Effect of pregnancy malaria on T cells in immune organ of offspring mice / 中华地方病学杂志

Objective:To observe the influence of pregnant mice having malaria on T cell function of offspring mice, and to study the changes of cellular immune response in offspring mice exposed to malaria infection in uterus.Methods:Adult Kunming mice of clean grade were selected after mating, on the 14th day of pregnancy, pregnant mice were randomize assigned into experimental group ( n = 5) and control group ( n = 5) according to the method of random number table. Each mouse in the experimental group was intraperitoneally inoculated with 1 × 10 6 red blood cells infected with Plasmodium berghei ( P.b), and same volume of normal saline was given to control group. After birth, the changes of CD4/CD8 T cell subsets in their thymuses and spleens of the two group neonatal mice were analyzed by flow cytometry at day 0, 1, 3, 5 and 4-week-old. Then the 4-weeks-old neonatal mice were intraperitoneally inoculated with 1 × 10 6P.b. On the third day, the changes of CD4/CD8 T cells subsets in their thymuses and spleens were observed, respectively, and the immune response of spleen cells stimulated by P.b antigen or mitogen [concanavalin A (Con A)] was detected. Results:Compared with the control group, the proportions of CD3 +CD4 +CD8 - T cells in thymus and spleen of the offspring of the experimental group (0, 1, 3, 5 days) were higher ( P < 0.05), while the proportions of CD3 +CD4 -CD8 + T cells in thymus were lower ( P < 0.05). For 4-week-old offspring and after infection of P.b, the proportions of CD3 +CD4 +CD8 - T cells in thymus and spleen of the experimental group were both significantly higher than those of control group ( P < 0.05), in contrast, the proportions of CD3 +CD4 -CD8 + T cells in thymus and spleen were both significantly lower than those of control group ( P < 0.05). The spleen cells of 4-week-old mice were stimulated by P.b antigen or mitogen ConA in vitro, compared with the control group, there were no significant differences in the proportions of CD3 +CD4 +CD8 - T cells and CD3 +CD4 -CD8 + T cells in the experimental group ( P > 0.05). Conclusion:During pregnancy, the maternal infection of P.b could significantly affect the ratio of CD4/CD8 T cell subsets in thymus and spleen of offspring mice; and could change the cellular immune response of offspring to P.b infection.

Source analysis of lymphocytes secreting interleukin-22 from spleen of mice infected with Trichinella spiralis at the early encapsulated stage / 中华地方病学杂志

Objective To investigate the cell origin of interleukin (IL)-22-secreting cell of mice infected with Trichinella spiralis (T.spiralis) at the early encapsulated stage.Methods Twelve Balb/c mice were divided into the infected group and the control group according to body weight by random number table.The infected mice were intragastrically administrated with 300 muscle larvae of T.spiralis,and the control mice were given the same amount of normal saline.The IL-22-secreting cell subsets in mouse splenic lymphocytes were detected by flow cytometry at the fourth week after infection.Results The proportion of IL-22-secreting cells in splenic lymphocytes of T.spiralis infected mice was increased when compared with control group [(0.88 ± 0.25)％ vs (0.28 ±0.17)％,t =-4.899,P ＜ 0.05].There was no significant difference between the proportion of CD3+IL-22+ cells and CD3-IL-22+ cells in the splenic lymphocytes of the infected group [(0.29 ± 0.17)％ vs (0.51 ± 0.17)％,t =-2.195,P ＞ 0.05],and the percentage of CD3-IL-22+ cells were similar between the infected group and the control group [(0.51 ± 0.17)％ vs (0.44 ± 0.22)％,t =-0.600,P ＞ 0.05].The proportion of CD3+IL-22+ cells in the infected group was significantly higher than that in the control group [(0.29 ± 0.17)％ vs (0.07 ± 0.06)％,t =-3.068,P ＜ 0.05],and the percentage of CD4+IL-22+ T cells and γδTCR+IL-22+ T cells were obviously increased in CD3+ lymphocytes [(1.28 ± 0.54)％ vs (0.16 ± 0.07)％,(0.33 ± 0.22)％ vs (0.02 ± 0.00)％,t =-4.997,-3.342,P ＜ 0.05].Conclusions The proportion of IL-22-secreting splenic lymphocytes is increased in mice infected with T.spiralis at the early encapsulated stage.The rise is caused by increased numbers of IL-22-secreting CD3 + lymphocytes,especially CD4+ T cells and γδT cells.

