Table of Content

30 August 2003, Volume 21 Issue 4

Previous Issue    Next Issue

论著

Study on CD4~+ Cells Deletion Mechanism in Experimental Alveolar Echinococcosis

LIFu-rong;SHIYou-en;SHIDa-zhong;DAVuitton;PSCraig

2003, 21(4):  2-202. 

Asbtract ( )   PDF (412KB) ( )  

Related Articles | Metrics

　Objective To study the possible mechanism of CD4~+ T cells deletion in mice with alveolar echinococco- sis, particularly on the relationship between Echinococcus multilocularis infection and apoptosis of T lymphocyte subsets. Methods BALB/c mice were infected with E. multilocularis and uninfected mice were used as control group. CD4~+ T cell and CD8~+ T cells were separated 12 weeks and 25 weeks after infection. Purified CD4~+ and CD8~+ T cell subsets were cul- tured in complete medium and stimulated with EmAg, anti-CD3 mAb, rIL-2, mouse rTNFα and PWM respectively. After 16 h of incubation, cells were collected and assessed by electron microscopy. DNA fragmentation was observed by eletrophoresis, stained by TUNEL assays and PI, analyzed by flow cytometry. Results CD4~+ and CD8~+ T cells in 25 weeks experiment group presented chromatin condensation, lost nuclear membrane integrity, and formed exocytoplasmic vacuolization. DNA ladder was observed by agarose gel eletrophoresis, and the appearance of DNA fragments was equiva- lent to approximately 200 bp. None of these appearances were observed in control group in 12 weeks post infection and CD8~+ T cell in mice of 25 weeks post infection group. The apoptosis level of CD4~+ and CD8~+ T cells in 12 weeks post infec- tion group was not significantly different from the control group. While the apoptosis level of CD4~+ and CD8~+ T cells in- creased significantly in 25 weeks post infection group as compared with the control (P<0. 01). Higher apoptosis in CD4~+ T cells was observed than that of CD8~+ T cells. Apoptosis mainly appeared during S phase of cell cycle. Conclusion Apoptosis is a prominent causation of activation-induced CD4~+ T cell death in later period of E. multilocularis infection. In- crease of the death-promoter signals and decrease of the death suppresser signals may have been responsible, in part, for the apoptosis in CD4~+ T lymphocytes in the infected mice.


PCR in Evaluating the Effect of Allicin and Its Combination with SMZco on Murine Toxoplamosis

SHANLian-yu;YANGXiu-zhen;LIUPei-mei

2003, 21(4):  3-206. 

Asbtract ( )   PDF (309KB) ( )  

Related Articles | Metrics

　Objective To evaluate the effect of allicin alone or combined with SMZco on murine toxoplasmosis by a specific, rapid, and sensitive PCR technique. Methods 147 mice were infected with 2×10~4 tachyzoites intraperitoneally and divided into 5 groups at random. Each group was divided into two sub-groups except an untreated group. One sub- group was used to get samples for PCR test and the other for observing the survival duration. The therapeutic grouping was as follows: group A, a combination of allicin and SMZco administered orally for 7 days and continued by allicin alone till 21 days; group B, the combination administered for 14 days and continued with allicin till 21 days; group C, allicin alone for 21 days; group D, SMZco alone for 7 days; group E, untreated control. The dosage was: SMZco 400 mg/
kg·d and al- licin 35 mg/ (kg·d). PCR test was used to detect the parasites in samples of liver and blood from infected mice at 5, 10, 15, 20, 25, 30, 40 and 50 days after infection. Results Parasites were eliminated in the blood because no signal was seen in all the blood samples except for samples from group C at day 5 after infection. From day 10 after infection until the end of the experiment, no amplification of DNA was seen in all the samples. As for liver samples, signals were clear at day 5 post infection. From day 10 post infection till the day 50 post-infection, parasites were still detected, but the PCR products de- creased significantly than that of day 5 post-infection. Result showed that a combination of SMZco with allicin provided a significant protection. SMZco alone was also effective, but allicin alone was not. Conclusion When SMZco is used in combination with allicin, a much higher efficacy is received in the treatment of acute murine toxoplasmosis.


