Anti-osteosarcoma effect of antiserum against cross antigen TPD52 between osteosarcoma and Trichinella spiralis.

BACKGROUND: Trichinella spiralis (T. spiralis) is a parasite occurring worldwide that has been proven to have antitumour ability. However, studies on the antitumour effects of cross antigens between the tumour and T. spiralis or antibodies against cross antigens between tumours and T. spiralis are rare. METHODS: To study the role of cross antigens between osteosarcoma and T. spiralis, we first screened the cDNA expression library of T. spiralis muscle larvae to obtain the cross antigen gene tumour protein D52 (TPD52), and prepared fusion protein TPD52 and its antiserum. The anti-osteosarcoma effect of the anti-TPD52 antiserum was studied using cell proliferation and cytotoxicity assays as well as in vivo animal models; preliminary data on the mechanism were obtained using western blot and immunohistochemistry analyses. RESULTS: Our results indicated that TPD52 was mainly localized in the cytoplasm of MG-63 cells. Anti-TPD52 antiserum inhibited the proliferation of MG-63 cells and the growth of osteosarcoma in a dose-dependent manner. The tumour inhibition rate in the 100 µg treatment group was 61.95%. Enzyme-linked immunosorbent assay showed that injection of anti-TPD52 antiserum increased the serum levels of IFN-γ, TNF-α, and IL-12 in nude mice. Haematoxylin and eosin staining showed that anti-TPD52 antiserum did not cause significant pathological damage. Apoptosis of osteosarcoma cells was induced by anti-TPD52 antiserum in vivo and in vitro. CONCLUSIONS: Anti-TPD52 antiserum exerts an anti-osteosarcoma effect by inducing apoptosis without causing histopathological damage.

ROS-mediated NLRP3 inflammasome activation participates in the response against Neospora caninum infection.

BACKGROUND: Neospora caninum is an obligate intracellular protozoan that causes neosporosis, N. caninum infection is a major cause of abortion in cattle worldwide. Currently, specific treatment for neosporosis is not available. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a cytoplasmic protein complex that plays an important role in host defense against N. caninum infection, but the underlying mechanisms are poorly understood. METHODS: The reactive oxygen species (ROS) inhibitor and the ROS inducer, wild-type (WT) and NLRP3-deficient peritoneal macrophages or mice were used to investigate the role of ROS in NLRP3 inflammasome activation and controlling parasite burdens. ROS production, cell death and cell viability, production of inflammasome-mediated IL-1ß or IL-18, cleavage of caspase-1 and NLRP3 expression, as well as parasite burdens were detected. RESULTS: In vitro, N. caninum induced ROS generation in a dose-dependent manner in peritoneal macrophages. The pretreatment of ROS inhibitor N-acetyl-L-cysteine (NAC) significantly attenuated N. caninum-induced ROS production, LDH release, IL-1ß secretion and NLRP3 expression, whereas N. caninum proliferation was notably increased. In contrary, the ROS inducer pyrogallol (PG) significantly enhanced ROS production and NLRP3 inflammasome activity and decreased the parasite burden in N. caninum-infected peritoneal macrophages. NADPH-dependent ROS-mediated NLRP3 inflammasome activation induced by N. caninum can also be confirmed by using the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). However, the NAC or DPI pre-treatment or PG treatment did not significantly alter N. caninum-induced inflammasome activities and parasite proliferation in Nlrp3-/- peritoneal macrophages. In vivo, IL-18 releases in serum and parasite burdens in peritoneal exudate cells were significantly increased in PG-treated WT mice after infection with N. caninum; however, IL-18 productions and parasite burdens were not changed in PG-treated Nlrp3-/- mice. Furthermore, PG treatment in WT mice infected with N. caninum significantly decreased the mortality, weight loss and parasite burdens in tissues and histopathological lesions. CONCLUSIONS: Neospora caninum-induced NADPH-dependent ROS generation plays an important role in NLRP3 inflammasome activation and controlling parasites. The ROS inducer PG can control N. caninum infection mainly by promoting NLRP3 inflammasome activation. ROS-mediated NLRP3 inflammasome axis can be a potential therapeutic target for neosporosis.

Identification of a TRBD zinc finger-interacting protein in Giardia duodenalis and its regulation of telomerase.

