2010 Vol. 23, No. 5
Different Strategies for Preparation of Non-tagged rV270 Protein and Its Efficacy against Yersinia Pestis Challenge
2010, 23(5): 333-340.
Objective LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study. Methods A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co23 affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography. Results Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 106 CFU of Y. pestis virulent strain 141. Conclusion The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.
Interpretation Criteria for Standardized Western Blot for the Predominant Species of Borrelia Burgdorferi Sensu Lato in China
2010, 23(5): 341-349.
Objective Western blotting (WB; immunoblotting) is a widely used tool for the serodiagnosis of Lyme borreliosis (LB), but so far, no generally accepted criteria for its performance and interpretation have been established in China. The present study was designed to determine the criteria for standardized Western blot for the predominant species of Borrelia burgdorferi sensu lato in China, in which WB was produced with strain PD91 as the representative strain attributed to predominant genospecies Borrelia garinii of Borrelia burgdorferi sensu lato. Methods Approximately 13 bands between 14 and 100 kD were differentiated for strain PD91 by using Gel-Pro analysis software. In a study with 631 serum samples (taken from 127 patients with Lyme borreliosis and 504 controls), all observed bands were documented. To establish criteria for a positive WB result for strain PD91, receiver operating characteristic (ROC) curves were used. Results The following interpretation criteria were recommended: for IgG, at least one band of P83/100, P58, P39, P30, OspC, P17, P66, and OspA; for IgM, at least one band of P83/100, P58, OspA, P30, OspC, P17 or P41. In addition, syphilis, leptospirosis and other related diseases should be excluded when the positive band is P41 in IgM. For IgG criteria, the sensitivity is 73.2%, the specificity is 99.4% and Youden index is 0.726; for IgM criteria, the sensitivity is 50.6%, the specificity is 93.1% and Youden index is 0.437. Conclusion Standardization of WB assays is necessary for comparison of results from different laboratories. Moreover, the criteria of other genospecies of Borrelia burgdorferi sensu lato should be determined in the future to complete the criteria of WB for the diagnosis of the Lyme disease in China.
Recombinant E.coli LLO/OVA Induces Murine BMDCs Maturation via TLR4 and NOD1 Receptor and Promotes Specific Cytotoxic T Cell Immunity
2010, 23(5): 350-356.
Objective To explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells (BMDCs) and T lymphocytes in vitro. Methods After BMDCs stimulated by E.coli LLO/OVA, their Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) receptor signalling pathway were examined by superarray hybridization; and the priming effect of the vaccine activated BMDCs on CD43T and CD83T was determined by [3H]thymidine uptake and ELISA, the tumor cytotoxic effect of activated CD83F cells was determined by cytotoxic assay. Results After BMDCs were activated by E. coil LLO/OVA via TLR4, NOD1 receptor and NF-κB signalling pathway, the expression of their surface molecules including MHC class Ⅰ, MHC class Ⅱ, CD40, CD80 and CD86 significantly up-regulated; the secretion of IL-12 and IFN-γ, increased also. The mature BMDCs stimulated the allergic CD43T and CD83T cells proliferation and their IL-2 and IFN-γ secretion, and the activated CD83T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro. Conclusion E.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.
Estrogen Receptor α and βExpressions in Hypothalamus-pituitary-ovary Axis in Rats Exposed Lactationally to Soy Isoflavones and Bisphenol A
2010, 23(5): 357-362.
