TNF-α) levels in CLP group showed a trend of elevation in the beginning, and decrease thereafter. It reached the peak at 36 hours (ng/L:1 422.67 ±72.19).The TNF-α level peak time of CLP+IMP group,CLP+rTsP53 group,CLP+IMP+rTsP53 group was advanced to 12 hours post CLP (ng/L:1376.29±44.67,929.36±40.42,809.61±22.61).At 24 hours post CLP, the interleukin-6 (IL-6) level of CLP group and CLP+IMP group reached the peak (ng/L:215.39 ± 16.05,191.63 ± 8.99).The peak time of CLP+rTsP53 group and CLP+IMP+rTsP53 was advanced to 12 hours post CLP (ng/L:113.01 ± 12.11,92.43±6.11).The level of IL-4,IL-10 in CLP group raised gradually to the highest at 72 hours (ng/L:366.25 ±24.25,923.14±30.36).The IL-4 and IL-10 levels of CLP+IMP group raised to their maximum value at 12 hours and 24 hours respectively (ng/L:281.47±16.33,555.67±13.57),then declined. The IL-4 and IL-10 levels of CLP+rTsP53 group and CLP+IMP+rTsP53 group gradually ascended their peak value at 72 hours [IL-4 (ng/L) was 453.14±18.53,410.43 ± 15.75,IL-10 (ng/L) was 1 185.61 ± 16.74,1 006.77 ± 36.91,respectively].From 12 hours, the pro-inflammatory cytokines levels of CLP+IMP+rTsP53 group were significantly less than those of CLP+IMP group and CLP+rTsP53 group.④ At 24 hours post CLP, compared with mice in CLP,CLP+IMP, or CLP+rTsP53 group, mice in CLP+IMP+rTsP53 group had slighter ultra structure injuries in the microvilli, cell junction and mitochondria of small intestinal mucosa epithelial cells. Conclusions: The levels of pro-inflammatory cytokines were reduced and the levels of anti-inflammatory eytokines were escalated by intervention of rTsP53 protein combining with IMP boosted in polymierobial septic mice serum, and enhanced the survival rate of the mice. The injection of rTsP53 protein alone had no protective effects on polymicrobial septic mice,because the amount of bacteria in mice blood was augmented

Prognosis of sepsis induced by cecal ligation and puncture in mice improved by anti-Clonorchis Sinensis cyclopholin a antibodies.

BACKGROUND: Cyclophilin A (CyPA), a ubiquitously distributed intracellular protein, is thought to be one of the important inflammatory factors and plays a significant role in the development process of sepsis. In the form of cytokine, CyPA deteriorates sepsis by promoting intercellular communication, apoptosis of endothelial cells and chemotactic effect on inflammatory cells. In our previous study, cyclophilin A of Clonorchis sinensis (CsCyPA), a type of excretory-secretory antigen, could induce the patients infected with Clonorchis sinensis to produce specific anti-CsCyPA antibodies. In this study, we investigated whether anti-CsCyPA antibodies could cross-react with CyPA and then play a protective role against sepsis, just like other anti-cytokine antagonists. METHODS: The mice model with sepsis was established with cecal ligation and puncture (CLP). Fifty mg/kg purified anti-CsCyPA antibodies were injected via the caudal vein 6 h after the CLP operation, and persistent observation was performed for 72 h. Blood samples and tissues were collected at 6 h, 12 h, 24 h, 48 h and 72 h after CLP. Cytokines in serum were measured by ELISA. Lung and mesentery tissues were stained with hematoxylin-eosin. Endothelial cells (ECs) isolated from murine aorta were co-cultured with CyPA of mice (MuCyPA) and anti-CsCyPAs for 24 h, then, viability was measured by Cell Counting Kit-8. RESULTS: Anti-CsCyPA antibodies could combine with MuCyPA and inhibit its peptidyl prolyl isomerase (PPIase) activity. In the antibodies treatment group, blood coagulation indicators including PT, aPTT, D-dimer and platelet count were obviously more ameliorative, the proinflammary factors like IL-6, TNF-α, IL-1ß were significantly lower at 12 h and 24 h after surgery and the viability of ECs was significantly improved compared to those in the control group. Furthermore, the survival rate was elevated, ranging from 10.0 % to 45.0 % compared to the control group. CONCLUSIONS: These antibodies may have a favorable effect on sepsis via inhibition of enzymic activity or protection of endothelial cells.

