Animals ; Cattle ; microbiology ; Microbial Sensitivity Tests ; Minisatellite Repeats ; Mycobacterium bovis ; drug effects ; genetics

OBJECTIVETo evaluate four candidate variable number tandem repeat (VNTR) loci for genotyping Mycobacterium tuberculosis complex strains.

METHODSGenomic sequences for two M. tuberculosis strains (CCDC5079 and CCDC5180) were generated, and using published sequence data, four candidate VNTR loci were identified. The VNTRs were used to genotype 225 Chinese clinical M. tuberculosis complex strains. The discriminatory power of the VNTRs was evaluated using BioNumerics 5.0 software.

RESULTSThe Hunter-Gaston Index (HGI) for BJ1, BJ2, BJ3, and BJ4 loci was 0.634, 0.917, 0.697, and 0.910, respectively. Combining all four loci gave an HGI value of 0.995, thus confirming that the genotyping had good discriminatory power. The HGI values for BJ1, BJ2, BJ3, and BJ4, obtained from Beijing family strain genotyping, were 0.447, 0.878, 0.315, and 0.850, respectively. Combining all four loci produced an HGI value of 0.988 for genotyping the Beijing family strains. We observed unique patterns for M. bovis and M. africanum strains from the four loci.

CONCLUSIONWe have shown that the four VNTR loci can be successfully used for genotyping M. tuberculosis complex strains. Notably, these new loci may provide additional information about Chinese M. tuberculosis isolates than that currently afforded by established VNTR loci typing.

Cluster Analysis ; Genotyping Techniques ; Humans ; Minisatellite Repeats ; Mycobacterium bovis ; genetics ; Mycobacterium tuberculosis ; genetics

The bacilli Calmette-Guerin (BCG) Tokyo-172 strain was considered to exhibit good protective efficacy with a low rate of unfavorable side effects. However, we describe a rare case of BCG osteomyelitis developed in an immunocompetent host who was given with BCG Tokyo-172 vaccine on the left upper arm by multipuncture method. A 9-month-old girl presented with progressive inability to move her right elbow and had radiographic evidence of septic elbow combined with osteomyelitis of right distal humerus. A biopsy from the site revealed chronic caseating granulomatous inflammation, positive for BCG Tokyo-172 strain on the multiplex polymerase chain reaction. The child had to undergo second surgical debridements and oral antituberculosis chemotherapy. There were no sequelae after 2 yr of follow-up. This complication, although uncommon, should be considered in the appropriate clinical setting.
Antitubercular Agents/therapeutic use ; BCG Vaccine/*adverse effects ; DNA, Bacterial/genetics ; Female ; Humans ; Infant ; Magnetic Resonance Imaging ; Multiplex Polymerase Chain Reaction ; Mycobacterium bovis/genetics/*isolation & purification ; Osteomyelitis/drug therapy/*etiology/*microbiology/surgery

Bacillus Calmette-Guerin (BCG) has been traditionally used as a vaccine against tuberculosis. Further, intravesical administration of BCG has been shown to be effective in treating bladder cancer. Although BCG contains a live attenuated strain of Mycobacterium bovis, complications such as M. bovis BCG infection caused by BCG administration are extremely rare. Here, we report a case of BCG infection occurring after intravesical BCG therapy. A 67-yr-old man presented with azotemia and weight loss. He had been diagnosed with bladder cancer 4 yr back, and had undergone transurethral resection of the bladder tumor and intravesical BCG (Tice strain) therapy at that time. An acid-fast bacterial strain was isolated from his urine sample. We did not detect Mycobacterium tuberculosis protein 64 (MPT-64) antigen in the isolates obtained from his sample, and multiplex PCR and PCR-reverse blot hybridization assay indicated that the isolate was a member of the M. tuberculosis complex, but was not M. tuberculosis. Finally, sequence analysis of 16S ribosomal RNA and DNA gyrase, subunit B (gyrB) suggested that the organism was M. bovis or M. bovis BCG. Although we could not confirm that M. bovis BCG was the causative agent, the results of the 3 molecular methods and the MPT-64 antigen assay suggest this finding. This is an important finding, especially because M. bovis BCG cannot be identified using common commercial molecular genetics tools.
Administration, Intravesical ; Aged ; BCG Vaccine/administration & dosage/*adverse effects ; DNA Gyrase/genetics ; Humans ; Male ; Mycobacterium Infections/*diagnosis/etiology ; Mycobacterium bovis/genetics/*isolation & purification ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S/genetics ; Urinary Bladder Neoplasms/*therapy

OBJECTIVETo detect the effects of Polyporus polysaccharide (PPS), Bacillus Calmette-Guerin (BCG), and their combination on the nuclear factor kappa B (NF-κB) signaling pathway associated-gene expression and investigate the molecular mechanisms of the toxic-reducing effect of PPS in coordination with BCG against bladder cancer.

