[Overexpression of Toxoplasam gondii ROP18 by Tet-on Lentivirus Expression System].

OBJECTIVE: To construct a 293T mutant cell line over-expressing ROP18 of Toxoplasma gondii by Tet-on lentivirus expression system. METHODS: Rop18 gene of T. gondii was amplified by PCR, and inserted into a lentiviral vector pLVCT-tTR-KRAB. The recombinant plasmid pLVCT-tTR-KRAB-ROP18 (6 µg) and 293T human embryonic kidney cells were co-transfected with psPAX2 (4 µg) and pMD2.G (2 µg) for the packaging. The result of co-transfection was evaluated by fluorescence microscopy. At 48 h and 72 h after co-transfection, the supernatant of the packaging lentivirus was collected for the 293T cell infection. The doxycycline (DOX) was added into the medium to induce the ROP18 expression in 293T cells. The ROP18 fusion expression was observed under fluorescence microscope and detected by RT-PCR after induction. RESULTS: PCR product of the gene fragment encoding ROP18 was 960 bp. The recombinant plasmid pLVCT-tTR-KRAB-ROP18 was identified by PCR and restriction enzyme digestion. Green fluorescence was observed in 293T cells at 48 h post-transfection. Bright green fluorescence was observed in 293T cells at 24 h after DOX induction. RT-PCR results showed that a 960 bp specific band (ROP18 gene) was detectable in 293T cells. CONCLUSION: 293T cell line stably expressing ROP18 is established with Tet-on lentivirus expression system.

Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/ß-catenin pathway.

Slit2 is often overexpressed in cancers. Slit2 is a secreted protein that binds to Roundabout (Robo) receptors to regulate cell growth and migration. Here, we employed several complementary mouse models of intestinal cancers, including the Slit2 transgenic mice, the ApcMin/+ spontaneous intestinal adenoma mouse model, and the DMH/DSS-induced colorectal carcinoma model to clarify function of Slit2/Robo1 signaling in intestinal tumorigenesis. We showed that Slit2 and Robo1 are overexpressed in intestinal tumors and may contribute to tumor generation. The Slit2/Robo1 signaling can induce precancerous lesions of the intestine and tumor progression. Ectopic expression of Slit2 activated Slit2/Robo1 signaling and promoted tumorigenesis and tumor growth. This was mediated in part through activation of the Src signaling, which then down-regulated E-cadherin, thereby activating Wnt/ß-catenin signaling. Thus, Slit2/Robo1 signaling is oncogenic in intestinal tumorigenesis.

[Construction of beta-hydroxyacyl-acyl carrier protein dehydratase mutant of Toxoplasma gondii by tetracycline inducible expression system].

OBJECTIVE: To construct a beta-hydroxyacyl-acyl carrier protein dehydratase (FABZ) mutant of Toxoplasma gondii with tetracycline inducible expression system. METHODS: The fabz gene was amplified from T. gondii genomic DNA, and then used to construct the tetracycline inducible expression vector pTetO7-Sag1-FABZ-Ty-DHFR. The vector was transfected into TATi strain by electroporation. The FABZ defective mutant was selected by pyrimethamine and limitting dilution assay. The expression of Ty-tagged mutant was detected by Western blotting. 5 x 10(5) tachyzoites of FABZ defective mutant were cultured in HFF in the presence of anhydrotetracycline (ATc, 1 microg/ml) for 24 h and 48 h, respectively. The expression of Ty-tagged FABZ protein in the mutant was detected by Western blotting. RESULTS: The mutant could express the transit peptide (t-FABZ) and mature FABZ (m-FABZ) with the Ty-epitope tag. After ATc added in culture medium for 24 h and 48 h, the expression of t-FABZ in the mutant decreased significantly (P<0.05). CONCLUSION: The FABZ mutant is constructed with a tetracycline inducible expression system.

Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems.

In this study, we evaluated four methods to separate and purify Toxoplasma gondii tachyzoites from in vivo and in vitro culture systems, including trypsin digestion, purification with a 3-µm filter, CF-11 cellulose purification, and Percoll purification. Our results indicate that both purification with a 3-µm filter and CF11 cellulose purification methods remove leukocytes or HeLa cells, and can therefore be used as candidate methods for the purification of in vivo and in vitro culture products. Trypsin digestion had a high tachyzoite recovery rate, but 22.35% of leukocytes and 69.64% of HeLa cells remained in the purified products. Percoll solution [30% (v/v)] also had a high tachyzoite recovery rate, but 3.44% of leukocytes and 61.61% of HeLa cells remained in the purified products. The 40% Percoll solution was also a candidate method for purifying tachyzoites from in vivo culture products, with a 65.45% tachyzoite recovery rate and without leukocytes.

