Prevalence and fundamental researches of prevention and treatment of toxoplasmosis in China: an overview / 中国血吸虫病防治杂志

Toxoplasma gondiiis an important zoonotic pathogen and its infection has a significant impact on human health and animal husbandry. This review presents the research progresses in the epidemic, genotype, pathogenicity, diagnosis, treatment and vaccine development of Toxoplasma and toxoplasmosis in China, so as to provide the reference for the study of the pathogen and the disease in the country.

Study on genotype-associated HPV infection in cervical lesions / 中华疾病控制杂志

Objective To explore the association of genotypes of human papillomavirus (HPV) with cervicitis, cervical intraepithelial neoplasia (CIN) and carcinoma in situ of cervix. Methods A total of 464 patients with cervical biology admitted to Hefei women and child health care hospital from October, 2014 to October, 2015 were selected. Among them, there were 242 cases of cervicitis, 222 cases of CIN (76 of group Ⅰ, 71 of group Ⅱ, and 66 of group Ⅲ), and 9 cases of cervical cancer. Hybrid chip technology was used to detect cervical secretions of patients, and 21 kinds of HPV DNA were typed according to histopathological biopsy. Results The HPV infection was found in 464 patients with cervical lesions. Among them, 354 cases (76.3%) had HPV infection with 232 cases (65.5%) of single HPV infection and 122 cases (34.5%) of multiple infections included. The rate of HPV infection was 64.9% in the group of cervicitis, while the rate was 86.8% in group I of CIN and in group II of CIN, the rate of HPV infection was 87.3%. Surprisingly, the HPV infection rate in group III of CIN was as high as 90.9%. The infection rate of HPV in the patients with CIN was significantly higher than those with cervicitis (P<0.001). All patients with cervical cancer were infected with HPV. Conclusions Persistent infection of high-risk HPV subtypes increases the hazard of cervical tumor and CIN. Therefore, genotyping of HPV DNA is helpful for screening and prediction of cervical cancer.

The preparation, identification and physicochemical properties of anti-Porphyromonas gingivalis IgY / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To obtain egg yolk antibody in hen eggs laid by hens immunized with the protein of Porphyromonas gingivalis (Pg). To generate, purify IgY against Pg (anti-Pg-IgY) and identify its specificity.</p><p><b>METHODS</b>PgATCC33277 was cultured under standard anaerobic conditions and harvested after proliferation. Then Pg was extracted by sonication until the cell pellets were shattered completely. After centrifugatiton, the supernatant was collected. Five-month-old Roman hens were immunized for egg antibody production. The antibody was inoculated intramuscularly and subcutaneously in the breast from multiple spot with 1.0 ml of a vaccine consisting of oil-adjuvant protein which was mixed with 1 ml protein of Pg and 1 ml Freund's adjuvant complete every 10 days, for 4 times. The eggs were collected after the first immunization and stored at 4°C. The anti-Pg-IgY was extracted and purified. The protein concentration was tested by bicinchoninic acid (BCA), the specificity of IgY analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), the titre of IgY and its physicochemical character were evaluated by indirect enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>The concentration of obtained anti-Pg-IgY was 2.05 g/L. SDS-PAGE analysis of the anti-Pg-IgY showed that the molecular weight of IgY was consistent with the theoretical value. Protein of anti-Pg-IgY appeared approximately 5 days after the first immunization, and reached the peak at 50 - 55 days. Antibody titres reached 1:100 000. Each egg produced more than 10 mg IgY, and its purification was up to 95% as well.</p><p><b>CONCLUSIONS</b>Layer hens immuned by Pg may provide specific IgY of high titre and high concentration. The antibody has high purity and is heat, acid and alkali-resistant.</p>

Cloning and efficient prokaryotic expression of soluble stage-specific antigen cC1 from Cysticercus cellulosae / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone the coding gene of the stage-specific antigen cC1 from Cysticercus cellulosae and express high levels of soluble cC1 in E.coli.</p><p><b>METHODS</b>The cC1 gene was amplified from Cysticercus cellulosae by RT-PCR and cloned into pMD18-T vector, followed by subcloning into the prokaryotic expression plasmid pET28a. The recombinant plasmid was transformed into E.coli BL21(DE3) and the expression conditions were optimized. The expressed product was purified by Ni(+)-affinity chromatography, analyzed by high-performance liquid chromatography (HPLC), and identified with SDS-PAGE and Western blotting.</p><p><b>RESULTS</b>The fragment length of the amplification product by RT-PCR was 1056 bp. Comparison of the amplified gene sequence with the cC1 gene in Genbank identified a samesense point mutation at 423 position in the gene cloned into the expression plasmids. After a 6-h induction with 0.05 mmol/L IPTG at 37 degrees celsius;, the expression of the 40 kd soluble fusion protein exceeded 60% of the total bacterial protein, and the fusion protein was recognized by Cysticercus-infected human sera. The purity of the fusion protein was about 94% after purification by affinity chromatography.</p><p><b>CONCLUSION</b>The stage-specific antigen cC1 from Cysticercus cellulosae has been successfully cloned and the soluble protein efficiently expressed in E.coli, which provides the basis for its further study and application.</p>

