Table of Content

30 April 2001, Volume 19 Issue 2

Previous Issue    Next Issue

论著

Effect of Immunostimulatory DNA Sequence on the Production of Th1 and Th2 Cytokines Induced by Dermatophagoides farinae Allergen in vitro

ZHOUCheng;YAOHangping;WENTinghuan;SUNCuiru

2001, 19(2):  1-67. 

Asbtract ( )   PDF (227KB) ( )  

Related Articles | Metrics

　Objective To invesigate the immunoregulatory effect of immunostimulatory DNA sequence (ISS ) on the production of Th1 and Th2 cytokines induced by mite allergen in PBMC of the patients with mite allergic asthma in vitro . Methods PBMC from the patients with allergic asthma and normal controls were isolated and cultured in vitro stimulated by ISS and Dermatophagoides farinae allergen (Df). IL 12, IFN γ and IL 5 in the cell supernatants were detected by ELISA. Df specific IgE in sera of patients were assayed by fluorescent enzyme immunoassay. Results PBMC from both the patients and normal controls stimulated by ISS plus Df produced a significant increase in the level of both IFN γ and IL 12 compared with non ISS and Df stimulations, whereas IL 5 was decreased. Moreover, the levels of IFN γ, IL 12 produced were significantly higher in normal controls than in the patients, on the contrary, IL 5 was down regulated. It was also shown that the level of IL 12 produced by PBMC of the patients with ISS plus Df stimulation correlated positively with that of IFN γ. Conclusion ISS not only promotes the expressions of Th1 cytokines but also downregulates the production of Th2 cytokines induced by Df in both allergic and non allergic individuals, indicating its potential application in the immunotherapy of mite allergy.


Screening and Sequence Analysis of the Adult Stage-specific Gene of Trichinella spiralis

YANGJing;ZHUXinping*;ZHANGXinmei;YANGYapingZHOULei;GAOXin;LIUMingyuan;BoireauPascal

2001, 19(2):  2-71. 

Asbtract ( )   PDF (290KB) ( )  

Related Articles | Metrics

　Objective To obtain the sequence of the adult stage specific gene of Trichinella spiralis . Methods An adult T. spiralis cDNA library was screened using an adult T. spiralis stage specific probe. The gene sequence was analyzed by DNAstar software and GenBank database. Results A cDNA clone of 1 629 bp was obtained by screening the library with an adult stage specific probe. The clone contained one open reading frame composed of 1 464 bp encoding 487 amino acids. GenBank searching revealed that no identical cDNA was found. Two zinc fingers located at the positions within amino acids 207-270. Some possible antigen epitopes were predicted within the amino acids 52-64,108-116,137-163 and 226-260. Conclusion A novel stage specific gene of adult T. spiralis encoding the protein containing certain predicted antigen epitopes was obtained.


Exogenous Nitric Oxide Induces Apoptosis in Tachyzoites of Toxoplasma gondii

LINJing;HUJian-shi;LINJian-yin

2001, 19(2):  3-75. 

Asbtract ( )   PDF (412KB) ( )  

Related Articles | Metrics

　Objective To explore whether NO is able to induce apoptosis in Toxoplasma gondii tachyzoites.Methods Apoptosis induced by NO in T.gondii tachyzoites was investigated by TUNEL (terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling ) method, electron microscopy and agarose gel electrophoresis. Results NO donor, sodium nitroprusside (SNP),was found to induce apoptosis in Toxoplasma gondii tachyzoites in a time- and dose-dependent manner by TUNEL detection. N-acetylcysteine, a NO scavenger, could inhibit SNP-induced apoptosis in the tachyzoites while potassium ferricyanide could not induce apoptosis in the tachyzoite. Electron macroscopy showed that SNP-treated tachyzoites possessed typical morphological features of apoptosis, including chromatin condensation below the nuclear membrane, nuclear pyknosis, and formation of apoptotic body.Agarose gel electrophoresis revealed that SNP-treated tachyzoite DNA fragment exhibited characteristic "DNA ladder" after 15 to 20 h. Conclusion SNP, NO donor, might induce apoptosis in T.gondii tachyzoites in terms of characteristic morphological and biochemical features.