Experience and analysis of excellent works of human parasitology in nation-al medical colleges and universities / 中国血吸虫病防治杂志

Teaching competition is an effective way for college and university teachers to improve their teaching skills. Based on the teaching practice and experience in medical parasitology,this paper discusses several key issues in teaching competition including topics,teaching designs and teaching methods. It provides references for the teachers in department of parasitology of universities and colleges to improve the quality of classroom teaching.

Effect of exogenous nitric oxide on antioxidants from mice infected with Trichinella spiralis / 中国血吸虫病防治杂志

Objective To study the effect of exogenous nitric oxide donor sodium nitroprusside(SNP)on antioxidant en?zymes activities and lipid peroxidation of mice infected with Trichinella spiralis. Methods BALB/c mice were infected with T. spiralis separated by the digestion method. Forty?two days post?infection,the peripheral blood and hepatic tissue from the in?fected or normal mice were collected. Then 4 groups were set:liver homogenate from infected mice+SNP(Group A),liver ho?mogenate from normal mice+SNP(Group B),peripheral blood from infected mice+SNP(Group C),and peripheral blood from normal mice+SNP(Group D). The final concentrations of SNP in each group were set as 0(blank control),2,5,10μmol/L and 30μmol/L,respectively. After reacting with SNP at 37℃for 30 min,the superoxide dismutase(SOD),glutathi?one peroxidase(GSH?Px),catalase(CAT)activities,and malondialdehyde(MDA)concentration were examined and com?pared. Results The levels of SOD,CAT,GSH?Px and MDA concentration in the liver and the blood from the mice infected with T. spiralis were significantly higher than those of the normal ones(all P < 0.05). When reacted with 10 μmol/L and 30 μmol/L SNP,the SOD,GSH?Px,and CAT activities in Group A and B decreased significantly(all P<0.05),while the liver MDA concentration reacted with 2-30μmol/L SNP increased obviously(all P<0.05). As reacted with 30μmol/L SNP,the ac?tivities of blood SOD,GSH?Px,and CAT in Group C and D decreased,while the MDA concentration in blood still increased (all P<0.01). When the SNP concentration was in the range of 2-30μmol/L,there were a negative correlation between the SNP concentrations and SOD,GSH?Px,and CAT activities,as well as a positive correlation with the MDA concentration in the liver and blood from the mice infected with T. spiralis(all P<0.05). Conclusions T. spiralis infection could cause oxidative damage to mice,and increase SOD,GSH?Px,and CAT activities. Nitric oxide released from SNP can decrease antioxidase activ?ities,and inhibit the antioxidant capacity of mice infected with T. spiralis.

Anti-tumor effect of Plasmodium yoelii infection on melanoma in mice / 中国血吸虫病防治杂志

Objective To explore the anti-tumor effect of 17XL strains of Plasmodium yoelii(P.y)infection on melanoma in mice. Methods B16F10 tumor cells were axillarilly injected into the right flank of 20 C57BL/6 mice to establish tumor-bearing mouse models. The next day,the mice were randomly divided into a P.y infection group and control group,10 mice each group. Each mouse of the P.y infection group was intraperitoneally injected with 1×106 red blood cells including 20% P.y infection red blood cells,and each one of the control group were intraperitoneally injected with 1×106 normal red blood cells of C57BL/6 mice. The time of tumor formation of the mice in the two groups was observed and the tumor volumes were measured. Results The time of tumor formation in the P.y infection group[(11.30 ± 0.21)d]was significantly later than that in the control group [(10.40 ± 0.22)d](P < 0.05). From the tumors could be accurately measured to the study end point,both the tumors of mice in the two groups were growing,and the tumor volumes of mice in the P.y infection group were significantly less than those in the control group at each time point(all P < 0.05). The growth rate of tumors in the P.y infection group[(71.10 ± 6.29)mm3/d]was significantly slower than that in the control group[(302.80 ± 49.94)mm3/d](P < 0.05),and the growth rates of tumors every day in the P.y infection group were significantly slower than those in the control group(all P < 0.05). Conclusion The P.y in-fection can delay the occurrence of tumor and inhibit the growth of melanoma.

Cloning,expression and identification of gametocyte specific protein Pfgdv1 of Plasmodium falciparum / 中国血吸虫病防治杂志

Objective To clone a gametocyte specific protein Pfgdv1 of Plasmodium falciparum,express and identify re?combinant Pfgdv1 protein in vitro. Methods PCR was performed to amplify Pfgdv1 from P. falciparum DNA which was got from the patient who was infected with P. falciparum,and the PCR product was inserted into pET28a(+)vector. pET28a?Pfg?dv1 recombinant plasmid was constructed and transformed into E. coli host BL21(DE3+). IPTG was used to induce the recombi?nant Pfgdv1 protein fused with His tag,and the protein was purified by His?NTA affinity chromatography. The recombinant pro?tein was identified by SDS?PAGE and Western blotting. Results The PCR product of Pfgdv1 gene was about 1.65 kb,meeting the expectation of predicted fragment size. The recombinant protein was about 67 kDa,which could be recognized by His?Tag monoclonal antibody. Conclusion The Pfgdv1 gene of P. falciparum is successfully cloned,and the recombinant Pfgdv1 pro?tein is expressed,thereby providing an opportunity for further study on transmission blocking vaccine.

