[Application of denaturing high perfomance liquid chromatography in detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis].

OBJECTIVE: to explore the value of denaturing high performance liquid chromatography (DHPLC) in detection of rpoB mutations in rifampin-resistant M. tuberculosis, in order to establish a convenient and rapid approach to screening rpoB gene mutations in M. tuberculosis. METHODS: rifampin resistance-determining region of rpoB gene in M. tuberculosis was amplified by PCR and further analyzed by DHPLC to screen mutations at optimized denaturation temperature (65.4 degrees C), which utilized heteroduplex formation between wild-type and mutated DNA strands to identify mutations. The PCR products from strains with different chromatographic profiles were sequenced further to evaluate the sensitivity and specificity. RESULTS: there were 46 M. tuberculosis strains including 42 rifampin resistance strains and 4 rifampin sensitive strains. From these strains, 15 different chromatographic profiles were produced by DHPLC. Combined with the results of gene sequencing, it was shown that strains with different chromatographic profiles had distinct mutations. Except D108 and D24 which had same chromatographic profiles but different genetic polymorphisms, all other strains showed consistent chromatographic profiles with genetic polymorphisms, i.e. if they had identical chromatographic profiles, their genetic polymorphism were the same; but if they had different chromatographic profiles, their genetic polymorphism were also different. CONCLUSION: DHPLC is a simple, efficient, high-throughput and automatic method with high sensitivity and specificity, which may be useful in the rapid detection of rpoB gene mutation in M. tuberculosis.

[Dimo-thylidioctyl ammonium bromide-BCG polysaccharide nucleic acid adjuvant enhanced the immunogenicity of a Mycobacterium tuberculosis subunit vaccine].

OBJECTIVE: To investigate the activity of a novel adjuvant consisting of dimethyldioctyldecyl ammonium bromide (DDA) and BCG polysaccharide nucleic acid (BCG-PSN). METHODS: BCG-PSN was extracted by hot-phenol method, and combined with DDA and Mycobacterium tuberculosis fusion antigen AMM (Ag85B-MPT64(190-198)-Mtb8.4) to formulate the Mycobacterium tuberculosis subunit vaccine. Mice were immunized subcutaneously with a 2-week interval between the immunizations (0.2 ml/dose), and humoral and cell-mediated immunity were detected by ELISA and ELISPOT respectively. RESULTS: With the stimulation of Ag85B in vitro, the number of antigen specific IFN-gamma producing spleen lymphocytes were 222 +/- 79, 259 +/- 85, 230 +/- 64 per million respectively in the mice immunized with AMM + DDA + BCG-PSN, AMM + DDA, and BCG. Spleen lymphocytes in these 3 groups produced higher levels of IFN-gamma compared to the groups with the adjuvant of IFA or BCG-PSN alone or without adjuvant upon stimulation with Ag85B (t = 2.923-7.118, P < 0.05). Furthermore, the adjuvant consisting of DDA and BCG-PSN increased the ratio of Ig(2a)/IgG1 than DDA alone (0.125 vs. 0.025). Combined with AMM, the adjuvant DDA and the one consisting of DDA and BCG-PSN induced higher level of immunity than incomplete Freund's adjuvant (IFA), NaCl, and BCG-PSN alone. CONCLUSION: Mycobacterium tuberculosis subunit vaccine AMM + DDA + BCG-PSN induced a strong Th1-type immune response, and DDA + BCG-PSN, especially DDA promoted the immune response of the M. tuberculosis subunit vaccine in mice.

[Analysis of the genotype of intraspecies of common Mycobacteria based on 16S-23S rRNA gene internal transcribed spacer sequences].

OBJECTIVE: To analyze the genotypes of intraspecies of common mycobacteria. METHODS: The genotypes of 94 strains of mycobacteria from DSMZ, as compared with 12 reference strains, were studied by 16S-23S rRNA internal transcription space (ITS) sequence analysis. RESULTS: The sequencing of 16S-23S rRNA ITS of the 106 strains of common mycobacteria were completed. All the mycobacteria could be discriminated to several genotypes except 4 M. intracellulare, 4 M. avium, 6 M. marinum and 2 M. malmoense which were identical to their reference strains. CONCLUSIONS: 16S-23S rRNA ITS sequence analysis is a reliable method to discriminate mycobacteria in interspecies even in intraspecies.

Silencing of ENO1 by shRNA Inhibits the Proliferation of Gastric Cancer Cells.