[Preliminary study on the immunological characteristics of permissive and non-permissive hosts infected with Schistosoma japonicum].

OBJECTIVE: To study the difference among immune responses of three kinds of experimental animals with different susceptibility to the infection of Schistosoma japonicum, and preliminarily explore the mechanism of the immune response in permissive and non-permissive hosts. METHODS: Twelve animals of each kind of rodents, C57BL/6 mice, Sprague Dawley (SD) rats and Microtus fortis, were randomly divided into the infected group and uninfected group each with 6 animals. In infected groups of C57BL/6 mice, SD rats, and M. fortis, each animal was infected with 20, 200 and 1000 cercariae of S. japonicum, respectively. 42 d later, all rodents were sacrificed. Adult worms in portal vein and granulomas in liver were observed and the sera were collected. The levels of cytokines IL-10 and IFN-gamma as well as serum IgG, IgG2a, and IgG1 were detected by ELISA. RESULTS: At the 42th day post infection, worms in portal vein and liver granulomas were observed in C57BL/6 mice and SD rats, but not in M. fortis. The level of IL-10 in the sera of SD rats [(2.21 +/- 0.12) pg/ml] was significantly higher than that in the sera of M. fortis [(1.64 +/- 0.39) pg/ml] and C57BL/6 mice [(0.10 +/- 0.04) pg/ml] (P<0.01). IL-10 in the sera of M. fortis was also significantly higher than that in the sera of C57BL/6 mice (P<0.01). IFN-gamma in the sera of SD rats [(0.21 +/- 0.11) pg/ml] was significantly higher than that in the sera of M. fortis [(0.11 +/- 0.03) pg/ml] and C57BL/6 mice [(0.09 +/- 0.02) pg/ml] (P<0.05), but no difference between M. fortis and C57BL/6 mice (P>0.05). The levels of IgG (1.53 +/- 0.31), IgG1 (1.48 +/- 0.44) and IgG2a (0.41 +/- 0.11) in SD rats were significantly higher than that in the sera of M. fortis (0.48 +/- 0.14, 0.15 +/- 0.03 and 0.12 +/- 0.061) (P<0.01). The levels of IgG (1.21 +/- 0.16), IgG1 (0.88 +/- 0.31) in C57BL/6 mice were significantly higher than that in the sera of M. fortis (P<0.01). IgG1 antibody is the predominant subclass in the three kinds of rodents. The levels of IL-10, IFN-gamma and antibody subclass IgG, IgG1, IgG2a in all non-infected rodents were not detected. CONCLUSION: IL-10 in non-permissive hosts, which is an essential agent in the regulation of Th2 immune response, is higher than that in permissive host It may play an important role in the resistance to schistosome in the non-permissive hosts.

[Effects of oral type II collagen on serum antibody and the cytokine cxpression in Peyer's patches].

AIM: To explore the effects of oral type II collagen (CII) on the morphology, cytokine expressions of Peyer's patches(PP)and the levels of serum specific IgG, IgA, IgM. METHODS: CII was orally administrated to Kunming mice in continuous 10 days at different dosage. The CII or adjuvant immunization was given at 11 d and 21 d. The blood and Peyer's patches were collected at 11 d, 21 d and 31 d. The PP hyperplasy was observed by light microscope after HE staining. The fluorescent real time RT-PCR was used to detect the mRNA expressions of IL-17, TNF-α, IFN-γ and TGF-ß1 in PP lymph node. The serum specific IgG, IgA, IgM contents were detected by ELISA. RESULTS: After oral administration of CII for 10 d, the PP lymph node hyperplasia was active and the cap-shape structure could be seen clearly in high dose group, the serum IgA could be detected, the gene expressions of IL-17, TNF-α and IFN-γ were inhibited. After the CII initial immunity, the IgA, IgM, IL-17 levels were descended and TGF-ß1 level was increased in the experiment groups as compared with control group(P < 0.05 or P < 0.01). After the CII booster, IgA was notably increased in high dose group(P < 0.05), in experiment groups IgM was still suppressed (P < 0.05 or P < 0.01) and TGF-ß1 levels were higher than control group(P < 0.05). In adjuvant immunization groups the cytokine expressions were similar to CII immunization groups, the differences of serum specific IgG, IgA, IgM could not be observed as compared with control group. CONCLUSION: The oral administration of CII can increase the serum specific IgA and suppress the gene expressions of IL-17, TNF-α, IFN-γ in the Peyer's patches. It can still have inhibitory action on the serum specific IgA, IgM and IL-17 gene expressions after CII immunization. The results indicate that the changes of the serum specific antibodies and cytokine gene expressions play an important role on treating rheumatoid arthritis by oral CII to induce immune tolerance.