BACKGROUND: Giardia duodenalis causes giardiasis, with diarrhea as the primary symptom. The trophozoite proliferation of this zoonotic parasite is mainly affected by telomerase, although the mechanism of telomerase regulation has not been thoroughly analyzed. METHODS: This study was performed to identify the telomerase RNA-binding domain (TRBD)-interacting protein in G. duodenalis and its regulation of telomerase. Interaction between TRBD and interacting proteins was verified via pulldown assays and co-immunoprecipitation (co-IP) techniques, and the subcellular localization of the protein interactions was determined in vivo via split SNAP-tag labeling. The hammerhead ribozyme was designed to deplete the mRNA of TRBD-interacting proteins. RESULTS: Using TRBD as bait, we identified zinc-finger domain (ZFD)-containing proteins and verified it via pulldown and co-IP experiments. Protein-protein interaction occurred in the nuclei of 293T cells and both nuclei of G. duodenalis. The hammerhead ribozyme depleted ZFD mRNA levels, which reduced the reproduction rate of G. duodenalis, telomerase activity and telomere length. CONCLUSIONS: Our findings suggest that ZFD may regulate telomere function in G. duodenalis nuclei.

Effect of temperature, pH, physical and chemical factors on germination rate of the chlamydospores of the nematophagous fungus Duddingtonia flagrans.

This study mainly investigated the effects of environmental factors on the germination/dormancy, sporulation and resistance of Duddingtonia flagrans chlamydospores. Results showed that the germination temperature of chlamydospores was >10°C and ≤35°C. After the chlamydospores were treated at -20, -40 and -80°C for 12-24 h, they still had the ability to germinate. The chlamydospores germinated at pH 3-13 but did not germinate at pH 1-2 and pH 14. The chlamydospores could tolerate ultraviolet rays for 720 min, but visible light irradiation for 24 h significantly reduced their germination rate. The chlamydospores did not germinate under anaerobic conditions. After the chlamydospores were cultured on water agar (WA) containing 5, 10 and 20% NaCl, their germination rate was significantly inhibited. Once NaCl was removed, the chlamydospores almost completely recovered their germination ability. Among the nine kinds of additives used in the study, 0.3% arginine significantly promoted spore germination (P < 0.05) but 1% trehalose and 1% glycerine significantly inhibited spore germination during incubation from 24 h to 48 h (P < 0.05). This work indicated that D. flagrans chlamydospores are highly resistant to environmental variations and so could be used for biocontrol of animal parasites.

Morphological variability, molecular phylogeny, and biological characteristics of the nematophagous fungus Duddingtonia flagrans.

This study aims to investigate the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Duddingtonia flagrans isolates. We isolated 13 isolates of D. flagrans and found features that have never been reported before, such as two to three septa incluing club-shaped conidia. Meanwhile, we conducted molecular phylogenetic analysis of the seven isolates and tested the radical growth of the isolates under different pH values, temperatures, and media. The capturing ability against infective larvae (L3) of Cooperia spp. in yak was detected in vitro. Finally, one isolate was selected for scanning electron microscopy (SEM) to investigate the trap formation process. The fungal sequence was obtained and submitted to GenBank (Accession no. KY288614.1, KU881774.1, KP257593.1, KY419119.1, MF488979.1, MF488980.1, and MF488981.1), and the tested isolates were identified as D. flagrans. Except for three isolates, the radial growth of the other isolates on 2% corn meal agar and 2% water agar exhibited faster growth than on other media. The fungus could not grow at 10 and 40°C but grew within 11 to 30°C. Moreover, it did not grow at pH 1-3 and 13-14, but instead at pH 4-12. In the in vitro experimental, L3s were reduced by 94.36%, 88.15%, and 91.04% for SDH035, DH055, and F088, respectively. SEM results showed that at 8 hr post addition of nematodes, some of the latter were captured. In the later stages of the interaction of the fungus with nematodes, a large number of chlamydospores were produced, especially on the predation trap. Results of the present study provided information about the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Arthrobotrys flagrans isolates before administering them for biocontrol.

Intestinal microbes influence the survival, reproduction and protein profile of Trichinella spiralis in vitro.