Studies on Synthesis and Degradation of Collagen at Transcription Level in Liver Fibrosis of Rabbits with Schistosomiasis japonica

CHENFeng;CAIWei-min;CHENZhi;LIURong-hua

2001, 19(2):  4-79. 

Asbtract ( )   PDF (301KB) ( )  

Related Articles | Metrics

　Objective To study the synthesis and degradation of collagen at the transcription level during liver fibrosis in rabbits with schistosomiasis japonica. Methods New Zealand rabbits infected with Schistosoma japonicum cercariae were served as animal models of liver fibrosis. The liver specimens were collected through operations at 4, 6, 8, 10, 12, 16, 20, 24 and 28 weeks after infection. TypeⅠcollagen, type Ⅲ collagen, type Ⅳ collagen, MMP-1 and MMP-9 mRNA levels of liver tissue were detected by RT-PCR plus dot blotting, and the size of egg granulomas and the degree of liver fibrosis were measured by histopathological examinations. Results TypeⅠcollagen, type Ⅲ collagen, type Ⅳ collagen, MMP-1 and MMP-9 mRNA levels increased simultaneously in the early stage after the infection, mostly reaching their peaks at 10 weeks after infection. Compared with normal controls, type Ⅰ collagen, type Ⅲ collagen, type Ⅳ collagen, MMP-1 and MMP-9 mRNA levels increased by 12.0 , 11.0 , 6.6 , 10.0 and 11.0-fold, respectively, coinciding with the changes of egg granulomas. Thereafter, both collagen and collagenase mRNA levels decreased. TypesⅠ, Ⅲ and Ⅳ collagen mRNA levels declined to 2-fold to 3-fold compared with normal controls ( P <0.05), while MMP-1 and MMP-9 mRNA levels declined to normal level ( P >0.05) at 28 weeks. This study showed that the synthesis and degradation of collagen remained dynamic balance in the early stage of schistosomiasis, while in the later stage the metabolism of collagen synthesis was higher than that of collagen degradation. Conclusion It was confirmed at the transcription level that when the metabolism of collagen synthesis was higher than that of collagen degradation in rabbits with schistosomiasis japonica, liver fibrosis might be produced.


Expression and Immunocompetence Characterization of Plasmodium falciparum Lactate Dehydrogenase

WUYingsong;LIMing;DONGWenqi;LIYingjie

2001, 19(2):  5-83. 

Asbtract ( )   PDF (398KB) ( )  

Related Articles | Metrics

　Objective To express lactate dehydrogenase (LDH) gene of Plasmodium falciparum FCC1/HN in the E. coli TG1 and analyse its immunocompetence. Methods The LDH gene of the P. falciparum was specifically amplified by polymerase chain reaction, and the recovered gene fragment was cloned into pGEX 4T 1 vector for expression of fusion protein with glutathione S transferase(GST). The recombinant plasmid was transformed into the E. coli TG1. Four mice (Kunming strain) were immunized with purified expressed protein(antigen) and the polyclonal antibodies were collected. The immunocompetence of recombinant protein was analysed by ELISA and Western blot. Results The LDH gene of P. falciparum was successfully expressed in the E. coli TG1. The expressed protein exhibited a specific reaction with immune sera obtained from rabbits immunized with P. falciparum . The specific humoral responses were induced in mice and the titer of the specific antibody was 1∶16 by two dimensional diffusion assay. Conclusion The LDH gene of P. falciparum has been successfully expressed in the E. coli TG1 and the expressed protein has high antigencity.


Studies on the Activity and Immunohistochemistry of Heme Oxygenase in Schistosoma japonicum

LIUWen-qi;LIYong-long;AndreasRuppel

2001, 19(2):  6-86. 

Asbtract ( )   PDF (242KB) ( )  

Related Articles | Metrics

　Objective To explore the activity of heme oxygenase and immunolocate the enzyme in the adult worms of Schistosoma japonicum .Methods Microsomal protein was isolated from the homogenate of adult S.japonicum , heme degradation and effect of different pH conditions and buffers on degrading reaction were investigated by incubating microsomal protein with hemin. The slices of whole worm and cells of S.japonicum were prepared, distribution of HO in schistosome was studied by immunofluorescent and alkaline phosphatase(AP) -immunocytochemical assays.Results Microsomal protein of adult worms can degrade the heme in vitro , the activity being 56.7 nmol bilirubin/(mg·min).The optimal pH was 8.7. Immunofluorescent and AP-immunocytochemical assays revealed that the HO distributed dispersively in the worm, and located in cytoplasm.Conclusion The presence of HO was firstly proved in S.japonicum .