Objectives This paper aims to investigate the uterotrophic activities of lactational exposure to combination of soy isoflavones (SIF) and bisphenol A (BPA) and to examine estrogen receptor a (ERα) and estrogen receptor β (ERβ) expressions in hypothalamus-pituitary-ovary axis and uterus. Methods Maternal rats that were breeding about 8 litters were randomly divided into four groups with seven dams in each group.Dams in different treatment groups received corn oil (control),150 mg/kg BW of SIF,150 mg/kg BW of BPA or combination of 150 mg/kg BW of SIF and 150 mg/kg BW of BPA,respectively,from postnatal day 5 to 11 (PND5-11) by gavage.On PND12 and PND70,10 female litters were killed and hypothalamus,pituitary,ovary and uterus were collected.ERα and ERβ expressions in these organs were detected with Western blotting assay.And vaginal opening time and estrus cycle were examined in animals fed for PND70. Results On PND12,the relative uterine weight of rats treated with ISF or BPA or their combination was significantly higher than that of untreated rats (P<0.05).But the relative uterine weight of rats in the co-exposure group was slightly lower than that in the group only exposed to SIF or BPA.On PND 70,however,the relative uterine weight in each treatment group was not statistically different from that in the control group (P>0.05).Vaginal opening time and estrus cycle in groups treated with SIF or BPA or their combination were similar to those in the control group (P>0.05).Exposure to SIF or BPA or their combination could up-regulate or down-regulate ERa and ERβ expressions in hypothalamus,pituitary,ovary and uterus on PNDI2 and PND70.These regulation patterns for ERα and ERβ were different in different organs at different time points. Conclusion Lactational exposure to ISF or BPA or their combination could induce uterotrophic responses in neonate rats,which disappeared in later life.But these data fail to suggest a possibility for synergic actions between SIF and BPA.It was also demonstrated that the uterotrophic effects of SIF and BPA exposure might,at least,involve modification of ERα or ERβ expressions in the hypothalamus-pituitary-ovary axis.
Genome-wide and Interaction Linkage Scan for Nonsyndromic Cleft Lip with or without Cleft Palate in Two Multiplex Families in Shenyang,China
2010, 23(5): 363-370.
Objectives To identify the loci involved in nonsyndromic cleft lip with or without cleft palate (NSCL/P) in Northern Chinese people in Shenyang by using genomewide and interaction linkage scan. Methods Two multiplex families in Shenyang from North China were ascertained through probands with NSCL/P.Blood of every member was drawn for DNA extraction and analysis.Genotypes were available for 382 autosomal short tandem repeat (STR) markers from the ABI Prism Linkage Mapping Set version 2.5.Linkage between markers and NSCL/P was assessed by 2-point parametric LOD scores,multipoint-heterogeneity parametric LOD scores (HLODs),and multipoint nonparametric linkage score (NPL). Results The initial scan suggested linkage on Chromosomes 1,2,and 15.In subsequent fine mapping,1q32-q42 showed a maximum multipoint LOD score of 1.9(empirical P=0.013) and an NPL score of 2.35 (empirical P=0.053).For 2p24-p25,the multipoint NPL increased to 2.94 (empirical P=0.007).2-locus interaction analysis obtained a maximum NPL score of 3.73 (P=0.00078)and a maximum LOD score of 3 for Chromosome 1 (at 221 cM) and Chromosome 2 (at 29 cM). Conclusion Both parametric and nonparametric linkage scores greatly increased over the initial linkage scores on 1q32-q42,suggesting a susceptibility locus in this region.Nonparametric linkage gave a strong evidence for a candidate region on chromosome 2p24-p25.The superiority of 2-locus linkage scores compared to single-locus scores gave additional evidence for linkage on 1q32-q42 and 2p24-p25,and suggested that certain genes in the two regions may contribute to NCSL/P risks with interaction.
Calcium Overload Is A Critical Step in Programmed Necrosis of ARPE-19 Cells Induced by High-Concentration H2O2
2010, 23(5): 371-377.