Efficacy of ginkgolic acids against Cryptosporidium andersoni in cell culture.

Cryptosporidium is a worldwide waterborne parasite and the treatment is a severe problem in immunocompromised patients. In this study, we used the in vitro culture system to evaluate the anti-Cryptosporidium activity of ginkgolic acids (GAs), nitazoxanide (NTZ), garlicin (GAR), and artemether (ART). The growth of Cryptosporidium andersoni in HCT-8 cells was determined by real-time PCR assay. When exposed to 5.00 µg/ml GAs or 10.00 µg/ml NTZ for 48 h, the number of C. andersoni in cultures was on a very low lever, but the number of parasites did not significantly decrease when exposed to GAR and ART. Our results indicate that GAs could be a potential drug for the treatment of cryptosporidiosis.

[Stable green fluorescent protein expression in Toxoplasma gondii mutant].

OBJECTIVE: To construct a Toxoplasma gondii mutant for stably expressing green fluorescent protein (GFP), and establish method to determine the rate of mutant-infected HeLa cells. METHODS: Freshly lysed-out tachyzoites of T. gondii RH strain were transfected with plasmid ptubP30-GFP/sag-CAT. Stable transformants were selected with chloramphenicol and limited dilution. The expression of GFP in mutant tachyzoite was determined by RT-PCR and fluorescence microscopy. When infected with 1 x 10(4)-1 x 10(7) mutant tachyzoites respectively for 24 h, the total number of HeLa cells with green fluorescence was determined by fluorescent microscope in 10 high-power fields, and the rate of HeLa cells with parasitophorous vacuole was determined by flow cytometry. RESULTS: Untransfected tachy-zoites were killed by chloramphenicol, while the stable transformants showed resistance to chloramphenicol. The expression of GFP gene was detected by RT-PCR. The P30-GFP transfectants displayed fluorescence outside the parasite. The rate of mutant-infected HeLa cells increased with the rise of the number of mutant for infection. When infected with 1 x 10(4)-1 x 10(7) tachyzoites, the numbers of HeLa cells with fluorescence were (14 +/- 6), (133 +/- 45), (332 +/- 93) and (443 +/- 90), and the rates of infected cells were (0.49 +/- 0.09)%, (8.76 +/- 0.50)%, (21.0 +/- 21.49)%, and (39.00 +/- 3.47)% by flow cytometry, respectively. CONCLUSION: T. gondii mutant with GFP tag is constructed, which provides a new method to determine the proliferation when cultured in host cells.

[Real-time PCR in analyzing DNA extraction from Cryptosporidium oocysts].

OBJECTIVE: To compare the quality and quantity of DNA extracted from Cryptosporidium oocysts by different methods. METHODS: Cryptosporidium oocysts were treated with different kinds of lysis buffers from USA Promega (Promega) and Shanghai Generay (Generay) commercial DNA extraction kits, 2% Triton X-100 and 5% guanidine thiocyanate. The oocysts were then broken down by freeze-thawing, proteinase K and sonication. Genomic DNA was purified using the commercial kits or Chelex-100. Real-time PCR technique was used to determine the copies of Cryptosporidium oocyst wall protein (COWP) gene. The Promega commercial DNA extraction kit was used as control. RESULTS: The Promega kit resulted in a higher copy number of COWP gene [(6.45-9.86) x 10(6)] than that of Generay commercial DNA kit [(2.38-3.69) x 10(6)], 5% guanidine thiocyanate [(1.27-21.29) x 10(5)] or 2% Triton X-100 [(2.06-866.70) x 10(3)] , respectively. The method of freeze-thawing plus proteinase K plus sonication provided the highest copy number of COWP gene. CONCLUSION: The method of freeze-thawing + proteinase K + sonication is most effective. The effect of DNA extraction by Generay kit and 5% guanidine thiocyanate is similar to that of Promega kit.

[Identification of calf-origin Cryptosporidium bovis Shanghai isolate by nested PCR].