Analysis of global gene expression profiles suggests a role of acute inflammation in type 3C diabetes mellitus caused by pancreatic ductal adenocarcinoma.

AIMS/HYPOTHESIS: Pancreatic ductal adenocarcinoma (PDAC) can cause type 3C diabetes, known as PDAC-associated diabetes mellitus (PDAC-DM), but the mechanism is unknown. This study aimed to reveal the mechanism. METHODS: PDAC lesions from patients with or without PDAC-DM (n = 4 in each group) were individually profiled for 23,512 mRNAs with microarrays. Bioinformatic analysis and in vivo and in vitro assays were then conducted. RESULTS: We determined that 2,778 genes were differentially expressed; over-representation of ten genes was validated with quantitative RT-PCR. The analysis of gene ontology showed that the differentially expressed secretory genes were related mainly to inflammation. High levels of a marker of inflammation (C-reactive protein [CRP]) and an inflammatory mediator (TNF super-family member 13 [TNFSF13]) were found in the serum of patients with PDAC-DM. After surgical resection of PDAC lesions, CRP and TNFSF13 levels significantly decreased (p < 0.01). Furthermore, we found that the levels of TNFSF13 in PDAC lesions and TNFSF13 and CRP in serum were significantly correlated with the diabetic status of patients with PDAC-DM (p < 0.01). Assays in vivo showed that after exposure to an inhibitor of inflammation (celecoxib), the fasting blood glucose level in the mouse model of PDAC-DM dramatically decreased from 6.9 ± 0.1 to 5.6 ± 0.1 mmol/l in 2-4 days (p < 0.01). CONCLUSIONS/INTERPRETATION: We found that acute inflammation was involved in the pathogenesis of PDAC-DM. We contend that acute inflammation is a potential target for the diagnosis and treatment of PDAC-DM.

Proteomic identification of potential Clonorchis sinensis excretory/secretory products capable of binding and activating human hepatic stellate cells.

Epidemiological and experimental evidence demonstrated that Clonorchis sinensis is an important risk factor of hepatic fibrosis and cholangiocarcinoma. C. sinensis excretory/secretory products (CsESPs) are protein complex including proteases, antioxidant enzymes, and metabolic enzymes, which may contribute to pathogenesis of liver fluke-associated hepatobiliary diseases. However, potential CsESP candidates involved into hepatic fibrosis and cholangiocarcinoma still remain to be elucidated. In the present study, we performed proteomic identification of CsESP candidates capable of binding and activating human hepatic stellate cell line LX-2. Immunofluorescence analysis confirmed the interaction of CsESPs with LX-2 cell membrane. LX-2 cells could be stimulated by CsESPs from 24 h post incubation (p < 0.05). Specifically, 50 µg/ml of CsESPs showed the strongest effect on cell proliferation in methyl thiazolyl tetrazolium (MTT) assay which could also be demonstrated by flow cytometry analysis (p < 0.01). Furthermore, expression level of human type III collagen in LX-2 cells treated with CsESPs was significantly higher than that in control cells measured by molecular beacon and semiquantitative reverse transcription (RT)-PCR approaches (p < 0.01). Finally, CsESPs before and after incubation with LX-2 cells were subjected to two-dimensional gel electrophoresis (2-DE) analysis and matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Nine proteins with abundance change above threefold were Rho GTPase-activating protein, mitochondrial cytochrome c oxidase subunit Va, α-enolase, phospholipase C, interleukin-15, insect-derived growth factor, cytochrome c oxidase subunit VI, DNAH1 protein, and kinesin light chain. Taken together, we identified potential CsESP candidates capable of binding and activating human hepatic stellate cells, providing more direct evidences that are previously unknown to accelerate strategies for C. sinensis prevention.

Comparison of two serpins of Clonorchis sinensis by bioinformatics, expression, and localization in metacercaria.