METHODSAfter T739 cells were treated with PPS, BCG and their combination, the changes in mRNA and protein expression of inhibitor of kappa B kinase beta (IKKβ), NF-κB subunit p65 (NF-κB p65), intracellular adhesion molecule 1 (ICAM1) and chemokine (C-c motif) ligand 2 (CCL2) in bladder cancer cell line T739 were determined by relative quantitative real-time PCR, Western blot, and flow cytometry (FCM). NF-κB p65 DNA-binding activity in T739 cell was detected by biotinylated probe-ELISA, and NF-κB p65 nuclear expression in T739 cell was observed by immunohistochemistry.

RESULTSCompared with the T739 control group, the mRNA expression of IKBKB (IKKβ), Rel A (NF-κB p65), ICAM1 and CCL2 in T739 cells treated with BCG were increased obviously (Ratio>2.0), as well as the expression of IKKβ, CCL2 and ICAM1 proteins. Meanwhile, NF-κB p65 DNA-binding activity and NF-κB p65 nuclear expression in T739 cells treated with BCG were up-regulated significantly (P<0.05). Compared with the control, the increased expression in T739 cells were simultaneously down-regulated after PPS treatment, except for ICAM1 protein expression. With cells treated with a combination of BCG and PPS, the expression of genes associated with the NF-κB signaling pathway, such as IKBKB, ICAM1 and CCL2, were all down-regulated compared to the BCG group, as well as Rel A mRNA expression, NF-κB p65 DNA-binding activity and NF-κB p65 nuclear expression.

CONCLUSIONSPPS could inhibit the over-activation of the NF-κB signaling pathway induced by BCG in bladder cancer cells and accordingly attenuate the adverse reactions to BCG therapy.

Cell Line, Tumor ; Cell Nucleus ; drug effects ; metabolism ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Mycobacterium bovis ; drug effects ; NF-kappa B ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Polyporus ; chemistry ; Polysaccharides ; pharmacology ; Signal Transduction ; drug effects ; Urinary Bladder Neoplasms ; genetics ; microbiology ; pathology

BACKGROUNDChronic hepatic inflammation is characterized by the accumulation of lymphocytes as a consequence of increased recruitment from the blood and retention within the tissue at sites of infection. CXC chemokine ligand 16 (CXCL16) mRNA has been detected in both inflamed and normal liver tissues and is strongly upregulated in the injured liver tissues in a murine model. The aim of this study was to investigate the effect of cefodizime on CXCL16 mRNA of liver tissues in mice with immunological hepatic injury.

METHODSThe murine model of immunological hepatic injury was induced by Bacillus Calmette Guerin and Lipoposaccharide. The mice with immunological hepatic injury were randomly assigned to the model group, the cefodizime group and the ceftriaxone group. The three groups were continuously given agents for seven days and CXCL16 mRNA of liver tissue was determined and contrasted with the control group treated by normal saline. Reverse transcription-polymerase chain reaction was used to assay CXCL16 mRNA levels in liver tissues.

RESULTSThe expressions of CXCL16 mRNA were significantly higher in the model group and the ceftriaxone group than in the control group and the cefodizime group (P < 0.05), indicating the mice in the model group and the ceftriaxone group were immunodeficient. There was no statistical difference in the expressions of CXCL16 mRNA between the control group and the cefodizime group. Similarly, no statistical difference in the expressions of CXCL16 mRNA between the model group and the ceftriaxone group was detected (P > 0.05).

CONCLUSIONCefodizime effectively reduces the infiltration of lymphocytes into liver tissues and alleviates the liver damage by decreasing CXCL16 mRNA in liver tissues in mice with immunological hepatic injury.

Animals ; Cefotaxime ; analogs & derivatives ; therapeutic use ; Chemokine CXCL16 ; Chemokine CXCL6 ; genetics ; Chemokines ; Lipopolysaccharides ; toxicity ; Liver ; drug effects ; metabolism ; microbiology ; Mice ; Mycobacterium bovis ; physiology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