Objective Oxidative stress plays an important role in retinal pigmental epithelium (RPE) death during aging and the development of age-related macular degeneration.Although early reports indicate that reactive oxygen species (ROS) including H2O2 can trigger apoptosis at lower concentrations and necrosis at higher concentrations,the exact molecular mechanism of RPE death is still unclear.The purpose of this study was to investigate the molecular pathways involved in RPE death induced by exogenous ROS,especially at higher concentrations. Methods Cultured ARPE-19 cells were treated with H2O2 at different concentrations and cell viability was measured with the MTT assay.Cell death was morphologically studied by microscopy using APOPercentage assay and PI staining.Furthermore,the impact of oxidative stress on ARPE-19 cells was assessed by HO-1 and PARP-1 Western blotting and by the protection of antioxidant EGCG.Calcium influx was determined using the fura-2 calcium indicator and the role of intracellular calcium overload in ARPE-19 cell death was evaluated following cobalt treatment to block calcium effects. Results H2O2 reduced the viability of ARPE-19 cells in a concentration-dependent manner,which was presented as a typical s-shaped curve.Cell death caused by high concentrations of H2O2 was confirmed to be programmed necrosis.Morphologically,dying ARPE-19 cells were extremely swollen and lost the integrity of their plasma membrane,positively detected with APOPercentage assay and PI staining.24-hour treatment with 500 μmol/L H2O2 induced remarkable up-regulation of HO-1 and PARP-1 in ARPE-19 cells.Moreover,antioxidant treatment using EGCG effectively protected cells from H2O2-induced injury,increasing cell viability from 14.17%±2.31% to 85.77%±4.58%.After H2O2 treatment,intracellular calcium levels were highly elevated with a maximum concentration of 1200nM.Significantly,the calcium channel inhibitor cobalt was able to blunt this calcium influx and blocked the necrotic pathway,rescuing the ARPE-19 cell from H2O2-induced death. Conclusions At high concentrations,H2O2 induces ARPE-19 cell death through a regulated necrotic pathway with calcium overload as a critical step in the cell death program.
2010, 23(5): 378-383.
Objective To investigate the potential of Gomphidius viscidus,a kind of ectomycorrhizal fungi,for phytoremediation of anthracene in soil.Methods Absorptioe changes of micro-habitat were studied in detail.Conclusion Ectomycorrhizal plants have a strong potential for remediation of polycyclic aromatic hydrocarn characteristics of both active and inactivated mycelia.Results A high calculated adsorption capacity of 1 886.79 mg/g and 1 515.15 mg/g at 25 ℃,pH 6.0 for active and inactivated mycelia respectively,was obtained based on Langmuir model.The ANT biosorption was more ideally characterized by the Langmuir model than by the Freundlich model.The biosorption of anthracene to biomass was extremely fast and could be modeled with pseudo-second order adsorption kinetics.Moreover,ectomycorrhizal mycelia demonstrated a strong ability to adjust the physiological process to get adapted to the change of micro-habitat.
Assessment of Comparative Genomic Hybridization Experiment by an in situ Synthesized CombiMatrix Microarray with Yersinia pestis Vaccine Strain EV76 DNA
2010, 23(5): 384-390.
Objective The quality of microarray data influences the accuracy of comparative genomic analyses to a large extent.To ensure that the results obtained by using an in situ synthesized microarray are accurate,data quality is to be assessed by evaluating the melting temperature(Tm)of probes,probability of false synthesis rates,and fragmentation of labeled targets.Methods DNA from the Yersinia pestis vaccine strain EV76 was used for microarray analyses.Microarray results were confirmed by PCR.Statistical and bioinformatics methods were employed to perform microarray data analyses and evaluation.Results Correlation coefficients of the three datasets were above 0.95 after two-time stripping and hybridization with a labeled DNA with the size of fragmentation being 200 bp-2 kb,which showed that the hybridization results were highly reproducible.Correlation coefficients were lower with the values ranging from 0.87 to 0.92 between the datasets generated from hybridization with different sizes of the labeled DNA fragment.For the relationship between Tm and signal intensity,there was a different distribution of Tm in the lowest 300 or 3 000 probes with a range of 70 ℃-72 ℃ and the highest 300 or 3 000 probes with a range of 72 ℃-74 ℃.Conclusion The results of this study suggest that the initial microarray design may affect the accuracy of final analyses and that the probe Tm and the size of the labeled fragment may be the two factors of the greatest importance.
HIV-Specific IL-2+ and/or IFN-γ+ CD8+ T Cell Reponses during Chronic HIV-1 Infection in Former Blood Donors
2010, 23(5): 391-401.
