A new method for screening latent tuberculosis infection in Beijing army recruits / 军事医学

Objective To investigate the Mycobacterium tuberculosis infections in 2014 among Beijing army recruits, and evaluate a new method for screening latent tuberculosis infections.Methods A total of 194 army recruits were subjected to chest X-ray examination purified protein derivative(PPD) skin test, antibody detection, and interferon gamma release assay(IGRA) by ELISA combined with recombinant protein CFP10-ESAT6 and latent infection protein Rv2628.Results The positive rates of PPD skin test and antibody test were 49.7% and 15.5%, respectively.The latent infection rate of IGRA test was 22.2% in 194 cases after CFP10-ESAT6 stimulation.After stimulation of latent tuberculosis infection(LTBI) with Rv2628, IFN-γ level was significantly higher than that in healthy control group (P0.05).There was no significant difference between antibody negative and positive groups after stimulation by CFP10-ESAT6 and Rv2628 (P>0.05).The area under the ROC curve of Rv2628 diagnosis of tuberculosis infection was 0.84.When Youden index was 0.621,the specificity was 94.7% and sensitivity was 67.4%.ConclusionCombined detection of antigens Rv2628 and CFP10-ESAT6 specific IFN-γ values can be potentially used for differential diagnosis of active or latent tuberculosis infections.

Prediction and analysis of T-cell and B-cell epitopes of Mycobacterium tuberculosis Rv2657c protein / 实用医学杂志

Objective To predict the epitopes of Mycobacterium tuberculosis Rv2657c protein, to understand its immunogenicity Methods The T?cell and B?cell epitopes of Mycobacterium tuberculosis Rv2657c protein were predicted by DNAStar software package. The homology of Rv2657c amino acid sequence with the human protein sequences was prepared using Blast method, then the CTL epitopes were predicted using SYFPEITHI supermotif method, BIMAS quantitative motif method and NetCTL prediction method, and the Th epitopes were predicted by RANKPEP and SYFPEITHI supermotif prediction method. Results The prediction using DNAStar software package showed that Rv2657c protein had 5 B?cell epitopes and 6 T?cell epitopes. The protein had 6 CTL epitopes and there were 38 Th epitopes. Conclusion Rv2657c protein has both B?cell epitopes and T?cell epitopes. It may be a candidate target antigen for the studies of vaccine and diagnosis of tuberculosis.

Prokryotic expression of CFP-10 protein of Mycobacterium tuberculosis / 中国医师杂志

Objective To construct recombinant plasmid containing CFP-10 gene of Mycobacterium tuberculosis(MTB).Methods The gene fragment of CFP-10 was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA and cloned to pET-32a(+) vector.The recombinant plasmid pET-32a-CFP-10 was transformed into E.coli BL21 (DE3) and induced by IPTG.Results CFP-10 gene fragment was amplified from genomic DNA of Mycobacterium tuberculosis H37Rv strain,and thepET-32a(+) prokaryotic recombinant plasmid was constructed successfully.The recombinant protein was expressed with the induction of IPTG.Conclusions The prokaryotic expression vector for CFP-10 was successfully constructed and the recombinant protein was highly expressed in E.coli BL21 (DE3),which lays a foundation for its subsequent immunological function study.

Correlation between genetic polymorphisms of interleukin-1A/1B and susceptibility to tuberculosis / 中华微生物学和免疫学杂志