α-Enolase is a significant subunit of enolase and acts as a glycolytic enzyme responsible for catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the anaerobic glycolysis pathway. The research about their role is known little in tumor invasion and metastasis. This research analyzed the effect of α-enolase in proliferation and progression of human gastric cancer. The constructed PLKO.1-ENO1 shRNA vector was transfected into 293 T cells and used to infect gastric cancer cells, MKN45, by using lentivirus method. Negative controls were generated by infection with viruses containing empty vector PLKO.1-scramble-shRNA by the same protocol and using wild-type MKN45 cells as blank control. The silencing effect was confirmed by reverse transcription polymerase chain reaction and Western blotting at messenger RNA and protein levels, respectively. Cell proliferation and chemosensitivity were tested by methyl-thiazolyl-tetrazolium assay. Cell apoptosis was tested by flow cytometry. The cell line α-enolase short hairpin RNA stabling silence α-enolase was successfully constructed. In the α-enolase short hairpin RNA cell lines, messenger RNA and protein expression of α-enolase were significantly lower than those in negative control and blank control groups. The proliferation and clone formation ability were significantly inhibited, cell apoptosis was increased significantly, and the inhibition rate of chemotherapy drugs was increased ( P < .05). Our data provide strong evidence that α-enolase short hairpin RNA interference vector can effectively suppress the proliferation and increase chemosensitivity of MKN45 cells, which may provide a novel gene therapy for gastric cancer.

Chitosan microspheres enhance the immunogenicity of an Ag85B-based fusion protein containing multiple T-cell epitopes of Mycobacterium tuberculosis.

To develop novel delivery system for tuberculosis (TB) subunit vaccine, biodegradable chitosan microspheres were prepared and used to deliver a fusion protein, Ag85B-MPT64(190-198)-Mtb8.4 (AMM for short), made from three Mycobacterium tuberculosis genes. AMM-loaded microspheres were first characterized for their morphology, size, zeta potential, loading efficiency, and in vitro release of AMM. C57BL/6 mice were immunized at weeks 1, 3 and 5 subcutaneously with AMM formulated in chitosan microspheres, in incomplete Freund's adjuvant (IFA), or in phosphate-buffered saline (PBS), respectively. Three weeks after the last immunization, humoral and cell-mediated immune responses were examined. It was shown that the microspheres bound AMM quite efficiently (loading efficiency: >99%). AMM-loaded chitosan microspheres were observed as aggregated shapes with the average particle size of 5.78+/-0.65 microm and zeta potential of 32.77+/-1.51 mV. In vitro release studies revealed that only small amount of antigen was released in 16 days. Following subcutaneous administration, splenocytes immunized with AMM in chitosan microspheres produced higher levels of IFN-gamma compared to administration of AMM in PBS upon stimulation with Ag85B and synthetic peptide MPT64(190-198). The levels of Ag85B-specific IgG (H+L), IgG1 and IgG2a in sera of mice immunized with AMM in chitosan microspheres were also higher than those with AMM in PBS. These results indicate that chitosan microspheres when used as a carrier for fusion protein AMM could elicit strong humoral and cell-mediated immune responses.

[BCG cell wall proteins enhance clearance of Pseudomonas aeruginosa in rat lungs].

OBJECTIVE: To test the function of BCG cell wall proteins in enhancing the clearance of Pseudomonas aeruginosa in rat lungs. METHODS: The BCG cells were broken by supersonic technique. The cell wall proteins were isolated by discontinues sucrose density-gradient centrifugation, and fractionated by Sephadex G-150 chromatography. The relative molecular mass of the isolated proteins was analyzed by SDS-PAGE. The hBD-1 gene mRNA expression in the SPC-A-1 cells was identified by Northern blot and RT-PCR. The cell wall component was injected intraperitoneally to the rats. Forty eight hours later, 5 X 10(6) CFU of P. aeruginosa ATCC27853 or Staphylococcus aureus ATCC25923 were inoculated via trachea. The bacteria colony in the supernatant of the homogenated lungs of the rats was counted 24 hours after the inoculation. RESULTS: Two protein components of BCG cell wall were fractionated. The relative molecular mass of component 2 was in the range of 18 X 10(3) -30 X 10(3). The hBD-1 mRNA expression detected by Northern blot was markedly enhanced by the stimulation of heat-inactivated BCG whole cells. The BCG-induced hBD-1 mRNA expression in the SPC-A-1 cells detected by RT-PCR was mainly contributed by fraction 2 of the BCG cell wall proteins. The bacteria decreased significantly in the lungs of the rats with the injection of BCG component 2 (n=8, P<0. 01). CONCLUSION: The fraction (relative molecular mass is 18 X 10(3) -30 X 10(3)) of BCG cell wall proteins improve the defense of rat lungs against P. aeruginosa infection.