OBJECTIVE: To identify a strain of Cryptosporidium in the feces of naturally infected calf in Shanghai. METHODS: Stool sample was examined by modified acid-fast staining. The size and morphology of the oocysts were microscopically determined. Genomic DNA was extracted from the oocysts isolated from feces of a naturally Cryptosporidium-infected calf. According to the sequence of Cryptosporidium 18S rRNA gene, two pairs of primers were designed and synthesized. The PCR products was amplified by nested PCR and sequenced in double directions. Homology searches were done over the Web using the program Blast. Phylogenetic tree was constructed with NJ method by MEGA4.0 software. RESULTS: Oocysts of the Shanghai isolate were round or elliptical with a size of (5.6 +/- 0.49) microm x (5.2 +/- 0.51) microm. Nested PCR resulted in fragments of approximately 810 bp, and the 18S rRNA nucleotide sequence had 100% identity with C. bovis from Brazil (GenBank Accession No: 151935628). This isolate was clustered in the same clade with C. bovis from Brazil. It showed an identity of 99% with the sequences of C. bovis from Qinghai Province of China, Mongolia, USA, and Tunisia. CONCLUSION: The calf-origin Cryptosporidium derived from Shanghai has been identified as C. bovis.

Toxoplasma gondii: a simple Real-time PCR assay to quantify the proliferation of the apicoplast.

A Real-time quantitative PCR assay to quantify the Toxoplasma gondii apicoplast was studied. Primers were designed to amplify a 305bp product specific to T. gondii apicoplast. Standard curves were generated for both T. gondii apicoplast DNA and genomic DNA, and were used to compute the relative concentration of apicoplast DNA copies in the test samples. The results indicated that the copies of T. gondii apicoplast DNA was significantly low when exposed to ciprofloxacin, clindamycin and azithromycin for 48h in the second infectious cycle. Our study shows that the Real-time PCR technique is a simple and quick technique for screening the anti-apicoplast drugs.

Effect of select medium supplements on in vitro development of Cryptosporidium andersoni in HCT-8 cells.

We evaluated the effect of fetal calf serum (FCS), glucose, ascorbic acid, calcium pantothenate, folic acid, and insulin on the growth of Cryptosporidium andersoni in human colon tumor (HCT-8) cells. After being incubated for 48 h, the proliferation of parasites was determined by real-time polymerase chain reaction (PCR) assay, and the development of C. andersoni was observed by transmission electron microscopy (TEM). Ten percent FCS was the best concentration for C. andersoni culture. Glucose, ascorbic acid, and insulin had a significant effect on the growth of C. andersoni when added into 10% FCS RPMI 1640. Calcium pantothenate had no significant effect and folic acid had the inhibited effect. We also observed the stages of trophozoite, macrogamont, microgamont, type I meront, type II meront, and sporozoite of C. andersoni in HCT-8 cells by TEM. Our results indicated that the best medium for C. andersoni was 10% FCS RPMI 1640 medium containing 50 mM glucose, 50 microg/ml ascorbic acid, and 0.3 U/ml insulin. Real-time PCR could provide a quick and precise technique to determine the proliferation of parasites. Cultivation of C. andersoni in HCT-8 cells will facilitate the study of interactions between parasites and host cells as well as provide a reliable system for evaluating anticryptosporidial compound efficacy.

[DNA purification methods of Cryptosporidium oocysts].

PCR technique can especially detect trace amount of Cryptosporidium oocysts. The preparation of template DNA is important for PCR detection. Before DNA purification, the oocysts should be purified and splitted. This paper summarizes the ordinary purification and splitting methods for Cryptosporidium oocysts.

Anti-Toxoplasma gondii activity of GAS in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: The sarcotesta of Ginkgo biloba is a Chinese herbal medicine used for treating toxoplasmosis, a serious disease requiring treatment with antibiotics that can have serious side effects. AIM OF THE STUDY: To investigate the anti-Toxoplasmagondii activity of ginkgolic acids (GAs) isolated from the Ginkgo biloba sarcotesta in Toxoplasmagondii-infected human foreskin fibroblast (HFF) cells in vitro. MATERIALS AND METHODS: The safe concentration of GAs for HFF cells was determined by methyl thiazolyl tetrazolium (MTT) cell proliferation assay. The presence of Toxoplasmagondii was measured by [3H]-thymine deoxyriboside ([3H]-TdR) and [3H]-leucine ([3H]-Leu) incorporation, as well as Giemsa staining. The positive control was the commonly used and highly effective antibiotic azithromycin. RESULTS: Light microscopy revealed that most HFF cells were infected after 4h of exposure to Toxoplasmagondii. After 48 h of exposure to either GAs or azithromycin, Toxoplasmagondii DNA and protein synthesis were minimal, there were no visible parasites in HFF cells, and the HFF cells had no significant morphological changes. CONCLUSIONS: These results demonstrate that GAs have significant anti-Toxoplasma activity with low toxicity to HFF cells, suggesting that GAs could be an alternative treatment for toxoplasmosis.

Molluscicidal activity against Oncomelania hupensis of Ginkgo biloba.