Objective To study the correlation between genetic polymorphisms of interleukin (IL)-1A/1B and susceptibility to tuberculosis (TB).Methods Genetic polymorphisms of IL-1A and IL1 B in 1032 TB patients and 1008 non-TB patients were analyzed using PCR-MassARRAY method.The correlation between genetic polymorphisms of IL-1A/1B and susceptibility to TB was statistically analyzed.Results Two tag SNPs of IL-1A and three tag SNPs of IL-1B were screened for the study.There were differences in the allele frequencies of rs2853550 and rs3783526 between TB group and non-TB group (P=0.047and P =0.034,respectively).IL-1 B SNP1 rs2853550 (P =0.025,OR =1.302,95 ％ CI =1.034-1.640,TC vs.CC) was found to be highly associated with TB,while the other SNPs showed no significant correlations with TB.Furthermore,IL-1B SNP1 rs2853550 [P=0.019,OR=1.308,95％ CI=1.045-1.638 for (TC+TT) vs.CC] in the dominant model conferred significant risk for TB,but IL-1A SNP2 rs3783526 [P=0.000,OR=0.764,95％ CI =0.591-0.988 for GG vs.(AA+GA)] in the recessive model showed protective effects against TB.The haplotype ‘TG’ in the IL-1B block showed a higher risk for TB compared with the common ‘ CA’ haplotype (P=0.032,OR=1.265,95％ CI=1.020-1.567).The diplotypes containing ‘ GA’ haplotype in IL-1A block and ‘ TG’ haplotype in IL-1B block were major risk factors for TB (for onecopy,adjusted P=0.014,OR=1.403 and 95％ CI=1.072-1.836; adjusted P=0.013,OR=1.339 and 95％ CI=1.063-1.688,respectively),but the diplotype with ‘CG’ in IL-1B block played a protective effect against TB (for two-copy,P=0.006,OR=0.664 and95％ CI=0.494-0.891).Conclusion The genetic polymorphisms of IL-1B rs2853550 might be closely associated with TB,but the GG genotype of IL1 A SNP rs3783526 might have the characteristic of anti-TB.

The relationship between carboxylesterase 1 gene polymorphisms and susceptibility to antituberculosis drug-induced hepatotoxicity / 中华内科杂志

Objective To study the relationship between the genetic polymorphisms of carboxylesterase 1 gene (CESI) and the susceptibility to antituberculosis drug-induced hepatotoxicity (ATBDIH).Methods Genetic polymorphisms of CES1 in 473 tuberculosis patients with or without hepatotoxicity (200∶ 273) after antituberculosis chemotherapy were analyzed by PCR-MassArray.Results In4 tags of CES1 single nucleotide polymorphism (SNP),the frequency of the rs1968753 allele had statistical difference between the hepatotoxicity group and the no-hepatotoxicity group ( P =0.0236 ).The characteristics of anti-hepatotoxicity had been shown relationship with rs8192950 ( P =0.044,OR =0.649,95％ CI =0.426-0.989,AC/AA ) and rs1968753 ( P =0.048,OR =0.556,95％ CI =0.311-0.995,GG/AA).The diplotypes with ‘ CGC' haplotype exhibited significant protection against hepatotoxicity at one copy (P=0.048,OR=0.654,95％CI=0.430-0.996).Conclusions The genetic polymorphisms of CESI might have significant association with ATBDIH.SNP rs8192950 AC genotype and rs1968753 GG genotype might be the candidates for risk prediction of ATBDIH.

Induction of myelogenous leukemia cells with histone deacetylase inhibitors through down-regulating the Daxx protein expression / 华中科技大学学报（医学）（英德文版）

The effects of two different histone deacetylase (HDAC) inhibitors, sodium butyrate (NaB) and trichostatin A (TSA),on apoptosis of human leukemic cells in vitro and the molecular mechanisms were investigated. The experiments were divided up 5 groups: control group, NaB group, TSA group, NaB+Z-VAD-FMK group and TSA+Z-VAD-FMK group. The apoptosis rate was determined by morphological analysis and flow cytomytry. The expression of Daxx, Bcl-2, and Bcl-xl proteins was detected by Western blot. NaB and TSA could induce the apoptosis of HL-60 and K562 cells, and Z-VAD-FMK caused a marked decrease in apoptosis induced by HDAC inhibitors. HDAC inhibitors could down-regulate the expression of Daxx protein, but had no significant influence on the expression of Bcl-2 and Bcl-xl proteins. The results suggested that NaB and TSA induce distinct caspase-dependent apoptosis of human leukemic cells through down-regulating the expression of Daxx protein in vitro.

Therapeutic effects of Ag85A plasmid DNA vaccines in a mouse model of multi-drug resistant Mycobacterium tuberculosis infection / 中华微生物学和免疫学杂志