The interactions between intestinal microbes and parasitic worms play an essential role in the development of the host immune system. However, the effects of gut microbes on Trichinella spiralis are unknown. The aim of this work was to explore microbe-induced alterations in the survival and reproduction of T. spiralis in vitro. To further identify the proteins and genes involved in the response of nematodes to microbes, quantitative proteomic analysis of T. spiralis was conducted by iTRAQ-coupled LCMS/MS technology and quantitative real-time-PCR was used to measure changes in mRNA expression. The results showed Lactobacillus acidophilus, and especially Lactobacillus bulgaricus, significantly enhanced the survival and reproductive rates of nematodes. Salmonella enterica, and especially Escherichia coli O157:H7 (EHEC), had opposite effects. Genetic responses were activated mainly by EHEC. A total of 514 proteins were identified and quantified, and carbohydrate metabolism-related proteins existed in a higher proportion. These findings indicated that some gut bacteria are friendly or harmful to humans and in addition they may have similar beneficial or detrimental effects on parasites. This may be due to the regulation of expression of specific genes and proteins. Our studies provide a basis for developing therapies against parasitic infections from knowledge generated by studying the gut microbes of mammals.

Pentatrichomonas hominis: prevalence and molecular characterization in humans, dogs, and monkeys in Northern China.

Pentatrichomonas hominis is an anaerobic amitochondrial flagellated protist that primarily colonizes the large intestines of a number of species, including cats, dogs, nonhuman primates, and humans. The prevalence of this parasite in dogs, monkeys, and humans is, however, poorly understood. In this study, a total of 362 fecal samples including 252 dogs, 60 monkeys, and 50 humans from northern China were collected for an epidemiological survey of P. hominis infection.The average prevalence of P. hominis infection determined by nested PCR was 27.38% (69/252), 4.00% (2/50), and 46.67% (28/60) in dogs, humans, and monkeys, respectively. The prevalence was significantly higher in 6-month-old dogs (41.53%) and children (7.69%) than in older dogs (14.39%) and adults (0%) (P < 0.05). Sequencing of amplicons revealed that four variable positions separated sequences into three types, called CC1-3. CC1 was the most prevalent in the study population. This study determined that P. hominis infection is common in dogs, monkeys, and humans, especially in children and young dogs. Given the infection prevalence, P. hominis may pose a risk of zoonotic and anthroponotic transmission.

[In vitro effect of osthole on ultrastructure of Giardia lamblia].

Giardia lamblia trophozoites were cultivated axenically in TYI-S-33 modified medium containing 1.345 mg/ml of osthole (24 h IC50). The parasites were observed by scanning and transmission electron microscopes after treated with osthole for 24 h. The surface of the trophozoites treated with osthole was rough. The surface of ventral sucker and median body had obvious lesions, the cell membrane was damaged and the content spilled out. There were a lot of vacuoles in the cytoplasm. And the nuclear was severely deformed with a serrated edge and marginated nuclear chromatin. The microtubules of sucker had partially disintegrated.

Pentatrichomonas hominis: first isolation from the feces of a dog with diarrhea in China.

A trichomonad-like parasite isolated from canine fecal samples in Changchun, China was successfully cultivated in vitro using RPMI1640 medium supplemented with 10% heat-inactivated calf serum and antibiotics. These were then subjected to scanning and transmission electron microscopy for ultrastructural study. This parasite has four anterior flagella of unequal length, one independent flagellum, and one recurrent flagellum. It exhibits an anterior nucleus, a Golgi complex, an axostyle, food vacuoles, and hydrogenosomes. These features are consistent with the ultrastructural characteristics of previously described Pentatrichomonas hominis. Polymerase chain reaction and sequence analysis of three genetic loci, including ITS1-5.8S rRNA-ITS2, 18S rRNA, and EF-1α, were also used to compare these samples with other trichomonad species. Molecular identification was also consistent with P. hominis. This is the first time that isolation of P. hominis has been isolated from dog in China, although several other strains of P. hominis have been isolated from human samples.

Eimeria maxima: efficacy of recombinant Mycobacterium bovis BCG expressing apical membrane antigen1 against homologous infection.