In search for molluscicidal active components, petroleum ether, ethyl acetate, ethanol, and water extracts from Ginkgo sarcotesta were evaluated against the snail Oncomelania hupensis. The bioassay-oriented showed that the activity concentrates in the petroleum ether extract (LD(50) 7.81 ppm). Ginkgolic acids, isolated from the petroleum ether extract, exhibited strong molluscicidal activity (LD(50) 1.49 ppm), gingkgolic acid C15:1 having the strongest molluscicidal activity.

[In vitro culture of tachyzoites of Toxoplasma gondii RH strain in HeLa cells].

OBJECTIVE: To establish a stable and efficient in vitro culture model for tachyzoites of Toxoplasma gondii RH strain. METHODS: Tachyzoites were inoculated into HeLa cells to establish an in vitro culture system. The proliferation of tachyzoites was observed under microscope by the method of Giemsa stain. At the same time, the longterm tachyzoites maintenance in HeLa cells was established, and the effect of different temperature and time on the yield and motility of tachyzoites were observed. RESULTS: The RH strain tachyzoites were cultured and maintained in HeLa cells. Most HeLa cells were destroyed 96 h after inoculation. In the long-term culture system, the proliferation of tachyzoites was stable and its virulence to mouse showed no decrease. Furthermore, tachyzoites in this system proliferated by 5-20 times and (1-5) x 10(7) tachyzoites were harvested. When cultured in HeLa cells at 37 degrees C for 72h then at 25 degrees C for another 120 h, the tachyzoites proliferated by more than 40 times with a motility rate of over 90%. However, rare HeLa cells left in the medium were found. CONCLUSION: Tachyzoites of T. gondii RH strain can be subcultured in HeLa cells for a long time, and high proliferation rate of tachyzoites can be obtained from this in vitro culture system.

[Study on molluscicidal effect of the extracts from sarcotesta of Ginkgo biloba].

OBJECTIVE: To observe the molluscicidal activity and the fish acute toxicity of the molluscicides extracted from Ginkgo biloba sarcotesta by benzinum (EGSB) (with major component of ginkgolic acid), arecoline (ARE) and their combination. METHODS: Oncomelania hupensis snails were immersed in different concentrations of dry powder sarcotesta of Ginkgo biloba (DPGB), extract of Ginkgo biloba sarcotesta by water (EGSW) and EGSB by WHO recommended method for molluscicide screening to observe the molluscicidal activity, and also the inhibiting effect on the snails' climbing-up as well as acute toxicity to Brachydanio rerio. Niclosamide was used as control. RESULTS: The three extractions from Ginkgo biloba all showed molluscicidal activity, with EGSB as the best. Its 24 h LC50 and LC90 were 0.65 mg/L and 5.50 mg/L, and the 48 h LC50 and LC90 were 0.07 mg/L and 0.85 mg/L, respectively. The combination of EGSB and ARE showed better effect than EGSB alone. Its 24 h LC50 and LC90 were 0.26 mg/L and 0.56 mg/L respectively, a sharp decrease by 60% and 90% compared to EGSB (P<0.05). Under the concentration of 2.50 mg/L of EGSB, the rate of snails' climbing-up was 10%, while under the concentration of 0.16 mg/L of the EGSB+ARE combination, the rate was 8%. The inhibition on the snails' climbing-up of the combination was stronger than EGSB. The fish survived for 24 h and 10th respectively at the concentration of 1 x LC90 and 2 x LC90 of EGSB. Under the concentration of 2 x LC90 of the combination, only 50% of the fish died and no fish died at the concentration of 1 x LC90. The toxicity of the combination was lower than EGSB alone. CONCLUSION: EGSB shows an adequate molluscicidal activity and it is worth of further investigation.

[Detection of Pneumocystis carinii DNA in rats by PCR-ELISA].

OBJECTIVE: To establish a PCR-ELISA and evaluate its use in detecting DNA of Pneumocystis carinii (P. c) in rat model. METHODS: SD rats and Wistar rats were used in the experiment. P. c DNA from rat lung tissue and BALF was amplified by PCR. The amplified products were visualized by ethidium bromide (EB) staining after agarose gel electrophoresis or detected by ELISA. The results were compared with that by Giemsa stain. RESULTS: The positive rate in the two species of rats by the two methods was 96.4% and 100% in lung tissue, 96.4% and 100% in BALF, respectively, with no significant difference (P>0.05). Giemsa positive samples were all positive by PCR-ELISA. The negative control group had one positive by ELISA in lung tissue and BALF respectively. CONCLUSION: PCR-ELISA shows a high sensitivity and specificity in detecting the DNA of Pneumocystis carinii, which is a secure and easy use method.