Dendritic-cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN; CD209) has an important role in mediating adherence of Mycobacteria species, including M. tuberculosis and M. bovis BCG to human dendritic cells and macrophages, in which these bacteria can survive intracellularly. DC-SIGN is a C-type lectin, and interactions with mycobacterial cells are believed to occur via mannosylated structures on the mycobacterial surface. Recent studies suggest more varied modes of binding to multiple mycobacterial ligands. Here we identify, by affinity chromatography and mass-spectrometry, four novel ligands of M. bovis BCG that bind to DC-SIGN. The novel ligands are chaperone protein DnaK, 60 kDa chaperonin-1 (Cpn60.1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and lipoprotein lprG. Other published work strongly suggests that these are on the cell surface. Of these ligands, lprG appears to bind DC-SIGN via typical proteinglycan interactions, but DnaK and Cpn60.1 binding do not show evidence of carbohydrate-dependent interactions. LprG was also identified as a ligand for DC-SIGNR (L-SIGN; CD299) and the M. tuberculosis orthologue of lprG has been found previously to interact with human toll-like receptor 2. Collectively, these findings offer new targets for combating mycobacterial adhesion and within-host survival, and reinforce the role of DCSIGN as an important host ligand in mycobacterial infection.
Amino Acid Sequence ; Bacterial Adhesion ; physiology ; Bacterial Proteins ; genetics ; metabolism ; Cell Adhesion Molecules ; genetics ; metabolism ; Chromatography, Affinity ; Dendritic Cells ; metabolism ; microbiology ; Host-Pathogen Interactions ; genetics ; physiology ; Humans ; In Vitro Techniques ; Lectins, C-Type ; genetics ; metabolism ; Ligands ; Macrophages ; metabolism ; microbiology ; Mass Spectrometry ; Membrane Proteins ; genetics ; metabolism ; Models, Biological ; Molecular Chaperones ; genetics ; metabolism ; Molecular Sequence Data ; Mycobacterium bovis ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; metabolism ; pathogenicity ; Pulmonary Surfactant-Associated Protein A ; metabolism ; Receptors, Cell Surface ; genetics ; metabolism

New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules-triazoles-in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.
Antitubercular Agents ; chemistry ; isolation & purification ; pharmacology ; Arylamine N-Acetyltransferase ; antagonists & inhibitors ; chemistry ; Enzyme Inhibitors ; chemistry ; isolation & purification ; pharmacology ; High-Throughput Screening Assays ; Humans ; Mycobacterium bovis ; drug effects ; enzymology ; genetics ; Mycobacterium tuberculosis ; drug effects ; enzymology ; genetics ; Protein Conformation ; Structure-Activity Relationship ; Triazoles ; chemistry ; isolation & purification ; pharmacology

To improve the efficacy of peptide P277 in preventing autoimmune diabetes, heat shock protein 65 kD (HSP65) of Mycobacterium tuberculosis var. bovis was fused with linear polypeptide epitope of P277 and expressed as soluble protein in Escherichia coli. The fusion protein HSP65-P277 was purified by anion exchange column chromatography and then used to immunize prediabetic NOD mice with three ip inoculations in absence of adjuvants. Serum samples from the immunized mice were collected monthly and the concentration of blood glucose was measured. The study showed that administration of HSP65-P277 to NOD mice could prevent the development of diabetes more efficiently than the peptide P277 itself or HSP65. Fused to heat shock protein 65 of Mycobacterium tuberculosis could improve the efficacy of diabetes prevention of P277 in nonobese diabetic mice. The results suggest the fusion protein of HSP65-P277 would be useful for treating insulin-dependent diabetes mellitus.
Animals ; Bacterial Proteins ; biosynthesis ; genetics ; immunology ; Chaperonin 60 ; Chaperonins ; biosynthesis ; genetics ; immunology ; Diabetes Mellitus, Type 1 ; prevention & control ; Escherichia coli ; genetics ; metabolism ; Female ; Heat-Shock Proteins ; genetics ; immunology ; Immunization ; Mice ; Mice, Inbred NOD ; Mycobacterium bovis ; Peptide Fragments ; biosynthesis ; genetics ; immunology ; Random Allocation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Synthetic ; genetics ; immunology

Bovine tuberculosis (TB) is a major zoonosis that's caused by Mycobacterium bovis (M. bovis). Being able to detect M. bovis is important to control bovine TB. We applied a molecular technique, the variable number tandem repeat (VNTR) typing method, to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea. From 2003 to 2004, 59 M. bovis clinical strains were isolated from dairy cattle in Gyeonggi-do, Korea, and these cattle had tuberculosis-like lesions. Twenty-four published MIRUVNTR markers were applied to the M. bovis isolates and ten of them showed allelic diversity. The most discriminatory locus for the M. bovis isolates in Korea was QUB 3336 (h = 0.64). QUB 26 and MIRU 31 also showed high discriminative power (h = 0.35). The allelic diversity by the combination of all VNTR loci was 0.86. Six loci (MIRU 31, ETR-A and QUB-18, -26, -3232, -3336) displayed valuable allelic diversity. Twelve genotypes were identified from the 59 M. bovis isolates that originated from 20 cattle farms that were dispersed throughout the region of Gyenggi-do. Two genotypes [designation index (d.i.) = e, g] showed the highest prevalence (20% of the total farms). For the multiple outbreaks on three farms, two successive outbreaks were caused by the same genotype at two farms. Interestingly, the third outbreak at one farm was caused by both a new genotype and a previous genotype. In conclusion, this study suggests that MIRU-VNTR typing is useful to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea.
Animals ; Cattle ; DNA Primers/genetics ; *Genetic Variation ; Genotype ; Korea/epidemiology ; Minisatellite Repeats/*genetics ; Mycobacterium bovis/*genetics ; Prevalence ; Tuberculosis, Bovine/*epidemiology/*microbiology