Coccidiosis is one of the most important protozoan diseases and inflicts severe economic losses on the poultry industry. The aim of this study was to evaluate the capacity of Bacillus Calmette-Guerin (BCG) to deliver apical membrane antigen1 (AMA1) of Eimeria maxima to stimulate specific cellular and humoral immune responses in chickens. Day-old birds were immunized twice with rBCG/pMV261-AMA1, rBCG/pMV361-AMA1, or BCG via oral, intranasal, and subcutaneous routes and then orally challenged with homologous E. maxima sporulated oocysts. Gain of body weight, fecal oocyst output, lesion scores, serum antibody responses, numbers of splenocyte CD4(+) and CD8(+) T cells, and gut cytokine transcript levels were assessed as measures of protective immunity. Challenge experiments demonstrated that rBCG vaccination via intranasal or subcutaneous routes could increase weight gain, decrease intestinal lesions, and reduce fecal oocyst shedding, and the subcutaneous and intranasal routes were superior to the oral route based on the immune effects. Furthermore, intranasal rBCG immunization could also lead to a significant increase in serum antibody, the percentage of CD4+ and CD8+ T lymphocyte cells, and the levels of IL-1ß, IFN-γ, IL-15, and IL-10 mRNAs compared with the control group. These results suggested that intranasal rBCG immunization could induce a strong humoral and cellular response directed against homologous E. maxima infection. This study provides data for the use of rBCG to develop a prophylactic vaccine against coccidiosis.

Identification of a novel gene product expressed by Trichinella spiralis that binds antiserum to Sp2/0 myeloma cells.

To obtain novel antigen genes for use as an anti-tumor vaccine, a Trichinella spiralis cDNA expression library was constructed from muscle larvae RNA and screened with sera from Balb/C mice injected with Sp2/0 myeloma cells. One positive clone was obtained after three rounds of immunoscreening of the cDNA expression library and was subsequently excised in vivo using the ExAssist helper phage with SOLR strain. A full-length gene was amplified using 5'-RACE technology and analyzed by BLAST, Protein Analysis System of ELM, and DNAStar Software. The sequencing results showed that the fragment was 569 bp in length and contained an open reading frame. It was predicted that the full-length gene encoded 136 amino acids. This gene, TS2, contained four putative N-Arg dibasic convertase (nardilysine) cleavage sites, one peptide C-terminal amidation site, and one glycosaminoglycan attachment site. Six antibody epitopes were predicted by bioinformatic analysis.

[Cloning and prokaryotic expression of casein kinase II subunit beta gene fragment of Dirofilaria immitis].

OBJECTIVE: To clone and express the partial fragment of Csnk2b gene of Dirofilaria immitis in prokaryotic cells, and analyze the immunoreactivity. METHODS: The partial fragment of Csnk2b gene was amplified by PCR with a pair of specific primers. The PCR product was cloned into pMD18-T, and then sub-cloned to pGEX-4T-1 expression vector. The constructed plasmid pGEX-4T-1-Csnk2b was transformed into E. coli Rosetta (DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE and identified by Western blotting. RESULTS: The PCR product was about 700 bp. Enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pGEX-4T-1-Csnk2b was constructed. SDS-PAGE results showed that the relative molecular weight (M(r)) of the fusion protein (GST-Csnk2b) was about 45 000. GST-Csnk2b reacted positively with mouse anti-D. immitis serum. CONCLUSION: The partial Csnk2b gene has been expressed in prokaryotic expression system and shows immunoreactivity.

Giardia intestinalis: effects of Pulsatilla chinensis extracts on trophozoites.

Pulsatilla chinensis is a medicinal root plant that has been used to treat a wide range of disease conditions. Our study determined the antiprotozoal activity of various P. chinensis extracts and fractions against Giardia intestinalis including their effects on parasite growth, cell viability, adherence, and morphology. Ethyl acetate extracts (IC50 = 257.081 µg/ml) were the most active to inhibit the growth of G. intestinalis followed by aqueous extract (PWE), saponins, and n-butanol extract. The PWE and ethyl acetate extract inhibited G. intestinalis trophozoites adherence after 3 h of incubation and killed almost 50 % of the parasite population in a time-dependent manner. Changes in morphology, presence of precipitates in the cytoplasm, dissolved cytoplasm with large vacuole, break of flagella and ventral disk, membrane blebs, and intracellular and nuclear clearance of the treated trophozoites were observed by scanning and transmission electron microscopy. We demonstrated that P. chinensis induced these changes in G. intestinalis morphology and consequently has potential therapeutic use against giardiasis.

[Prokaryotic expression and identification of S-dsRNA gene from Cryptosporidium parvum virus].