Effect of endomorphin-1 on TLR2 and TLR4 expressions of dendritic cells from human blood / 中国药理学通报

Aim To observe the effect of endomorphin-1 (EM-1 )on TLR2 and TLR4 expressions of dendritic cells (DC)from human peripheral blood.Methods Monocytes isolated from human peripheral blood mono-nuclear cells were cultured in medium containing re-combinant human interleukin-4 and recombinant hu-man granulocyte macrophage colony stimulating factor. After six days of culture,the immature dendritic cells (imDC ) were divided into four groups,the control group (BLA group),EM-1 group,LPS group and LPS+EM-1 group.After 2 days of culture,the expres-sions of TLR2 and TLR4 were determined by fluores-cence activated cell sorter(FACS).The expressions of TLR2 and TLR4 at mRNA level in DC were detected by RT-PCR.Results The FACS results showed that the expressions of TLR2 and TLR4 in imDC were high-er,and their expressions were decreased with the mat-uration of DC.Compared with BLA group,the expres-sions of TLR2 and TLR4 in DC were down-regulated in EM-1 group (P0.05 ).mR-NA expressions of TLR2 and TLR4 on DC in LPS +EM-1 group were lower than those in LPS group (P<0.05 ).Conclusions EM-1 enables the down-regula-tion of the expressions of TLR2 and TLR4 on DC sur-face,the effects of EM-1 on immune function may be associated with TLR2 and TLR4 expressions on DC surface.

Immune response of purified Plasmodium falciparum antigen on human pe-ripheral blood T lymphocytes in vitro / 中国血吸虫病防治杂志

Objective To explore the effects of the purified Plasmodium falciparum(P. f)antigen on the T lymphocytes of the peripheral blood of healthy people in vitro. Methods The peripheral blood mononuclear cells(PBMCs)isolated from healthy donators were cultured with purified P. f antigen(5μg/ml)and normal RBC(nRBC)antigen(5μg/ml),in addition, the group in which only IL-2 added was set as a negative control. After cultured for 12 d,the corresponding antigens were used to re-stimulated the activated T cells in the two stimulated groups for 20 h. Then the cells were collected,the proliferations of the T cells labeled by CFSE were analyzed,and the secretion condition of IL-4 and IFN-γof the cells were detected by flow cytome-try. Results After amplification,the proliferative index(PI )of CD4+T cells that were stimulated with P. f antigen was signifi-cantly higher than those in the nRBC antigen group and the negative control group(both P0.05). Meanwhile,the percentages of the CD4+T cells se-creting IL-4+in the P. f antigen group was significantly higher than those in the other two groups(both P0.05). Conclusion The purified P. f anti-gen could effectively stimulate the proliferation of CD4+T cells in peripheral blood of healthy people,and the latter can play a role in immunoregulation through secreting IL-4 preferentially.

Construction and identification of recombinant Mycobacterium smegmatis vaccine expressing Cysticercus cellulosae cC1 antigen / 中国血吸虫病防治杂志

Objective To construct recombinant Mycobacterium smegmatis vaccine expressing Cysticercus cellulosae cC1 anti-gen. Methods The recombinant pET28a-cC1 plasmid was extracted and double digested by Xho I and BamH I restriction en-zymes,and shuttle plasmid pMV261 was extracted and double digested by Hind III and BamH I restriction enzymes. Both frag-ments were modified by Klenow fragment to form blunt end,then the large fragments of cC1 and pMV261 plasmid were purified and ligated by T4 ligase enzyme. The recombinant pMV261-cC1 plasmid was constructed and sequenced. Then the pMV261-cC1 plasmid was transformed into Mycobacterium smegmatis by the electrotransformation method. The recombinant cC1-Mycobacterium smegmatis was induced by heat and identified by the Western blotting method with the sera of cysticercosis patients. In addition, the growth states of the Mycobacterium smegmatis and the recombinant cC1-Mycobacterium smegmatis were compared and the growth curves were drawn. Results The restriction enzyme and sequencing results showed that the recombinant pMV261-cC1 plasmid was successfully constructed. After heat induction,a 40 kD band was showed by PAGE analysis of cC1-Mycobacterium smegmatis. The Western blotting results showed that the sera of cysticercosis patients could recognize the 40 kDa band,which sug-gested that cC1 protein was expressed in Mycobacterium smegmatis. Compared with the Mycobacterium smegmatis,the recombi-nant cC1-Mycobacterium smegmatis showed no significant difference in proliferation characteristics. Conclusion The recombi-nant cC1-Mycobacterium smegmatis vaccine has been successfully constructed.