Enhanced expression of the decoy receptor IL-13Ralpha2 in macrophages of Schistosoma japonicum-infected mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Type 2 cytokine interleukin (IL)-13 and its decoy receptor, IL-13 receptor (R) alpha2 appear to play a major role in tissue fibrosis of schistosomiasis and asthma. IL-13 is a key regulator of the extracellular matrix (ECM). It is known to signal to cells by binding to the IL-13Ralpha1, which then heterodimerizes with IL-4Ralpha. In contrast, IL-13Ralpha2 binds IL-13 with high affinity but does not signal. IL-13Ralpha2 is known to down-regulate granulomatous inflammation and prolong host survival in Schistosoma mansoni (S. mansoni) infection, but little is known about the location and expression level of IL-13Ralpha2 in the context of S. japonicum infection.</p><p><b>METHODS</b>We established S. japonicum-infected mouse models. Kinetic serum levels of IL-13Ralpha2 were examined with ELISA. IL-13Ralpha2 mRNA and protein of liver tissues were determined by PCR and immunoblotting analysis, respectively. Detection of IL-13Ralpha2 expression and location in macrophages was performed by TaqMan PCR and fluorescent immunocytochemistry technique, respectively.</p><p><b>RESULTS</b>A marked elevation of mRNA and protein expression of IL-13Ralpha2 was observed in mice during S. japonicum infection. An enhanced expression of IL-13Ralpha2 was further demonstrated in primary macrophages of murine schistosomiasis.</p><p><b>CONCLUSIONS</b>IL-13Ralpha2 in macrophages may be a critical contributor to pathogenesis of schistosomiasis. The data highlight the potential importance of cell signaling and antifibrotic gene therapeutics in T helper 2 cell (Th2)-mediated diseases.</p>

Cloning and expression of rotavirus SA11 VP7 and preparation of IgY antibodies against recombinant VP7 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To prepare eukaryotic expression of rotavirus (RV) SA11 capsid protein VP7, and to generate and purify yolk immunoglobulin (IgY) antibodies against the recombinant VP7 from Roman hens.</p><p><b>METHODS</b>MA104 cells were infected with the standard SA11 strain and the culture fluid was collected. A DNA fragment of 978 bp encoding SA11 VP7 was obtained by RT-PCR amplification from genomic RNA of RV SA11. The PCR products were ligated to pMD18-T vector following the confirmation by DNA sequencing and sub-cloned into pPICZalphaB. The recombinant pPICZalphaB-SA11 VP7 was transformed into E coli Top10. The plasmids were linearized by digestion of BstXI and transformed into Pichia pastoris X-33 through electroporation by DNA sequencing. The transformants were induced with methanol for expression. The cultural supernatant was subjected to SDS-PAGE and Western blotting. Fusion expression was purified through the column of affinity chromatography. IgY was identified and purified by SDS-PAGE and Western blotting from eggs of Roman hens immunized with recombinant SA11 VP7.</p><p><b>RESULTS</b>The RNA extracted from the RV culture fluid consisted of 11 bands visualized by silver staining. The expression vector pPICZalphaB-SA11 VP7 was constructed and the fusion protein in Pichia pastoris X-33 was harvested and purified. The recombinant SA11 VP7 with molecular weight of 40 200 was identified by Western blotting. The IgY antibodies against the recombinant SA11 VP7 were produced with a purity of 95 percent and yield of 10.2 mg per egg.</p><p><b>CONCLUSION</b>The preparation of IgY antibodies to recombinant SA11 VP7 might lay a foundation for the development of vaccines and diagnostic techniques.</p>

Influence of immunization dose schemes on immunoprotective response to recombinant signaling protein 14-3-3 of Schistosoma japonicum / 中华预防医学杂志

