[Research progress on the molecular pathogenesis of Trichomonas vaginalis].

Trichomonas vaginalis is one of the most common human sexually transmitted pathogens that colonize the urogenital mucosa. This paper reviews those factors in the molecular pathogenesis of the parasite, including cell adhesin, interaction with fibronectin and laminin, G-proteins, pore-forming protein and proteinases.

[Protein analysis of Trichomonas vaginalis treated by Pulsatilla chinensis in vitro].

Water extract of Pulsatilla chinensis (PWE) (0.625 mg/ml) was added to the medium for the culture of Trichomonas vaginalis. After 2 h and 4 h treatment, the content and component of soluble proteins in the parasites were analyzed by SDS-PAGE. A total of 28 protein bands was demonstrated. After treated with PWE for 2 h, the content of 5 protein bands increased and that of 4 bands decreased significantly. Four hours later, a new protein band appeared in the electrophoretogram, and the content of 5 bands increased and that of other 5 bands decreased.

[In vitro anti-Trichomonas vaginalis effects of a mixture of dihydroartemisinin and metronidazole].

OBJECTIVE: To observe the effect of a mixture of dihydroartemisinin and metronidazole on ultrastructure of Trichomonas vaginalis trophozoites in vitro for exploring trichomonacidal mechanism of the drug mixture. METHODS: The trophozoites were cultivated with liver extract solution medium that contained 2.5 x 10(6) parasites/ml. There were dihydroartemisinin 0.5 mg/ml and metronidazole 0.002 mg/ml in the experimental tubes of the drug mixture group. Groups of control (without drug), dihydroartemisinin (1 mg/ml) and metronidazole (5 mg/ml) were established and performed in the same experimental conditions. The parasites were observed by scanning and transmission electron microscopes after having treated with the drugs at 37 degrees for 3.5-5 h. RESULTS: Under scanning electron microscope, the cell membrane of T. vaginalis treated only with dihydroartemisinin for 35 h was damaged, part of pellicle peeled off. Although the surface of the trophozoites treated only with metronidazole for 5 h showed many small bubbles and hollows, the cell membrane looked integral. However, surface of the parasite exposed to the drug mixture for 3.5-4.2 h showed deep folds and cracks, the cell membrane was damaged and even peeled off. When the cell ruptured, the nucleus, axostyle, pelta and hydrogenosomes were exposed, and the cytoplasm spilled out. Transmission electron microscopy showed that the membrane system of the trophozoites treated only with dihydroartemisinin for 3.5 h was damaged considerably. The cytoplasm of damaged parasite spilled out. The cytoplasm of the parasite treated only with metronidazole for 3.5-5 h was damaged seriously. Vacuoles and crevices were visible in the cytoplasm. The cell membrane and the content of the parasites treated with the drug mixture for 3.5-4.5 h were damaged seriously. There were some vacuoles and crevices, dilated endoplasmic reticulum, injured and deformed hydrogenosomes in the cytoplasm. The cell organelles mostly disappeared. Crevices also existed in the nucleus. The nuclear membrane fractured and even disappeared. CONCLUSION: The acting targets of dihydroartemisinin and metronidazole to T. vaginalis trophozoite were different, and a combination of the two drugs shows stronger effect in killing the parasites.

[Killing effect of polymorphonuclear neutrophils on Trichomonas vaginalis].

OBJECTIVE: To study the killing effect of polymorphonuclear neutrophils (PMNs) on Trichomonas vaginalis. METHODS: The vaginal secretion from a patient with vaginitis was incubated in the liver infusion liquid medium to get T. vaginalis. One ml serum was collected from the patient and heated for 30 min at 56 degrees C to inactivate complement in serum, and was absorbed three times with the parasites at 0 degree C to make the serum free of antibodies. PMNs were separated from the patient's blood and purified with density gradient centrifugation and polymer accelerating sedimentation. NBT and safranin O were used to stain the sample. The interaction between PMNs and the parasites was observed under microscope. 300 trichomonads and 3x10(4) PMNs were incubated for 10, 20, 30, 40, 50, 60 minutes under the conditions of aerobic or anaerobic, with superoxide dismutase (SOD) and catalase (CAT) or without SOD and CAT, and with complement or without complement. They were then inoculated in solid medium for another five days under the anaerobic condition, and surviving organisms were enumerated. RESULTS: PMNs were observed to surround and kill a single trichomonad. In the petri-dish containing PMNs, the surviving rate of the parasites in anaerobic condition was 85%, only 3% in aerobic condition (P<0.01). SOD and CAT reduced the killing effect of PMNs, with a surviving rate of 98% and 94% respectively after 60 min incubation. Without SOD and CAT, the surviving rate is only 2% (P<0.05). PMNs in the serum without antibodies killed all the parasites, while the complement-inactivated serum fail to kill them. CONCLUSION: The trichomonacidal activity of PMNs relies on the presence of oxygen and complement in the serum of patient.

[Effect of dihydroartemisinin on the ultrastructure of Trichomonas vaginalis trophozoites in vitro].