OBJECTIVE: To clone and express S-dsRNA gene of Cryptosporidium parvum virus, and investigate the reactogenicity of the recombinant. METHODS: Total RNA was extracted from Cryptosporidium parvum and S-dsRNA gene was amplified by RT-PCR. The PCR product was cloned into pET-28a(+) expression vector. The recombinant plasmid pET-28a(+)-S was transformed into E. coli BL21 (DE3) and induced with IPTG. The expression situation of recombinant protein was analyzed by SDS-PAGE. Its reactogenicity was examined by Western blotting analysis. RESULTS: pET-28a (+)-S was identified by PCR and double endonuclease digestion. SDS-PAGE result showed that the recombinant protein (M, 37,000) was expressed in the form of inclusion body. High level expression of recombinant protein was found at 1 mmol/L IPTG condition after incubation at 37 degrees C for 4 h and reached up to 72.6% of the total protein. The protein was recognized by the antisera from mice immunized with antigens from Cryptosporidium parvum oocysts. CONCLUSION: The S-dsRNA gene of Cryptosporidium parvum virus has been expressed with adequate reactogenicity.

Selection and identification of a new adhesion protein of Cryptosporidium parvum from a cDNA library by ribosome display.

In this study, we described a novel display method to identify surface adhesion proteins of Cryptosporidium parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum was expressed on ribosome and selectively and specifically screened with intestinal epithelial cells (IECs) from newborn Cryptosporidium-free Holstein calves. Proteins were then enriched using a multi-step panning procedure. A new surface adherence protein of C. parvum was selected, named Cp20. Sequence analyses showed that Cp20 has a N-terminal signal peptide and four transmembrane regions. Indirect immunofluorescence assay (IFA) using an antibody specific for rCp20 demonstrated that the antibody specifically bound to the surface of sporozoites and oocysts. The recombinant plasmid pVAX1-Cp20 was constructed to examine the potential of the Cp20 gene as a target for specific preventive and therapeutic measures for cryptosporidiosis. The in vivo efficacies of the DNA vaccine was tested in BALB/c mice. The results indicated that the DNA vaccine elicited significant antibody responses and specific cellular responses when compared to control mice that received vector only or PBS. The DNA vaccine induced strong protective immune response against C. parvum and lower level of the oocysts shedding after challenge infection. This study suggested that Cp20 could serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.

Cell budding from pre-invasive tumors: Intrinsic precursor of invasive breast lesions?

Our previous studies showed that in patients with ductal carcinoma in situ (DCIS) of the breast, the tumor cells that overlie focal myoepithelial cell layer disruptions (FMCLDs) are generally arranged as finger-like projections that bud into the stroma. These budding cells have significantly more genetic instability and invasion-related gene expression, and less estrogen receptor (ER) expression, than their epithelial cell counterparts. This study aimed to assess these cells for potential molecular markers that are uniquely associated with cell adhesion and motility. Seventeen ER-positive DCIS cases were screened by immunostaining for ER, and 7 cases which harbored FMCLD lesions were used to examine the expression of the potential markers. Two cases with both DCIS and invasive lesions were selected for comparing the differences in molecular expression between these lesion types. The results showed that expression levels of talin, E-cadherin and focal adhesion kinase (FAK) in tumor cells overlying FMCLDs were higher than those within the corresponding duct. Integrin ß1 staining was detected only in a small number of the tumor cells overlying the FMCLDs. Vinculin staining was weak (18%) or not detected (82%), and no expression was found in the tumor cells within the corresponding duct or in the pure isolated DCIS. By contrast, the expression levels of talin, vinculin and integrin ß1 in the invasive tumors were distinctly higher than those in DCIS, and the expression of FAK and E-cadherin was lower. Using electron microscopy, we found that the tight junctions between tumor cells overlying the FMCLDs were reduced compared to the adjacent tumor cells in the lumen. These results indicate that the tumor cells overlying FMCLDs are likely to represent the specific precursors of invasive breast lesions. Our findings may also facilitate the identification of specific targets for further molecular profiling, which will more completely characterize this important cell population.

[Cloning and sequence analysis of a partial gene of Trichomonas vaginalis dsRNA virus].

A pair of degenerate primers were designed following the published nucleotide sequence of Trichomonas vaginalis virus(U08999, NC003873, NC003824, NC003834). Using the total nucleic acids extracted from the Trichomonas vaginalis as template, RT-PCR was performed with the primers to obtain a fragment of the TVV. The product was cloned, sequenced and compared with the sequences available in the GenBank. The size of the amplified gene was 1 454bp, which shares 82.9% sequence identity with the Trichomonas vaginalis virus T1.