Study on Diagnosis of Cerebral Cysticercosis by Antibody Detection and Imaging Techniques

LIANChen;LIUChen;ZHAOXue-hong;SHEJun-xia;ZHENGXiao-chun;LIZi-yin

2003, 21(4):  4-209. 

Asbtract ( )   PDF (247KB) ( )  

Related Articles | Metrics

　Objective To establish new criteria of antibody detection for the diagnosis of cerebral cysticercosis in combining with imaging examinations for reducing the missed and neglected diagnosis of the disease. Methods 1160 cases with adequate clinical data were collected for the study, among them all cases received antibody detection and comput- erized tomography
CT, 538 cases were examined by magnetic resonance imaging(MRI). Following the locations that cys- ticerci parasitized, the cases were grouped in four types of cerebral cysticercosis: 1 087 cases in brain parenchyma (93. 7%), 42 cases in brain ventricles (3. 6%), 22 cases in brain meninges (1. 9%), and 9 mixed cases (0. 8%). Accord- ing to the number of cysticerci showed by imaging analysis, cases involving parenchyma were further divided as subgroups of slight, moderate,and heavy infection with 1-2, 3-9 and over 10 parasites, with 552 cases(50. 8 %), 443 cases (39. 8%) and 102 cases (9. 4%) respectively. IHA and ELISA were used for detecting antibodies in the sera. Results 635 cases showed an IHA titer of 1: 8 and above (54. 7%), 700 cases (60. 3%) showed positive ELISA and 460 cases (39. 7%) showed weak positive. In the group of light infection (552 cases), 94. 7% showed an IHA titer of less than 1: 8, only 29 cases (5. 3%) with a titer of 1: 8 and above; 94 cases (17%) showed positive ELISA and 458 cases (83%) were weak positive. In the groups of moderate and heavy infections, all cases showed IHA titer of 1: 8 and over, and positive or weak positive ELISA. Conclusion Antibody titers are positively relevant to the intensity of Cysticercus infection. Most cases with light infection showed a low IHA titer (less than 1: 8) and a weak positive ELISA, a fact that these cases would have been missed by the immunological tests. Therefore, an integrated analysis of the results with immunological test and clinical imaging technique is important in diagnosing cerebral cysticercosis.


Polymorphism Analysis of Cytochrome C Oxidase 1 COI Gene in Necator americanus Collected from 5 Provinces in China

LITie-hua;GUOXiang-rong;HULing;XIAOShu-hua;XUEHai-chou;JohnHawdon

2003, 21(4):  5-213. 

Asbtract ( )   PDF (301KB) ( )  

Related Articles | Metrics

　Objective To detect and compare the COI gene sequences of Necator americanus collected from the provinces of Sichuan, Hainan, Yunnan, Hubei and Jiangsu, and to analyze the genetic diversity of the geographic isolates. Methods COI genes of N. americanus were amplified from the genomic DNA by PCR and sequenced. Results The COI gene sequences of N. americanus from five provinces were 97%-99% identical over 595 bp, and base variation oc- curred in 19 nucleotide sites in which the transition was more frequent than the transversion. The difference between the se- quences ranged from 1. 34% to 2. 18%. Conclusion The COI gene sequences show high identity among the geographic isolates of N. americanus with some difference at specific nucleotide sites.


cDNA Cloning and Location of Pagumogonimus skrjabini Cysteine Protease

WANGYing;ZHANGXi-lin;ZHANGYan-ling;DUANJian-hua;ZHANGJing-ru;HUANGFu-sheng

2003, 21(4):  6-217. 

Asbtract ( )   PDF (318KB) ( )  