Gene Point Mutation in the Dihydrofolate Reductase-thymidylate Synthase Gene of Plasmodium falciparum

ZHANGZaixingYANGYamingLIUHui

2001, 19(2):  7-89. 

Asbtract ( )   PDF (243KB) ( )  

Related Articles | Metrics

　Objective To investigate the gene point mutation in the dihydrofolate reductase thymidylate synthase (dhfr) gene of Plasmodium falciparum isolate from Yunnan Province strongly associated with pyrimethamine and cycloguanil resistance. Methods Nested PCR and restriction endonuclease digestion were applied to detect the gene mutation using dried blood filter paper collected from the fields in Yunnan Province. Results Different mutations were found in 4 amino acids at positions 16, 51, 108 and 164 of dhfr gene, particularly, Asn 108 and Ile 51, the mutaiton frequency being 94.1% and 90.1%, respectively. The frequency of the wild type genotype (3D7 type) Ser 108 appeared lower ( 9.1%) , while the frequency of the Ala 16 was high( 61.8%); the mutation type was very high, the ratio of HB3 type, 7G8 type/FCR3 type and Cambodian type was 1∶21∶7.5. Conclusion The investigation first demonstrated that Plasmodium falciparum Yunnan isolate dihydrofolate reductase thymidylate synthase gene(dhfr) at positions 16, 51 ,108 and 164 exhibited different degrees of point mutation. The frequency of mutation of the 7D8 type involved in pyrimethamine resistance was higher, while that of the FCR3 type involved in cycloquanil resistance was lower.


Cloning and Sequencing of Cytochrome c Oxidase II(COII) Gene of Three Species of Mosquitoes

HUANGZhaohui;WANGJinfu

2001, 19(2):  8-92. 

Asbtract ( )   PDF (204KB) ( )  

Related Articles | Metrics

　Objective To determine and compare the complete sequence of COII gene from Anopheles sinensis, Aedes albopictus and Culex quinquefasciatus and to identify the molecular homology among these mosquitoes. Methods The recombinant plasmids including COII gene from the three species of mosquitoes were obtained by the T A cloning technique,and the COII genes were sequenced and analyzed. Results The homology of the COII gene nucleotide sequence and the deduced amino acid sequence of COII among the three species of mosquitoes varied from 84.1% to 87.9% and from 85.1% to 89.5%, respectively. The C+G content of COII gene was 23.2%-24.9%, the frequency of transversion higher than that of transition. Conclusion Aedes albopictus and Culex quinquefasciatus are closely related species at their COII molecular level according to COII gene.


Investigation of Risk Factors for Development of Human Hydatidosis Among Households Raising Livestock in Tibetan Areas of Western Sichuan Province

WANGQian;QIUJiamin;PeterSchantz;HEJinge;AkiraIto;LIUFengjie

2001, 19(2):  9-96. 

Asbtract ( )   PDF (260KB) ( )  

Related Articles | Metrics

　Objective To identify factors influencing the development of hydatidosis in Tibetan areas of western Sichuan.Methods A questionnaire investigation was carried out to collect data on factors related to hydatidosis among households raising livestock.Results Analyses of data revealed that dogs rather than foxes were the most important source of AE transmission. People below 19 years old tended to suffering from CE rather than AE and people 19-38 years old, especially the females, were under an increasing risk of suffering from AE (female vs. male, OR=2 438, CI=1 317-4 514, P <0 05). Increased risks of both AE and CE prevalence associated with nomadic life, aging, playing with dogs,not protecting food from flies,and raising yaks or sheep.Conclusion In addition to age,sex,environmental conditions and hygienic behaviors, raising yaks or sheep and playing with dogs also increase the risk of suffering from hydatidosis as well in this area.


Application of Dot Immunogold Filtration Assay for Detecting Serum Antibodies in Clonorchiasis Patients

LIUDeng-yu;HUWen-qing;ZHANGHong-man

2001, 19(2):  10-99. 