Objective To study the therapeutic effects of Ag85A plasmid DNA vaccines in a mouse model of multi-drug resistant-(MDR-) Mycobacterium tuberculosis infection. Methods BALB/c mice were infected with Mycobacterium tuberculosis clinical strain HB361 with isoniazid and rifampin resist-ance by intratail-vein injection and were subsequently divided into 6 groups. At the third day after infection, the mice were treated with saline (group A), vector pVAX1 (greup B), rifampin (group C), vaccae (group D), Ag85A plasmid DNA vaccines (group E),rifampin and Ag85A plasmid DNA vaccines (group F) for 60 d. The lungs and spleens from the mice were taken and their pathological changes, weight and number of myeobacterial colony were examined at the third week after the end of treatment. Results At third week af-ter the end of treatment, the gross pathological observation and histopathological examination in lung showed that the lung lesions were limited, the profile of the alveoli was relatively clear, and normal structure could be seen in 2/3 areas of the lung sections in group D, E and F. The extent of lung lesion was 50% in group D,20% in group E and F. The pathological changes in group A, B, and C were more severer than those in group D, E and F. Compared with group A, the colony-forming units (CFU) in the lungs from mice in group D,E and F decreased 52%, 68%, 78%, respectively. The CFU in the spleens from mice in group D,E and F decreased 48%, 65%, 79%, respectively. Conclusion Ag85A plasmid DNA vaccines alone or Ag85A plasmid DNA vaccines along with chemotherapy have significant therapeutic effects on the mouse model of MDR-Mycobacterium tuberculosis infection.

Development of a diagnosis model for active pulmonary tuberculosis using mass spectrometry and pro-tein chip / 中华微生物学和免疫学杂志

Objective To develop a diagnosis model for active pulmonary tuberculosis. Methods The proteomic fingerprinting of 264 sera from active tuberculosis patients and controls were analyzed using the surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) and protein-chip technology. The peaks were detected and filtrated by Ciphergen PrnteinChip(R) Software (version 3.1.1). Using the Biomarker Pattern 5.0 software, a diagnostic model was developed for diagnosis of active tuberculosis. Re-sults Fifty protein peaks were significantly different between the patients with active pulmonary tuberculosis and the controls with overlapping clinical features (P<0.01). Five protein peaks at 4360, 3311, 8160, 5723, 15173 m/z were chosen for the system classifier and the development of diagnosis model 1. The model differenti-ated the patients with active pulmonary tuberculosis from the controls with a sensitivity of 83.0%, and a speci-ficity of 89.6%. The diagnostic accuracy was up to 86.4%. Three protein peaks at 5643, 4486, 4360 m/z were chosen for the system classifier and the development of diagnosis model 2. The model differentiated the pa-tients with active pulmonary tuberculosis from the controls with a sensitivity of 96.9%, and a specificity of 97.8%. The diagnostic accuracy was up to 97.3%. Conclusion It might be a new diagnostic test for the de-tection of sera from the patients with active pulmonary tuberculosis using SELDI-TOF-MS and protein chip.

The Clinical Preliminary Application and Analysis for Drug-Sensitive Test and Detecting Drug Resistance Genes to Mycobacterium Tuberculosis / 中国医师杂志

Objective To evaluate the clinic application value of detection of rPOB,katG and rPSL resistance genes to M tuberculosis by polymerase china reaction-single stranded conformation polymorphism (PCR-SSCP).Method The drug-resistance tests of M tuberculosis clinical isolates from 141 patients with the tuberculosis was analyzed by Bactec-960 rapid culture,drug-sensitive and PCR-SSCP techniques.Results As nearly As one of three patients with priliminary treatment had resistance to one kind of the drugs tested at least.The mutation rates of RFP,INH and SM drug-sensitive isolates were 6 5%,11 4% and 27 2% respectively.That of the drug-resistant isolates were 60 2%,45 7% and 62 7%.As drug-resistant concentration heightened,so mutation rate of genes increased.Conclusions Detecting the resistance genes was a new explore in guiding treatment,and multiple drug-sensitive test combinative use have cooperativity each other.It might become good test for clinical management.

embB mutations in Mycobacterium tuberculosis ethambutol-resistant isolates / 中华检验医学杂志

Objectives To understand the mutations of embB genes in M. tuberculosis isolates, and to evaluate their clinical value. Method 102 clinical isolates were identified for their mycobacterial species, and then analyzed their embB genes with PCR SSCP, PCR RFLP, and PCR direct sequencing. Results Mycobacterium tuberculosis strain H 37 R v was used as a control. 102 clinical isolates all had the same 16S rDNA SSCP profiles as M. tuberculosis . Forty one drug sensitive isolates had normal embB SSCP and RFLP profiles. Of 61 ethambutol resistant isolates, 23 (37.7%) displayed abnormal embB SSCP profiles. Eight isolates had abnormal RFLP profiles. All embB mutations situated at codon 306, whose EMB MICs were more than 20 ?g/ml. Eight isolates had ATG to ATA or ATT mutations at codon 306. Thirty isolates had ATG to GTG or CTG mutations at codon 306, whose EMB MICs were more than 30 ?g/ml. Conclusions Ethabutol resistances in some M. tuberculosis isolates were due to mutations on embB genes. PCR SSCP and PCR RFLP method might become a simple and rapid diagnostic test for genotypes of M. tuberculosis ethabutol resistance.