Plasmodium vivax specific peptides prediction and screening based on repeti-tive protein sequences and linear B cell epitope / 中国血吸虫病防治杂志

Objective To establish a method based on repetitive protein sequences and linear B cell epitope to predict and screen specific peptides of Plasmodium vivax. Methods A P. vivax protein sequence database was reconstructed based on Plas-moDB data,and a customized software for searching of repetitive sequences was used to count the repetition times of each 16 aa peptide in the whole database,and the highly repetitive peptides were chosen to predict the potential linear B cell epitopes. The re-petitive peptides with P. vivax specificity were selected as candidate antigen peptides to synthesize and to couple with KLH carrier protein for immunizing BALB/c mice. After the immunization,the antibody titers of the immunized mice were detected. Results The repetitive information of 16 aa peptides was analyzed by screening of the total 5 432 peptide sequences in the P. vivax data-base. A total of 22 peptides were identified as candidate peptides from the top 1 000 repetitive peptides by linear B cell epitope pre-diction on the BcePred website. Through clustering analysis and similarity comparison,five potential P. vivax specific peptides were selected,synthesized and then coupled with KLH to immunize the mice. The antibody titers of the immunized mice induced by the 5 peptides were all above 1:9 000. Conclusion The method for predicting and screening of specific peptides of P. vivax based on repetitive protein sequences and linear B cell epitope has been established successfully,and all the 5 peptides obtained by the method can induce the high titer antibody in mice.

Effect of PvMSP1 on differentiation,maturation and function of dendritic cells / 中国血吸虫病防治杂志

Objective To investigate the effects of Plasmodium vivax merozoite surface protein 1(PvMSP1)on differentia-tion,maturation and function of dendritic cells(DC)and the mechanisms of PvMSP1 on the activation of DC via toll like receptors (TLR). Methods DCs were incubated with different doses of PvMSP1(1.0,10.0,100.0μg/ml)in vitro. The changes of CD83, CD86,and HLA-DR on DC were detected by flow cytometry(FCM);the expressions of cytokine IL-10 and IL-12 of DC were mea-sured by ELISA;the expressions of TLR4 and TLR9 mRNA of DC were measured by RT-PCR;the proliferation induction to autol-ogous lymphocytes of DC was measured by MTT. Meanwhile,the untreated DC and LPS inducing DC were as the negative control and positive control,respectively. All the data were analyzed statistically. Results Compared with the untreated DC,the propor-tions of CD83,CD86 and HLA-DR on DC induced by LPS and PvMSP1 increased significantly(all P0.05). In the PvMSP1-treated group,the DC TLR4 mRNA production increased(P0.05);DC stimulated the proliferation of autologous lympho-cytes. Conclusion PvMSP1 enhances DC differentiation and maturation,and the mature DC induced by PvMSP1 has the ability of antigen presenting. The route for PvMSP1 inducing DC maturation might be TLR4 pathway rather than TLR9 pathway.

Construction, identification and immunoreactivity of Mycobacterium tuberculosis DNA vaccine pVAX1/ESAT-6 plasmid / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a Mycobacterium tuberculosis DNA vaccine pVAX1/ESAT-6 plasmid and investigate its immunoreactivity.</p><p><b>METHODS</b>The ESAT-6 gene fragment amplified from Mycobacterium tuberculosis genome was inserted into pVAX1 vector to construct the recombinant plasmid pVAX1/ESAT-6, which was identified by restriction enzyme digestion and sequencing. The recombinant plasmid was transformed into Hela cells using Sofast® Transfection reagent, and the cellular expressions of ESAT-6 mRNA and protein were analyzed by RT-PCR and immunofluorescence assay, respectively. The recombinant plasmid pVAX1/ESAT-6 was also transfected into mouse by electronic pulse method, and the mouse serum IFN-γ level and anti-ESAT-6 IgG antibody level were detected by ELISA, mouse lymphocyte proliferation assessed with flow cytometry, and IFN-γ-secreting lymphocytes counted using ELISPOT.</p><p><b>RESULTS</b>Double restriction-enzyme digestion and sequencing showed that the inserted fragment in the recombinant plasmid pVAX1/ESAT-6 was identical to ESAT-6 gene with an inframe insertion. RT-PCR yielded the target band as expected on agarose gel, and immunofluorescence assay of the transfected cells showed specific green fluorescence signals. The mice transfected with the recombinant plasmid showed significantly elevated serum level of anti-ESAT-6 IgG antibody and increased serum IFN-γ level, spleen cell proliferation, and number of IFN-γ-secreting lymphocytes.</p><p><b>CONCLUSION</b>The Mycobacterium tuberculosis DNA vaccine pVAX1/ESAT-6 plasmid we constructed can induce high levels of cellular and humoral immunoreactivity in mice.</p>