Objective Conflicting data have been generated from previous studies to determine which kind of relationship exists between HIV-1 specific CD8 T-cell responses and HIV-1 viral load or CD4 count over the course of infection.In this study,153 HIV-1 infected LTNPs were enrolled to investigate the role of HIV-1 specific CD8 T-cell responses in chronic HIV-1 infection among HIV-1 infected former blood donors.Methods The patients were stratified into three groups according to CD4 count:CD4≥500 cells/μL; 350 cells/μL≤CD4<500 cells/μL; CD4<350 cells/μL.PBMCs were isolated from the patients' anticoagulated blood samples.IL-2 and IFN-γ secretions of CD 8 T cells against 17 HIV-1 consensus B full peptide pools were analyzed by using ICS assay.Results An overall inverse correlation were observed between CD4 count and plasma viral load.Although no significant difference was observed during the comparisons of frequency/breadth of HIV-1 specific CD8 T cell responses,CD4 count stratification analysis showed that different correlation pattern existed in three strata: as for patients whose CD4 counts were less than 350 cells/μL,no significant correlations were identified between frequency/breadth of HIV-1 specific CD8 T cell responses and CD4 count/viral load; as for patients whose CD4 counts ranged from 350 cells/μL to 500 cells/μL,significant correlation was only observed between the response breadth of IL-2+IFN-γ+CD8 T cells and CD4 count; however,as for patients whose CD4 counts were more than 500 cells/μL,direct correlations were identified between IL-2+IFN-γ+/IL-2+/IFN-γ+CD8 T cells and viral load or CD4 count.Conclusions Universal consistent inverse correlation was only indentified between CD4 count and viral load.The relationship between HIV-1 specific CD8 T cell responses and CD4 count/viral load varied in different CD4 strata,which showed that better preserved CD4 T cells were correlated with better CD8 T cell functions.
2010, 23(5): 402-408.
Objective to explore dynamic characteristics of the HIV mother to child transmission(MTCT)epidemic in China.Methods A deterministic dynamic transmission model was used to determine the effect of key parameters on the likely long-term trends of the HIV MTCT epidemic in China.Matlab 7.0 was used to develop the model. Results The number of the susceptibles(S),the transmission rate(β),and the screening proportion(a)of HIV positive pregnant women have the greatest impact on the HIV MTCT epidemic in China.The growth of the MTCT epidemic in China could not be controlled only by decreasing the MTCT transmission rate.The prevalence of HIV positive women should be reduced and more pregnant women should be tested for HIV Conclusion Prevention of MTCT(PMTCT)should focus not only on the reduction of HIV transmission rates and incidences of HIV among women but also on the increase of HIV testing for pregnant women.The most cost-effective PMTCT means for China should be investigated in future studies.
A Life-skills-based HIV/AIDS Prevention Education for Rural Students of Primary Schools in China: What Changed? What Have We Learned?
2010, 23(5): 409-419.
Objective To evaluate a four-hour life-skills-based HIV/AIDS prevention curriculum among 5th grade students in rural primary schools of Hainan province. Methods The study included two stages.Stage one(September 2006-May 2007)was a pre-post-quasi experimental design;a total of 2 413 students aged 9 to 14 years from fifth grade classes of nine primary schools completed a baseline survey(1 720 students were in the intervention group,693 in the control group),and over 98% of them took part in a short survey.The experimental curriculum was provided to the intervention group.At stage two(September 2008),a cross-sectional questionnaire was administered to 6 923 students in 7th grade classes of eight middle schools in the same study sites.There were 1 437 students in the intervention group when the curriculum was conducted. Results Students tended to score higher in areas of HIV/AIDS related knowledge and attitudes,if they were younger than average,lived in the county seat, had access to the internet,and their parents had completed higher levels of education.Path analysis showed that,after controlling for characteristics such as family and community factors,the total effects of curriculum on knowledge in the short-term model increased remarkably compared with the baseline,and maintained major contributions to knowledge in the mid-term model.The positive effect of knowledge on attitudes was significantly improved in the short-term model as well. Conclusion A life-skills based curriculum can improve HIV/AIDS related knowledge and self-perceived level of life-skills among primary school students in rural areas in a short time,and these positive effects can still be observed at least 2 years post participation in the curriculum.