Asbtract ( )   PDF (209KB) ( )  

Related Articles | Metrics

　Objective To establish a new, fast,sensitive and specific assay for detecting antibodies in clonorchiasis.Methods Using Clonorchis sinensis adult worm antigen and SPA labelled with colloidal gold as color developing agent,a dot immunogold filtration assay (DIGFA) for detecting clonorchiasis antibodies was tested. Dot-ELISA was used as the parallel control.Results The positive rate of DIGFA and dot-ELISA in testing the sera from 119 proved cases were 96.4%(115/119) and 92.4%(110/119), respectively,the difference between the two assays being in significant ( P >0.05). The negative rate of DIGFA in healthy people was 100%(40/40). The cross reaction rate in 20 cysticercosis cases and 25 schistosomiasis cases were 5%(1/20) and 4%(1/25), respectively. Both coincidence rates comparing DIGFA with dot-ELISA were 90.9%(50/55). Conclusion DIGFA is as sensitive and specific as the dot-ELISA,and has the advantages of simplicity and without specific equipment.


Random Amplified Polymorphic DNA Analysis of Cryptosporidium Species and Strains

TIANZong-cheng;ZHANGXi-chen;WANGJing-min;YINJi-gang;LIJian-hua;CHENJian-bao;YANGJu

2001, 19(2):  11-102. 

Asbtract ( )   PDF (213KB) ( )  

Related Articles | Metrics

　Objective To analyse the genetic heterogeneity of 8 isolates of Cryptosporidium belonging to three species ( Cryptosporiduim muris,Cryptosporiduim parvum and Cryptosporidium meleagridis ) using RAPD technique.Methods DNA extracts of the 8 isolates were amplified by PCR using 26 out of 200 primers. The amplified products were separated by agar electrophoresis followed by staining with ethidium bromide.Results RAPD analysis revealed:① Obvious differences among species were found, the average genetic distance being 7.037;② Certain differences among different isolates of the same species were found,the average genetic distance being 5.162.Conclusion RAPD could differentiate the common and respective gene fragments of the 8 isolates of Cryptosporidium .


实验报道

Studies on Detecting Brugia malayi Larva in Mosquitoes by Polymerase Chain Reaction

WANGYing;DAIXiaodong;TIANXiaoguang;CUIYu;LIJie;YUANXiaodong;SUNDejian

2001, 19(2):  12-106. 

Asbtract ( )   PDF (467KB) ( )  

Related Articles | Metrics

　Objective To establish a specific, sensitive and simple assay for the detection of Brugia malayi larva in Anopheles sinensis .Methods Using a new DNA purification technique (Microcon 100) and two pairs of oligonucleotide primers (p1, p2 and p3,p4) suitable for detecting B malayi in seven areas in our country, the mosquito vectors infected by B malayi were detected by polymerase chain reaction(PCR).Results This PCR method could amplify separately a 322 basepair(bp) and a 155 bp DNA fragment and detect as few as 1/64 of one L 1 in 1 mosquito,the detectable limit was nearly 4 pg DNA of filarial larvae, and it could also detect 1 infected mosquito with one L 3 of B malayi in pools of up to 200 mosquitoes. In contrast,no such specific 322 bp or 155 bp DNA band was detected in Dilofilaria immitis and normal mosquito.Conclusion This PCR techique established for supervision of mosquito vector in B malayi endemic areas is specific,sensitive,and simple.


Studies on the Sex Difference in Proteins Between Male and Female Adult Worm of Schistosoma japonicum

ZHUJianguo;LINJiaojiao;WANHaibao;YUANChunxiu;LIMingZHAOFukun;ZHOUYuancong;WUXiangfu;CAIYoumin*

2001, 19(2):  13-109. 

Asbtract ( )   PDF (241KB) ( )  

Related Articles | Metrics

　Objective To explore the sex difference in proteins between male and female adult worm of Schistosoma japonicum . Methods Two electrophoresis was used to analyse the difference of protein between the male and female adult worm of S. japonicum (Chinese strain). Results Two dimensional electrophoresis analysis revealed that at the site of 43 kDa and an isoelectric point (pI) of 5.60-5.90 the male worm exhibited a band carrying a number of spots and dots, being longer and wider than that exhibited by the female worm. The female worm exhibited 7 specific dots. Conclusion The sex differences in proteins between male and female adult worms of S.japonicum are significant.