Related Articles | Metrics

　Objective To clone the cysteine protease cDNA fragment from Pagumogonimus skrjabini adults and lo- cate the tissue of the adult worm where cysteine protease is expressed. Methods The cysteine protease cDNA fragment was amplified by reverse transcription-polymerase chain reaction
RT-PCR with degenerated primers. The production was TA-cloned into the pUCm-T vector and sequenced. DNASIS program was used to analyse the nucleotide sequence and de- duce the amino acid sequence, which was aligned with the correlated parasite cysteine protease afterwards. The digoxin la- beled cRNA probe was synthesised by in vitro transcription with the cloned cDNA as template. The frozen sections of the adult worms were analysed by hybridization in situ to locate the gene expression. Results A 495 bp cDNA fragment was amplified by RT-PCR and sequenced. An amino acid sequence was deduced by DNASIS. Sequence analysis and align- ment showed significant homologies with the correlated parasite cysteine proteinases and conservation of Cys, His and Asn residues that from a catalytic triad. In the hybridization in situ analysis, intestinal epithelium was stained positively on trans- verse section of adult worms. Conclusion The cysteine proteinase cDNA fragment from Pagumogonimus skrjabini adults was cloned. There are some key sites which are correlated to the function of cysteine protease in the cDNA fragment. Cysteine protease is mainly expressed in intestinal epithelium of P. skrjabini.


Study on the Effect of H_2O_2 Against Acanthamoeba in vitro

ZHAOQun-fei;GAOXue-liang;QIANMin

2003, 21(4):  7-220. 

Asbtract ( )   PDF (236KB) ( )  

Related Articles | Metrics

　Objective To detect the effect of H_2O_2 on Acanthamoeba spp.. Methods By Wright's stain, quanti- tative culture, MTT assay and lactate dehydrogenase
LDH assessment, the in fluence of H_2O_2 on the morphological fea- ture, proliferation speed and the survival rate of Acanthamoeba was tested. Results At low concentration of 0. 125%, H_2O_2 can force the Acanthamoeba trophozoites into cysts irreversibly, and inhibit its proliferation. 1% H_2O_2 can directly de- stroy Acanthamoeba trophozoites. Conclusion H_2O_2 is effective in destroying Acanthamoeba. It is possible to be used as an ideal reagent for the prevention of Acanthamoeba keratitis.


Purification of Recombinant Schistosoma japonicum Signaling Protein 14-3-3 and Antibody Preparation

LIFeng;HUMin;SHENJi-long

2003, 21(4):  8-223. 

Asbtract ( )   PDF (268KB) ( )  

Related Articles | Metrics

　Objective To prepare and characterize the polyclonal and monoclonal antibodies
McAbs against Schis- tosoma japonicum(Sj) signaling protein 14-3-3. Methods Recombinant Sj14-3-3 protein was expressed and the gel slices were lyophilized. Rabbits were immunized with the ground gel powder to prepare polyclonal antibodies against Sj14-3-3. BALB/c mice were immunized with recombinant Sj14-3-3 purified by electroelution. The anti-rSj14-3-3 monoclonal anti- body was obtained by using hybridoma technique. Characterization of the antibodies was performed by enzyme linked im- munosorbent assay (ELISA) and Western blotting analysis. Results Purified recombinant Sj14-3-3 protein was ob- tained. The titers of immune sera from the animals were 1: 8-1: 64. The monoclonal antibody(McAb) against 14-3-3 antigen was obtained and its subclass was found to be IgG1. The McAb reacted strongly and specifically with the expressed Sj14-3-3 protein. Conclusion The anti-14-3-3 sera were obtained and the hybridoma cell lines which secreted stable monoclonal antibodies against Sj14-3-3 were established, which provides a new approach to study the role of Sj14-3-3 in the signal transduction of S. japonicum.


Study on the Thresholds of Malaria Transmission by Anopheles anthropophagus in Hubei Province

XIAZhi-gui;TANGLin-hua;GUZheng-cheng;HUANGGuang-quan;ZHENGXiang;WANGYi;HUANGXi-ping

2003, 21(4):  9-226. 

Asbtract ( )   PDF (235KB) ( )  