[Construction of the recombinant secretion type BCG-Eg95 vaccine of Echinococcus granulosus].

OBJECTIVE: To construct the recombinant secretion type BCG-Eg95 vaccine of Echinococcus granulosus (rsBCG-Eg95). METHODS: BCG-Ag85B signal sequence with 117 bp and Eg95 gene with 471 bp were amplified from the genome of BCG and pGEX-4T-Eg95 by PCR, respectively. BCG-Ag85B signal coding gene and Eg95 gene were cloned into E. coli-BCG shuttle-vector pMV261 to get the recombinant plasmid pSMEg95, which was confirmed by restriction endonuclease digestion, PCR amplification and gene sequencing. These recombinant plasmids were introduced into BCG by electroporation for the construction of rsBCG-Eg95 vaccine. The rsBCG-Eg95 positive clones were screened by Kan+ and identified by PCR amplification. RESULTS: BCG-Ag85B signal sequence coding gene and Eg95 coding gene were successfully cloned into pMV261, which was confirmed by restriction endonuclease digestion, PCR amplification and sequencing of the plasmid pSMEg95. The plasmids were introduced into BCG and confirmed as the recombinant secreting BCG-Eg95 vaccine of E. granulosus (rsBCG-Eg95). CONCLUSION: The recombinant secretion type BCG-Eg95 vaccine (rsBCG-Eg95) of E. granulosus with BCG-Ag85B signal sequence and Eg95 gene has been constructed.

Identification of Differentiation-Related Proteins in Gastric Adenocarcinoma Tissues by Proteomics.

There is a significant correlation between the degree of tumor differentiation and the survival of patients with gastric cancers. In this report, we compared proteomic differences between poorly differentiated gastric adenocarcinoma tissues and well-differentiated gastric adenocarcinoma tissues in order to identify differentiation-related proteins that may be closely correlated with differentiation of gastric cancer pathogenesis. We identified 7 proteins, of which calreticulin precursor, tapasinERP57 heterodimer, pyruvate kinase isozymes M1/M2 isoform M2, class Pi glutathione S-transferase, and chain A crystal structure of human enolase 1 were upregulated in poorly differentiated gastric adenocarcinoma compared with well-differentiated gastric adenocarcinoma, while myosin-11 isoform SM2A and actin alpha cardiac were downregulated. Two of them, pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 are enzymes involved in glycolytic pathway. The upregulation of pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 in poorly differentiated gastric adenocarcinoma was confirmed by Western blotting and immunohistochemistry. Furthermore, we observed 107 cases with gastric adenocarcinoma and found that the high expression of pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 correlates with tumor size (P = .0001 and P = .0017, respectively), depth of invasion (P = .0024 and P = .0261, respectively), and poor prognosis of patients. In conclusion, with this proteomic analysis, pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 were identified upregulated in poorly differentiated gastric adenocarcinoma comparing with well-differentiated gastric adenocarcinoma. The expression level of pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 was significantly correlated with overall survival. Some of them would be differentiation-related cancer biomarkers and are associated with tumor metastasis, invasion, and prognosis.

Molecular epidemiology of Mycobacterium tuberculosis in Gansu province of China.

BACKGROUND: Mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) and Beijing family typing based on detecting the deletion of RD105 sequence are two common genotyping methods used to study the molecular epidemiologic characteristics of Mycobacterium (M.) tuberculosis. We collected 218 strains of M. tuberculosis between 2004 and 2006 in the Linxia Hui Autonomous Prefecture of Gansu province in Northwest China. METHODS: MIRU-VNTR analysis and Beijing family typing based on detecting the deletion of RD105 sequence were used to type the 218 strains, and their typing power was evaluated to look for practical and efficient genotyping methods suitable for the region. RESULTS: The MIRU typing yielded 115 distinct genotypes, including 98 unique isolates and 17 different clusters containing 120 isolates (55.05%); the cluster rate was 47.25%. By detecting the deletion of RD105 sequence, 188 of 218 (86.23%) isolates belonged to Beijing family. Combination of Beijing family typing and MIRU typing yielded 118 distinct patterns, including 101 unique isolates and 17 clusters containing 117 isolates (54.13%). The largest cluster contained 58 strains with MIRU genotype of 223325173533 which contained 50 strains belonging to Beijing family and 8 strains belonging to non-Beijing family. CONCLUSIONS: The Beijing family strains occupied a large proportion and the Beijing family MIRU genotype 223325173533 is a dominant strain in Linxia of Gansu. Combining detecting the deletion of RD105 and MIRU typing together provides a simple, fast, and effective method which is low in cost and might be practical and suitable for M. tuberculosis genotyping in China.