Detection of Plasmodium vivax sporozoites-carrying mosquitoes using loop-mediated isothermal amplification (LAMP) / 中国血吸虫病防治杂志

Objective To establish a simple,convenient,quick and high sensitive method of loop-mediated isothermal amplification (LAMP) for detection of Plasmodium vivax-carrying mosquitoes.Methods The species conservative regions of P.v CSP gene were selected to design 2 pairs of primers which recognized 6 distinct regions.To evaluate the specificity of detection by LAMP,infected Anopheles,An.sinensis (An.s),Plasmodium falciparum (P.f),and healthy human blood DNA were selected as templates.To assess the sensitivity of detection,1.3×10~6,1.3×10~5,1.3×10~4,1.3×10~3,1.3×10~2,1.3×10~1 and 1.3×10~0 copies of P.v CSP plasmid DNA mixed with 1.0 μl An.s DNA were used as the templates of LAMP.The infected An.s DNAs were diluted with negative An.s DNA by 1:2,1:4,1:8,1:16,1:32,1:64,1:128 and 1:256 and then detected by LAMP to show the sensitivity of batch quantity detection.The applied value of this method was evaluated by detecting the same batch of 67 artificial infected An.s mosquitoes,and compared with the detection of microscopic examination and nested PCR in parallel.Results By using LAMP,the detection of infected An.s was positive,while the control samples were all negative.The limits of detection of different proportion dilutions of the mixture of P.v CSP plasmid DNA with An.s DNA were 1.3×10~2 copies.The limits of detection of different proportion dilutions of the mixture of infected An.s DNA with An.s DNA were 1:128.The positive rate of detecting the same batch of 67 artificial infected mosquitoes was 47.76% by LAMP,25.37% by the microscopic examination (X~2 = 7.24,P0.05).Compared with the test of the microscopic examination and then with a statistical analysis,the sensitivity of LAMP was 100%,which agreed well with the sensitivity of nested PCR (100%).Conclusion The method of LAMP is simple,convenient and high sensitive,and it is a potential method for detecting Plasmodium vivax-carrying mosquitoes in the field.

Sample related factors affecting short-term culture of erythrocytic Plasmodium vivax in vitro / 中国血吸虫病防治杂志

Objective To explore the sample reIated factors affecting the short-term culture of erythrocytic Plasmodium dvax in vitro.Methods The vivax malaria blood samples were collected from the patients with malaria in endemic areas,and then incubated with McCoy's 5A medium in an incubator containing 5%CO_2 at 37℃.The factors affecting the short-term culture of Plasmodium vivax were analyzed.Results Plasmodium vivax could finish one asexual cycle in the selected medium.By analyzing the culture results of 74 samples.it was found that the factors affecting the short-term culture included long time delaying al room temperature(>4 h),single stage(only parasites in ring stage were found),patients taking antimalarials,antibiotics or sulfonamides.and low parasitemia.Conclusion The sample related factors are important to the short-term culture of erythrocytic Plasmodium vivax in vitro.

Proportion variation of NK,?? T and Treg cells in peripheral blood of Plasmodium vivax malaria patients / 中国血吸虫病防治杂志

Objective To observe the changes of proportion of NK cells,?? T cells,CD4+CD25+T cells and their FOXP3-expressed sub-population in peripheral blood of acute Plasmodium vivax infected patients.Methods The proportions of NK cells,?? T cells,CD4+CD25+T cells and FOXP3-expressed sub-population within CD4+CD25+T cells were measured by flow cytometry(FCM)in peripheral blood of 25 patients with acute infection of Plasmodium vivax(AC)and compared with those of both 14 normal controls(NC)and 13 immune controls(IC).Results The proportion of NK cells in AC(8.48%)decreased significantly compared with that in NC(15.53%)and IC(17.69%)(P0.05).Conclusions During acute Plasmodium vivax infection,the proportion of NK cells decreases and the proportion of ?? T cells increases,and CD4+CD25+FOXP3+T cells decrease a little.