Related Articles | Metrics

　Objective To study the thresholds and potential of malaria transmission by Anopheles anthropophagus in Hubei Province and provide indicators for the disease surveillance, early warning, prevention and control in the locality. Methods From July to August 2001, field investigations on vectors and malaria situation were carried out in the village of Yanjiafan, Suizhou City, where the malaria incidence was high. The entomological investigations included the man-biting rate, the proportion of parous anophelines, the human blood index and the blood preference. The others included malaria incidence and parasite rate in human population, the intervals from episode to treatment of the cases, and collection of data on the mean temperature in the area. Based on the formula of basic reproductive rate, the critical man-biting rate was esti- mated. Results 92. 6%
63/68 of An. anthropophagus were found to have human blood meals, it occupied 91. 5% (97/106) of the mosquitoes in human dwellings, its human blood index and vectorial capacity were 12. 5 times (0. 50/ 0. 04) and 6. 5 times (0. 9448/0. 1449) higher than those of An. sinensis. The critical man-biting rate was 0. 2823 and the adjusted man-biting rate was 3. 5 times of its critical man-biting rate (0. 9892/0. 2823). The malaria incidence was 0. 65% (12/1844) and the parasite rate in pupils was 0. 51% (1/198). Conclusion A reduction of the adjusted man-biting rate of An. anthropophagus by 71. 5% is needed for interrupting malaria transmission by this vector in the study area.


临床研究

Factors Influencing the Development of Portal Hypertensive Gastropathy with Liver Fibrosis in Schistosomiasis

LOUYa-yi;WUWen-lin;LUQi-ming

2003, 21(4):  10-229. 

Asbtract ( )   PDF (292KB) ( )  

Related Articles | Metrics

　Objective To evaluate the factors influencing the development of portal hypertensive gastropathy
PHG with liver fibrosis in schistosomiasis japonica. Methods A retrospective study was executed on 196 hospitalized patients with schistosomiasis liver fibrosis (109 of them complicated with PHG) from 1998 to 2002. Endoscopic examina- tions were carried out for all the cases. The analysis was made with a comparison of the PHG incidence to the degree of esophageal varices and the degree of liver function according to Child Pugh's scores. Results With slight, moderate and severe degree of esophageal varices, the PHG incidence was 47. 7%, 54. 8%, and 59. 1% respectively (P>0. 05). With the Child pugh's classes of A, B and C, the PHG incidence was 56. 0%, 53. 3%, and 63. 6% respectively (P> 0. 05). With no surgical intervention, it was 51. 3%, and with splenectomy, only 50. 0%. With splenectomy plus an oper- ation of transection and an endoscopic sclerotherapy, it was 70. 6% and 85. 0%. The PHG incidence was significantly high- er in the group of splenectomy plus operation of transection and the group with endoscopic sclerotherapy than the group with no surgical intervention (P<0. 05). Conclusion The PHG incidence in schistosomiasis liver fibrosis has no relationship with the degree of esophageal varices and Child Pugh classes of liver function. However, splenectomy plus transection and endoscopic sclerotherapy may accelerate the PHG development.


实验报道

Cloning, Expression and Purification of the Major Surface Antigen P30 of Toxoplasma gondii

ZHENGDa-li;HUANGQing-ling;ZHANGTao;LINJian-yin

2003, 21(4):  11-233. 

Asbtract ( )   PDF (331KB) ( )  

Related Articles | Metrics

　Objective To obtain protein of the major surface antigen P30 of Toxoplasma gondii by molecular cloning. Methods The gene of P30
containing the whole P30 gene sequence, without the gene encoding signal peptide was obtained by polymerase chain reaction (PCR) using the primer designed according to the DNA sequence of P30. The recombinant plasmid was constructed using EcoR Ⅰ, Xho Ⅰ and was then transformed into E. coli Top10. The positive clones were identified by restriction enzymes and DNA sequence analysis. The fusion protein was induced by IPTG and purified by affinity chromatography using ProBond~(TM) Resin (a kind of nickel-charged sepharose resin) and was identi- fied by SDS-PAGE and Western blotting. Results The products of PCR, cleavage and link reaction were same as ex- pected and the sequence of inserted fragment in the recombinant plasmid was same as reported except one synonymy muta- tion. A 58 kDa fusion protein was induced by IPTG and was purified by chromatography. Conclusion Fusion protein containing Toxoplasma gondii P30 was achieved and was provided as experiment material for further research.


Factors Affecting the In vitro Microtest for Drug Sensitivity of Plasmodium falciparum

FENGXiao-ping;LIUDe-quan

2003, 21(4):  12-237. 