Diagnostic test of rifampicin resistance in Mycobacterium tuberculosis: a meta-analysis.

OBJECTIVE: To evaluate available evidence on the diagnostic value of radioactivity-free cultivation and detection technologies for rapid detection of rifampicin resistance in Mycobacterium tuberculosis. METHODS: A fully recursive literature search was conducted in PubMed, EMBASE, Biosis, Web of Science (all 1990-2010), CBMWeb (1978-2010), and Google Scholar. QUADAS items were used to evaluate the quality of included studies. Sensitivity, specificity, Summary receiver-operating curve SEN, SPE, SROC, and related techniques were used to assess the diagnostic value of radioactivity-free Mycobacterium tuberculosis cultivation and detection technologies. RESULTS: Six studies were included in the final analysis. The MB/BacT, BACTEC MGIT 960, and Manual MGIT systems were highly sensitive and specific for detecting rifampicin-resistant TB. The summary SEN and summary SPE of the MB/BacT and BACTEC MGIT 960 systems were 100%, 99%, 100%, and 96%, respectively. The SROC of the BACTEC MGIT 960 system was 0.9943. CONCLUSION: We recommended that the BACTEC 460 system be replaced by MB/BacT or BACTEC MGIT 960 as the final diagnostic test for rifampicin resistance in Mycobacterium tuberculosis. More studies are needed on the diagnostic value of other radioactivity-free cultivation and detection technologies to reliably determine their sensitivity and specificity for this bacterium.

Evaluation of a recombinant BCG expressing antigen Ag85B and PPE protein Rv3425 from DNA segment RD11 of Mycobacterium tuberculosis in C57BL/6 mice.

Antigen 85B (Ag85B) is an important immunodominant antigen of Mycobacterium tuberculosis, and is a very promising vaccine candidate molecule. Rv3425 is a member of the subgroup 3 of the PPE family, which does not exist in all BCG strains. In this study we constructed a new rBCG which included this united gene (Ag85B-Rv3425). The level of antigen-stimulated T cells expressing IFN-gamma was significantly higher in the C57BL/6 mice vaccinated with rBCG::Ag85B-Rv3425 than with BCG. In addition, the sera from mice immunized with rBCG::Ag85B-Rv3425 revealed an increase in the specific immunoglobulin G titers than that from mice immunized with BCG. Antigen specific IgG subclass analysis showed that rBCG::Ag85B-Rv3425 tended to facilitate IgG2a production, suggesting enhancement of predominant Th1 response which in turn may facilitate increased production of protective IFN-gamma. These results suggested that this rBCG::Ag85B-Rv3425 could be a strong vaccine candidate for further study.

Mycobacterium bovis bacille Calmette-Guérin (BCG) enhances human beta-defensin-1 gene transcription in human pulmonary gland epithelial cells.

AIM: To examine the stimulatory effect of bacille Calmette-Gu rin (BCG) cell wall components on human beta-defensin-1 (hBD-1) gene expression and analyze the response element in the 5'-flanking region of the gene. METHODS: BCG cell wall proteins were fractionated by Sephadex G-150 chromatography. Using reverse-transcription polymerase chain reaction (RT-PCR) and Northern hybridization analysis, hBD-1 mRNA expression was detected in a human pulmonary gland epithelial cell line SPC-A-1 cells. Progressive deletions of 5'-flanking region of hBD-1 gene were produced by PCR and ligated into promoterless chloramphenicol acetyltransferase (CAT) expression plasmid to construct pCAT reporter plasmids. Reporter gene expression was determined by ELISA. RESULTS: There was an obvious enhancement of hBD-1 mRNA expression after stimulation with heat-inactivated BCG whole cells (50 mg/L), or the cell wall components with a molecular weight of 18-30 kDa (3 mg/L) for 8 h. The upstream sequence between -314 bp and +54 bp had the inducible activity by BCG, which contained CCAAT/enhancer binding protein-beta (C/EBP beta), activator protein-1 (AP-1), and CP2 cis element. CONCLUSION: BCG cell wall components (18-30 kDa) can stimulate hBD-1 mRNA expression in pulmonary gland epithelial cells. The sequence (-314/+54) containing C/EBP beta, AP-1, and CP2 binding sites in the upstream of hBD-1 is involved in this induction.