THE RESEARCH OF FIVE DRUG-RESISTANCE GENES OF MYCOBACTERIUM TUBERCULOSIS IN 134 PATIENTS / 解放军医学杂志

M.tuberculosis strains isolated strains from sputum specimens of 134 patients were analyzed by PCR SSCP and traditional drug susceptibility tests. The results sbowed that the gene mutation rate of PZA, SM, RFP,INH and EMB resistance in those clinically isolated strains was 42 7%,71 7%,78 9%,68 6% and 43 9%, respectively. The gene mutation was in relation with drug resistance level of M. tuberculosis. The gene mutation rate was higher in high concentration resistance strains than in low concentration resistance strains.

Application of Gene Chip for Detecting Mutations of Rifampin-resistant Genes in Mycobacterium Tuberculosis Strains / 中国医师杂志

Objective To explore the feasibility of detecting the mutations of rpoB gene in rifampin-resistant Mycobacterium tuberculosis strains by gene chip. Methods The DNA chip was designed according to the sequence of Mycobacterium tuberculosis rpoB gene. The DNA fragments which contained hot mutation sites of rpoB gene were labelled with cy3 fluorescence, amplified by PCR technique and hybridized with gene chip. The DNA sequencing was used as the control. Results Eighteen of the Mycobacterium tuberculosis strains were detected for the mutations of rpoB gene by gene chip. Among them, one sensitive strain was identical with the type strain, and all of 17 strains with rifampin-resistant, rpoB mutations were detected. Of the 17 strains with rpoB mutations, 14 were detected as single point mutation, one was detected as double sites point mutations with the sites of 511 and 516, and two were detected as single point mutation with the sites of 518 and 526, respectively. Because the probe of 517 rifampin-resistant did not be dotted on the gene chip, only sites of 518 and 526 mutations were detected, which were consistent with the results of DNA sequencing. Conclusion Using the gene chip could detect mass rpoB gene mutation of rifampin-resistant with higher specialty and sensitivity, which could be used for clinical detection of rifampin-resistant strains and clinical medicine.

Rapid Detection of Mycobacterium Tuberculosis Streptomycin-resistant Genes by PCR-reverse Dot Blot Hybridization / 中国医师杂志

Objective To study the rapid detection of mycobacterium tuberculosis resistance to streptomycin by reverse dot blot hybridization technique. Methods The oligonucleotide probes of streptomycin-resistant genes (rpsL and rrs) were prepared and dropped on nitrocellulose membrane. The target DNA fragments of M. tuberculosis clinical isolates were labeled with biotin by PCR amplification, and then hybridized with the oligonucleotide probes on the membranes. PCR-SSCP and PCR-direct sequencing (PCR-DS) techniques were used to detect the target fragment of M.tuberculosis as control. Results In 53 M. tuberculosis clinical isolates, the consistent rate of three detection methods was 100%. Both the SSCP mapping of rpsL and rrs genes and the results of membrane hybridization in 9 drug-sensitive strains were identical to those in M. tuberculosis standard strain H37Rv. Of 44 streptomycin-resistant strains, 33 strains had AAG→AGG mutation at the codon 43 of rpsL gene, 6 strains had A→C mutation at the 513 site of rrs gene, 1 strain had A→T mutation at the 513 site of rrs gene, and the detection rate of the target genes mutation was 90 9%. In 53 M.tuberculosis clinical isolates, 40 resistant strains and 9 sensitive strains to streptomycin could be detected using dot blot hybridization and the consistent rate with the in vitro susceptibility test was 92 6%(49/53). Conclusion The reverse dot blot hybridization technique showed high sensitivity and specificity to detect Mycobacterium tuberculosis resistance to streptomycin. It possessed the simple and rapid characteristics, and could be used to detecte streptomycin-resistant M.tuberculosis clinical strains.