<p><b>OBJECTIVE</b>To discuss the optimal immunization dose by observing the immunoprotective effects of different doses of recombinant Schistosoma japonicum (Chinese strain) signaling protein 14-3-3 (rSj14-3-3).</p><p><b>METHODS</b>Sj14-3-3 gene was amplified by reverse transcriptase PCR (RT-PCR), subcloned into prokaryotic expression vector pET28a, then transformed into E.coli to express by inducing. Purified rSj14-3-3 was prepared through SDS polyacrylamide gel electrophoresis (SDS-PAGE), electroelution, dialysis, then BALB/c mice were divided into 5 groups and immunized in rSj14-3-3 protein followed by challenging infection (the 1st, 2nd, and 3rd groups were immunized in 50 microg, 100 microg and 300 microg antigen, respectively. The 4th, 5th groups were immunized in Freund's adjuvant and normal saline controls). After 6 weeks of challenging infection, the mice were killed and the worm and egg reduction rates were calculated. And the mice sera in different time were taken to examine the specific anti-Sj14-3-3 IgG.</p><p><b>RESULTS</b>rSj14-3-3 protein was expressed successfully. After immunizing and challenging, worm reduction was found to be 28.20% in the 1st group, 43.10% in the 2nd group, 40.00% in the 3rd group, respectively. Number of eggs in liver tissue was reduced by 41.80%, 57.50%, 55.70%, respectively. Compared the results of the tested groups to the controls, the differences were of significance by t-test (worm reduction rate: t = 6.8 in the 1st group, t = 8.7 in the 2nd group, t = 7.3 in the 3rd group, P < 0.01 in all tested groups. Egg reduction rate at the group's number above: t = 11.23, t = 11.54, t = 7.99, P < 0.01 in all tested groups). As compared the results between the tested groups by chi(2), the differences were of significance between the 1st and the 2nd groups (worm reduction rate: chi(2) = 8.96, P < 0.05; egg reduction rate: chi(2) = 15.69, P < 0.05), between the 1st and the 3rd groups, the differences were also of significance (worm reduction rate: chi(2) = 6.52, P < 0.05; egg reduction rate: chi(2) = 12.52, P < 0.05). The difference was not of significance between the 2nd and the 3rd groups (worm reduction rate: chi(2) = 1.20, P > 0.05; egg reduction rate: chi(2) = 0.93, P > 0.05). In all tested groups, total anti-Sj14-3-3 specific IgG rose markedly. IgG(1) and IgG(2a) subtypes were high, but IgG(2b) and IgG(3) were near the background in four subtypes tested.</p><p><b>CONCLUSION</b>Immunoprotection of rSj14-3-3 should have some relations with immunization dose, and the protection obtained from immunizing mice by using 100 microg antigen was the best.</p>

Construction of a novel Schistosoma japonicum DNA vaccine pBK-Sj14-3-3 and studies on its immunoprotection in mice / 中华预防医学杂志

<p><b>OBJECTIVE</b>To prepare Sj14-3-3 DNA vaccine and observe its immunoprotection against Schistosoma japonicum in mice.</p><p><b>METHODS</b>The Sj14-3-3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subcloned into eukaryotic expression vector pBK. The recombinant plasmid pBK-Sj14-3-3 was extracted, purified and inoculated into BALB/c mice by intramuscular injection. Mice were attacked by Schistosoma japonicum cercariae and then killed. Adult worm and egg were counted, respectively. Diameter of the egg granulomas in the liver of infected mice was measured.</p><p><b>RESULTS</b>Electrophoresis on 1% agarose gel showed that the product of RT-PCR and the inserted fragment of recombinant plasmid digested with EcoR I and Xho I had the same size, about 765 bp, confirming the latter was the 14-3-3 encoding gene by nucleotide sequencing. Adult worm load declined by 27%, average egg load of per gram (EPG) of the liver tissues by 79%, average egg production per couple of adult worm (EPWP) by 51%, and mean diameter of egg granulomas by 29% in vaccinated mice.</p><p><b>CONCLUSION</b>The recombinant plasmid pBK-Sj14-3-3 was successfully constructed, which had some immunoprotection against Schistosoma japonicum in infected mice, indicating its potential to be vaccine candidate molecule of Schistosoma japonicum.</p>

Longitudinal investigation and experimental studies on thelaziasis and the intermediate host of Thelazia callipaeda in Guanghua county of Hubei province / 中华流行病学杂志