Clonorchiasis, which has been an important public health problem in China, is caused by ingestion of raw or undercooked fish contaminated by live metacercaria. Therefore, preventing fish from infecting is of great significance for controlling the disease. SERPINs (serine protease inhibitors) are well known as negative regulators of hemostasis, thrombolysis, and innate immune responses. In the present study, two full-length sequences encoding SERPIN were identified from metacercaria cDNA library of Clonorchis sinensis (C. sinensis) and were denominated as CsSERPIN and CsSERPIN3, respectively. Bioinformatics analysis showed that the two sequences shares 35.9% identity to each other. Both of the sequences have SERPIN domain and the greatest difference between the two domains is the reactive centre loop. Transmembrane region was found in CsSERPIN3 while not in CsSERPIN. The expression of the two CsSERPINs was significantly higher at the life stage of metacercaria than that of adult. The transcription levels of CsSERPIN and CsSERPIN3 at metacercaria stage were 3.249- and 11.314-fold of that at adult stage, respectively. Furthermore, the expression of CsSERPIN was 4.32-fold of that of CsSERPIN3 at metacercaria stage. Immunobiochemistry revealed that CsERPIN was dispersed at subtegument and oral sucker of metacercaria, while CsSERPIN3 localized intensely in the tegument of metacercaria of C. sinensis inside of the cyst wall. All these indicated that the CsSERPINs play important roles at metacercaria stage of the parasite. CsSERPIN may take part in regulation of endogenous serine proteinase and CsSERPIN3 may be involved in immune evasion and be a potential candidate for vaccine and drug target for clonorchiasis.

Genetic diversity of Plasmodium vivax population in Anhui province of China.

BACKGROUND: Although the numbers of malaria cases in China have been declining in recent years, outbreaks of Plasmodium vivax malaria were still being reported in rural areas south of the Yellow River. To better understand the transmission dynamics of P. vivax parasites in China, the extent of genetic diversity of P. vivax populations circulating in Bozhou of Anhui province of China were investigated using three polymorphic genetic markers: merozoite surface proteins 1 and 3α (pvmsp-1 and pvmsp-3α) and circumsporozoite protein (pvcsp). METHODS: Forty-five P. vivax clinical isolates from Bouzhou of Anhui province were collected from 2009 to 2010 and were analysed using PCR/RFLP or DNA sequencing. RESULTS: Seven and six distinct allelic variants were identified using PCR/RFLP analysis of pvmsp-3α with HhaI and AluI, respectively. DNA sequence analysis of pvmsp-1 (variable block 5) revealed that there were Sal-I and recombinant types but not Belem type, and seven distinct allelic variants in pvmsp-1 were detected, with recombinant subtype 2 (R2) being predominant (66.7%). All the isolates carried pvcsp with VK210 type but not VK247 or P. vivax-like types in the samples. Sequence analysis of pvcsp gene revealed 12 distinct allelic variants, with VK210-1 being predominant (41.5%). CONCLUSIONS: The present data indicate that there is some degree of genetic diversity among P. vivax populations in Anhui province of China. The genetic data obtained may assist in the surveillance of P. vivax infection in endemic areas or in tracking potential future disease outbreak.

Schistosoma japonicum tegumental protein 20.8, role in reproduction through its calcium binding ability.

Schistosomiasis threatens thousands of millions of peoples' health every year in the world. Schistosoma japonicum, a pathogen of schistosomiasis, is covered by a lipid bilayer membrane which plays an important role in nutrient transport, signal transduction, interaction with host's immune system, etc. Thus, molecules in the tegumental membrane have gained more and more interest for understanding biological and pathological processes of schistosoma. In this study, we found a protein from S. japonicum cDNA library which has a 20.8 KDa molecular weight (SjTP20.8). Recombinant SjTP20.8 was produced and purified from Escherichia coli. The recombinant protein could be detected by S. japonicum-infected mice and human sera, and it had been found localizing in the tegumental membrane of S. japonicum in the section using immunofluorescence assay. In electrophoretic mobility shift assay, the protein could bind calcium iron in neutral condition. Result of cercariae challenge experiment indicates antibody against this protein can protect mice from chronic hepatic fibrosis. Our results indicate the S. japonicum tegumental protein 20.8 is crucial for the parasite's calcium absorbing and reproduction.

The serological diagnosis of human clonorchiasis by time-resolved fluoroimmunoassay based on GST2-specific IgG4 detection.