OBJECTIVE: To observe the effect of dihydroartemisinin (DHA) on the ultrastructure of Trichomonas vaginalis cultured in vitro. METHODS: The trophozoites of T. vaginalis were cultivated with liver extract solution medium containing 1 mg/ml dihydroartemisinin, and were then observed by scanning and transmission electron microscopes after the treatment for 2.5-4.0 h. RESULTS: The cell membranes of the trophozoites treated with DHA were damaged considerably. The surface of T. vaginalis showed deepening folds, hollow, and cracks. The nuclear membrane appeared fractures. There were a few of crevices in the nucleus and cytoplasm. Disordered and dilated endoplasmic reticulum, injured and deformed hydrogenosomes were found. Cytoplasm of the damaged parasites spilled over from torn place. After the cell membrane was peeled off, the nuclei, hydrogenosomes and pelta were exposed, which finally resulted in the death of the denatured parasites. CONCLUSION: Dihydroartemisinin can destroy membrane structure and organelles of Trichomonas vaginalis trophozoites, which leads to decomposition and necrosis of the parasites.

[Construction of prokaryotic expression vector of the fusion gene IFN-alpha1b/CSP II and expression in E. coli].

OBJECTIVE: To Construct the prokaryotic expression vector of the fusion gene IFN-alpha1b/CSP II. METHODS: IFN-alpha1b was amplified from the human genomic DNA by PCR and cloned into prokaryotic expression vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1/IFN-alpha1b was constructed. Circumsporozoite protein II (CSP II) was amplified from the Plasmodium falciparum genomic DNA by PCR and was cloned into the prokaryotic expression vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1/CSPII was constructed. IFN-alpha1b was cut from the recombinant plasmid pGEX-4T-1/IFN-alpha1b digested with BamH I and EcoR I and ligated with the recombinant plasmid pGEX-4T-1/CSP II also digested with BamH I and EcoR I. The recombinant prokaryotic plasmid pGEX-4T-1/IFN-alpha1b/CSP II was constructed. The fusion gene IFN-alpha1b/ CSP II was expressed in E. coli by IPTG. RESULTS: The prokaryotic expression vector pGEX-4T-1/IFN-alpha1b, pGEX-4T-1/CSP II and pGEX-4T-1/IFN-alpha1b/CSP II were identified by PCR, enzyme digestion and gene sequencing. The expressed fusion protein/IFN-alpha1b/CSP I in E. coli was identified by SDS-PAGE and Western blot. CONCLUSION: The prokaryotic expression vector of the fusion gene IFN-alpha1b/CSP II was successfully constructed, which was then expressed in E. coli.

[RAPD analysis on different isolates of Trichomonas vaginalis].

OBJECTIVE: To study genetic polymorphism of DNA on seven isolates of Trichomonas vaginalis. METHODS: The random amplified polymorphic DNA (RAPD) technique was performed to amplify genomic DNA of the seven T. vaginalis isolates, including Beijing 1, Beijing 2, Chengde, Tangshan, Jiujiang 1, Jiujiang 2 and Jiujiang 3. The DNA bands detected were analyzed by clustering analysis with SPSS software. RESULTS: The percentage of genetic similarity among the seven isolates was from 77.4% to 94.7%, showing a close genetic relationship among them. The percentages between the isolates of Beijing 1 and Tangshan, Jiujiang 1 and Jiujiang 2, Beijing 2 and Jiujiang 3 were 89.2%, 92.1% and 94.7% respectively, while that of Jiujiang 1 and Chengde was 77.4%, indicating a lower homology. CONCLUSION: There are a close genetic relationship and certain gene polymorphism among the seven T. vaginalis isolates; geographical origin plays little role to the genetic characteristics.

[Study on surface adhesion protein 33 gene sequence of different Trichomonas vaginalis isolates].

OBJECTIVE: To study genetic polymorphism of surface adhesion protein 33 (AP33) gene on the seven isolates of Trichomonas vaginalis. METHODS: PCR technique was performed to amplify AP33 gene from the seven isolates, DNA sequences were obtained from the AP33 gene of the isolates and phylogenetic tree was built. Minimal lethal concentrations (MLC) of metronidazole on the isolates were measured in vitro. RESULTS: Percentage of the similarity between 7 isolates and U87098 in GenBank was 98.2%-100%, which indicated a high homology and belonged to isotype isolates. There were four branches between Beijing 1 isolate and Tangshan isolate, Jiujiang 1 isolate and Jiujiang 2 isolate, Beijing 2 isolate and Jiujiang 3 isolate, Chengde isolate and U87098 isolate in phylogenetic tree, which showed a close genetic relationship respectively. No relativity was detected between geographical origin and genetic relationship. CONCLUSION: There is a close genetic relationship among the seven T. vaginalis isolates. MLC showed a difference between isolates which have close relationship.

[Isoenzyme analysis on different isolates of Trichomonas vaginalis].

OBJECTIVE: To study the biological types on the seven isolates of Trichomonas vaginalis from Beijing, Hebei-Tangshan, Hebei-Chengde and Jiangxi-Jiujiang in the mainland of China. METHODS: The samples were analyzed by PAGE, isoenzyme stain and cluster analysis. RESULTS: The isoenzyme systems used in the study included MDH, LDH, G-6-PD, PGI and PGM. No difference in the isoenzyme patterns of G-6-PD and PGI was found among the seven isolates. The MDH and LDH patterns of Beijing 1, Beijing 2, Jiujiang 3 strains were identical, while they were distinguishable from those of Chengde, Tangshan, Jiujiang 1, Jiujiang 2 isolates. The PGM pattern of Beijing 1 and Beijing 2 isolates were same but was different from that of the remainders. Gene tree was constructed according to the isoenzyme profiles. The results showed that there are differences in the patterns of the five isoenzymes between the isolates of Beijing 1, Beijing 2, Jiujiang 3 and other four isolates, and Jiujiang 3 was different from Beijing 1, Beijing 2 slightly. CONCLUSION: It seems reasonable to assume that there are at least three different biological types of Trichomonas vaginalis in China.