The Mutation of embB and pncA Genes in Drug-resistanct Mycobacterium Tuberculosis and Its Clinical Value / 中国医师杂志

Objective To investigate the mutation of embB and pncA genes in mycobacterium tuberculosis isolates, and assess its clinical value. Methods The drug-sensitivity of 106 clinical isolates of mycobacterial species was identified by the tranditional method, and then analyzed the mutation of their embB and pncA genes with PCR-SSCP. Mycobacterium tuberculosis strain H 37 RV was used as a control. Results The mutational frequency of pncA gene in 94 PZA-resistant mycobacterium tuberculosis was 44 6%(42/94). The mutational frequency of embB gene in 83 EMB-resistant isolates was 54 2%. The frequency of both genes mutation was 11 8%(11/94). Conclusion EMB and PZA resistances in some mycobacterium tuberculosis isolates were due to mutations of embB and pncA genes. PCR- SSCP method might become a simple and rapid diagnostic test for genotypes of EMB and PZA resistant mycobacterium tuberculosis.

Evaluation of protective effectiveness of two tuberculosis DNA vaccines in relation to pathological changes in lung, liver and spleen / 解放军医学杂志

Objective To evaluate the protective effectiveness of MPT64 and ESAT6 DNA vaccines against M. tuberculosis. Methods BALB/c mice were randomly divided into 14 groups and subjected to following treatments respectively, i.e. immunized with. ESAT6 (25?g)+MPT64 (25?g)(A), ESAT6(100?g)+IFN-?(100?g) (B), ESAT6 (75?g)+MPT64 (25?g)(C), ESAT6(100?g)+IL-12(100?g) (D), MPT64(100?g)+IL-12(100?g) (E), ESAT6 (25?g)+MPT64 (75?g)(F), MPT64 (100?g)(G), Pvax1 (H),ESAT6 (100?g)(I), ESAT6 (100?g)+MPT64 (100?g)(J), ESAT6 (50?g)+MPT64 (50?g)(K), MPT64(100?g)+IFN-?(100?g)(L), BCG(M ), and saline(N). Then they were infected with M. tuberculosis H37Rv via intravenous route. The pathological changes in the lung, liver, and spleen were observed after the infection. Results Eight weeks after the inoculation, there were only alveolar exudation and capillary hyperemia in the lung lesions in the mice of group N. In the mice of group M and J, main pathological changes included tuberculous granulomas consisting of numerous lymphocytes, macrophages, epithelioid cells and Langhans giant cells, and moderate hyperplasia in alveolar walls. The lung lesions of the other groups were similar, and both hyperplasia and exudate were found (A, B, C, D, E, G, H, I, K, L). No necrosis was found in all the above groups. There were hyperemia, dense lymphocytes infiltration in the portal area, and tuberculous granuloma in the liver in all the groups. No difference was found among all the groups. The pathological changes in spleen induced hyperplasia and fusion of splenic lymph nodule. The reactions in group M and J were stronger than that of the other groups. Conclusions MPT64 and ESAT6 DNA vaccines from M.tuberculosis could enhance immunity against M. tuberculosis, either they were used in combination with different dosages or with IL-12 or IFN-?. The vaccine used in group J showed the strongest effect in enhancing immunity, almost reaching that of combined use of BCG, IFN-? and IL-12.

The studies on therapeutic action of tuberculosis Ag85A DNA vaccines / 中国免疫学杂志

To study the therapeutic action of tuberculosis Ag85A DNA vaccines. Methods:BALB/c mice were infected by in-traperitoneal injection with M. tuberculosis H37Rv for 8 weeks, and then treated with the saline (A), plasmid vector (B), M. bovis BCG (C), M. vaccae (D), and Ag85A DNA vaccine (E). Ag85A-specific antibodies were determined by ELISA. The lungs, livers and spleens were taken and observed their pathological changes, weighted and performed mycobacterial cultures 2 or 5 monthes after treatment. Spleen cells were tested for proliferative responses.Results:Ag85A-specific antibodies increased in the mice of group E. At 2 months after immunothera-pies, the stimulation rates of spleen cells had no significant difference among each group. The numbers of viable bacteria in the lungs and spleens of therapeutic group were lower than those in the control group. The group C and B could be observed slight .lesion of lung. At 5 months after immunotherapies, the stimulation rates of spleen cells all increased significantly in the immunotherapeutic groups. The numbers of viable bacteria in the lung and spleen had no significant difference among each group. No lesions of lung could be observed in group E and D. Conclusion:The tuberculosis DNA vaccines seem to have some immunotherapeutic actions.