Due to its delayed fluorescence of a lanthanide chelate, high accuracy and low background the broad linear range, long fluorescent life-time and large Stoke's shift of europium chelates, the time-resolved fluorescence has been developed for higher sensitive immunoassay. In this article, a simple, sensitive and specific method-time-resolved fluoroimmunoassay (TRFIA) was adopted for immunoassay of clonorchiasis, and recombinant glutathione transferases 2 of Clonorchis sinensis (rCsGST2) was used as a diagnostic antigen. To evaluate this novel assay for clinical applications, 409 serum samples were investigated. The diagnostic accuracy of the antigen was evaluated by receiver-operating characteristic (ROC) analysis. The area under the ROC curve (AUC) was 0.965, 95% confidence interval (CI, 0.946, 0.985). To eliminate the random influence of ambient temperature, test parameters, photometric instruments and so on, the cut-off value was expressed as ratios between the fluorescence of sample and that of a well-defined negative control serum, and the deduced cut-off value was 9.3605. At the optimum cut-off criteria, the technique has a sensitivity of 95.80%, specificity of 93.60%. And the cross reactivity revealed that its cross reactivity with Schistosoma japonicum, round worm, hook worm, whip worm, and Toxoplasma gondii was 9.3, 8.3, 7.6, 9.8, and 5.0%, respectively. Kappa score of agreement between TRFIA and microscopic examination of stools was 0.892, P < 0.05. These combined results showed that our method is feasible and could be used for the clinical determination of clonorchiasis.

Dexamethasone down-regulates the expression of microRNA-155 in the livers of septic mice.

To investigate the expression of microRNA-155 (miRNA-155) in the livers of mice with lipopolysaccharide (LPS)-induced sepsis and to determine the role of dexamethasone (DXM) in the regulation of miRNA-155 expression, we pretreated mice with or without DXM prior to LPS exposure. Our study demonstrated that the expression of miRNA-155 and inflammatory factors increased in the liver tissues of mice with LPS-induced sepsis and that DXM down-regulated their expression in a dose-dependent manner. Moreover, DXM alone inhibited the expression of miRNA-155 to below the baseline level, but did not impact the expression of inflammatory factors, suggesting that the down-regulation of miRNA-155 by DXM may partially, but not completely, depend on the suppression of pro-inflammatory cytokines by DXM. Our data indicate that the overexpression of miRNA-155 in the livers of mice with LPS-induced sepsis may play an important role in the pathological processes of sepsis and that the down-regulation of miRNA-155 by DXM may be a novel mechanism regulating inflammation and immunity.

Low Divergence of Clonorchis sinensis in China Based on Multilocus Analysis.

Clonorchis sinensis, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of C. sinensis in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, act, tub, ef-1a, cox1, cox3, nad4 and nad5 [4.986 kb]) was employed to explore the intra-species genetic construction of C. sinensis in China. Two hundred and fifty-six C. sinensis isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs) showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C) showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in cox1, cox3, nad4 and nad5, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of C. sinensis infection in mummified desiccated feces, these data point to an ancient origin of C. sinensis in China. In conclusion, we present a likely phylogenetic structure of the C. sinensis population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking C. sinensis infection worldwide. Thus, the present study improves our understanding of the global epidemiology and evolution of C. sinensis.

An antigenic recombinant serine protease from Trichinella spiralis induces protective immunity in BALB/c mice.

In this study, we report the cloning and characterization of a cDNA encoding a Trichinella serine protease gene (TspSP-1.3) from GenBank. The recombinant TspSP-1.3 protein (rTspSP-1.3) was expressed in an Escherichia coli expression system and purified with Ni-affinity chromatography. Real-time quantitative PCR analysis revealed that TspSP-1.3 was expressed at significantly higher levels in muscle larvae and adult worms than in newborn larvae. TspSP-1.3 was detected in excretory-secretory proteins of Trichinella spiralis with western blotting. Immunization with the rTspSP-1.3 antigen induced humoral immune responses, which manifested as elevated specific anti-rTspSP-1.3 IgG and IgE antibodies and a mixed Th1/Th2 response. To determine whether purified rTspSP-1.3 had good antigenicity and could be a vaccine candidate for the control of T. spiralis infection, we immunized BALB/c mice with rTspSP-1.3 and subsequently challenged the mice with T. spiralis larvae. The results showed that mice vaccinated with rTspSP-1.3 exhibited an average reduction in the muscle larvae burden of 39 % relative to the control group. These results suggest that TspSP-1.3 could be a novel vaccine candidate for controlling Trichinella infection.