<p><b>OBJECTIVE</b>To verify houseflies Musca spp. as the intermediate host of Thelazia callipaeda and reveal epidemiological situation of thelaziasis in Hubei province.</p><p><b>METHODS</b>Dogs eyes infected with T. callipaeda, 400 houseflies Musca and 259 fruitflies Amiota okadai in the city of Laohekou city (previously named as Guanghua county) of Hubei province had been investigated since September 2000. The newborn larvae of T. callipaeda from Laohekou suburbs were fed to houseflies Musca and A. okadai. Larvae used for the study were isolated from female T. callipaeda in laboratory and the susceptibility to houseflies Musca and A. okadai was observed.</p><p><b>RESULTS</b>Twenty-one dogs from Laohekou, the original epidemic areas of thelaziasis were examined and 7 positive dogs in 21 (33.3%) and 11 T. callipaeda (9 females and 2 males) were identified. From 1975 to 2000, no thelaziasis cases were found through retrospective surveys. These 200 houseflies Musca and 135 A. okadai were dissected for examination but showed all negative with the infection. However, newborn larvae of T. callipaeda were used to experimentally infect 112 houseflies Musca and 84 A. okadai and all infected flies were examined on the 20th day after inoculation. As a consequence, houseflies Musca failed to be infected but 9 in 84 (10.7%) A. okadai were positive. 26 infective larvae of T. callipaeda were obtained and 21 of them were inoculated into right eye of one rabbit. The female worm began to produce newborn larvae in 37 days after infection and 3 adult T. callipaeda (two females and one male) were obtained.</p><p><b>CONCLUSIONS</b>Fruitflies A. okadai from Hubei province were susceptible to T. callipaeda, which was similar to the result of experimental studies in Anhui province. This survey further confirmed that A. okadai was the intermediate host of T. callipaeda but not houseflies Musca. Infective resources (adult dogs, for instance) had been under controlled thus human thelaziasis had been eradicated in this rural area.</p>

Effects of HBV infection on hepatic fibrosis and level of Th1/Th2 cytokines in the patients with Schistosomiasis japonica / 中华检验医学杂志

Objective The levels of Thl cytokines(IL-10 and IL-13)and Th2 cytokines(INF-? and TNF-?)were determined in the sera of patients with Schistosomiasis japonica in order to find the relationship between cytokines and severe hepatic fibrosis(HF)in schistosomiasis.Methods A total of 358 patients with advanced Schistosomiasis japonica were examined by ultrasound.68 HBsAg negative patients were chosen randomly as experimental control.Among them,39 patients were found to have mild HF and 29 were severe HF.The sera levels of Thl and Th2 cytokines were determined with ELISA.Results Among these 358 patients,83(23.2%)were HBsAg positive.Neither earlier nor severer hepatic fibrosis was noted in the patients who had been simultaneously infected with HBV than those only infected with schistosomiasis. There was a significant difference between mild[ 1.60(1.30-12.14)ng/L]and severe[ 4.20(1.43- 52.07)ng/L]HF patients in the level of IL-10(Z=-3.907,P0.05)was found in level of IFN-?,between severe[3.12(1.38-66.14)ng/L]and mild[5.87(1.33-216.33)ng/ L]HF subjects.Our observation did not reveal any obvious difference of TNF-? between severe[ 2.48(0.79 -19.86)ng/L]and mild[ 2.28(0.67-15.72)ng/L]HF groups.Conclusions Patients infected with advanced shistosomiasis may become more susceptible to HBV.The results of the present investigation showed that a high level production of IL-13 was associated with severe HF.

Studies on the aberrant methylation of 14-3-3 sigma gene in human breast cancer / 中华检验医学杂志

Objective To observe the methylation and expression of 14-3-3 sigma gene in human breast cancer.Methods 40 breast cancer tissues and 18 mammary gland tissues with benign lesions were analyzed by methylation specific PCR(MSP),RT-PCR,and Western-blot(WB)so as to detect the methylation status and expression of 14-3-3 sigma mRNA or protein.Results Methylation of 14-3-3 sigma gene was detectable in 85%(34/40)of patients with breast cancers.RT-PCR showed negative in 12.5% of breast cancers(5/40),WB also indicated that 14-3-3 sigma was not detected in 32 of 40 breast carcinomas (80%).Furthermore,both RT-PCR and WB were negative in 30 of 34 positive cases by MSP.While methylation of 14-3-3 sigma was not detectable and its expression was demonstrated by RT-PCR and WB among 18 cases of benign breast diseases.These evidences proposed that methylation of 14-3-3 sigma gene had great relevance with its silence.Conclusion Methylation and loss of expression in 14-3-3 sigma gene were high frequent events in breast cancers.And methylation of 14-3-3 sigma gene might be related to its loss.