Asbtract ( )   PDF (281KB) ( )  

Related Articles | Metrics

　Objective To explore factors influencing the results of in vitro microtest for drug sensitivity of Plas- modium falciparum
Pf. Methods Handy media, microplates predisposed with antimalarial drug, cultured Pf parasites (FCC-1/HN isolate) and blood samples from patients were used to evaluate the factors influencing the in vitro determination of drug sensitivity of Pf. Results Liquid medium and lyophilized medium stored at 4℃ for 2 months and 1 year re- spectively could keep their effect unchanged. The effect of the drug-coated plates was not changed within the following peri- od of storage: plates coated with chloroquine and piperaquine stored at 4℃ for 2 years and 6 months respectively; plates coated with pyronaridine and artesunate stored at 4℃ for 3 months. The adhesive paper of the sealed plate could be un- sealed once only. The plastic plate must be harmless to the growing of parasites. The drug liquid should not be stored over 2 wk at 4℃, otherwise the drug concentration was changed. Parasites tested were at synchronous ring stage, with a densi- ty of 1 000-80 000/μl blood, stored at room temperature for 1 h, and at 4℃ for 48 h. Operation needed to follow strictly the standard technical procedure. Conclusion Drug plates, media, adhesive paper, parasites and operation technique can affect the result of in vitro microtest for drug sensitivity of P. falciparum. Standardized materials and operational proce- dure should be used to guarantee a reliable result of the test.


Study on Diagnosis of Schistosomiasis by ELISA Using Periodate-treated Soluble Egg Antigen

HUANGYue-long;YIXin-yuan;ZENGXian-fang;ZHANGRan;YUANShi-shan

2003, 21(4):  13-241. 

Asbtract ( )   PDF (272KB) ( )  

Related Articles | Metrics

　Objective To improve SEA-ELISA, an immunodiagnostic assay for schistosomiasis. Methods Solu- ble egg antigen
SEA of Schistosoma japonicum was treated with sodium periodate (SP) in order to oxidate its glycosylated epitopes. ELISA using the treated SEA was then performed to detect specific antibodies to SEA in the sera of schistosomi- asis patients. Results Serum samples were tested by ELISA using SEA treated with sodium periodate (SP-SEA- ELISA), including 64 sera from cases with chronic schistosomiasis japonica, 119 sera from normal individuals in non-en- demic area, 34 sera from patients with clonorchiasis, 33 sera of paragonimiasis cases and 36 sera from patients with cysticer- cosis. The results showed that its sensitivity (98. 4%) was similar to that of the routine SEA-ELISA (100. 0%) (P> 0. 05) and the specificity is higher than that of the SEA-ELISA (P<0. 05). SP-SEA-ELISA showed a higher negative rate (89. 0%) for sera of schistosomiasis patients 12 months post-treatment than that of the SEA-ELISA (42. 1%). Conclu- sion Use of SP-SEA can increase the specificity of ELISA, reduce cross-reactivity with serum samples from cases infected with other parasites and improve its value in evaluating therapeutic efficacy.


Detection of Trichomonas vaginalis with Direct Immunofluorescence Assay

TIANYong-hong;XIONGCheng-liang;GUANHuang-tao;PANGXue-bing;JIANGChang-fu

2003, 21(4):  14-244. 

Asbtract ( )   PDF (226KB) ( )  

Related Articles | Metrics

　Objective To detect Trichomonas vaginalis Tv by direct immunofluorescence assay (DFA) and dis- cuss its clinical significance. Methods The parasites at a concentration of 3×10~5 cells/L were fixed by acetone on slides which were then blocked by 1% BSA (bovine serum albumin) or 10% BSA or 10% NCS (newborn calf serum) respective- ly, incubated with different dilution of polyclonal goat anti-Tv IgG (1: 20-1: 2 560) for different incubation time (15, 30, 45, 60, 90, 120 min). 120 clinical vaginal specimens were examined by direct immunofluorescence assay, the wet mount method and the in vitro cultivation. Results Blocked by 1% or 10% BSA, incubated at 37℃ for 45 min with a titer 1: 160 of polyclonal antibody were the optimal conditions for direct immunofluorescence assay. Its sensitivity and specificity were 87. 9% and 98. 6% respectively in comparison with the in vitro cultivation method. Conclusion Direct im- munofluorescence assay is a useful alternative to the wet mount method which shows a lower sensitivity.