This study was conducted to amplify the cfpl0-esat6 fusion gene by SOE and insert into the integrating shuttle plasmid pMV361 to form the recombinant plasmid. Then another recombinant plasmid was constructed by insertinga-A g signal sequence of BCG. The two recombinant plasmids were introduced into BCG and the induced products from recombinant BCG were analyzed. In conclusion,the successful construction of rBCG expressing the fusion protein CFP10-ESAT6 will be the base of the development of novel Mycobacterium tuberclosis vaccines.
Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Mycobacterium bovis ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Tuberculosis Vaccines ; biosynthesis

Although an immune response to bacillus Calmette Guerin (BCG) has often been associated with antitumor activity, the action mechanism(s) of intravesical BCG therapy for prophylaxis and treatment of superficial bladder cancer is not clearly understood. In an attempt to evaluate the roles of tumor necrosis factor (TNF)-alpha and lymphotoxin (LT) in the antitumor activity, TNF-alpha productivities by peripheral blood monocytes, serum levels of TNF-alpha, and LT productivities by peripheral blood lymphocytes were studied in superficial bladder cancer patients after six intravesical administrations of BCG. TNF-alpha productivities by peritoneal macrophages of guinea pigs were also studied after six intravesical administrations of BCG. The maximum TNF-alpha productivities by peripheral blood monocytes of superficial bladder cancer patients were seen after the fourth week of administration of BCG, and the serum TNF-alpha levels were also slightly increased after intravesical BCG administration in the superficial bladder cancer patients. LT productivities by peripheral blood lymphocytes of superficial bladder cancer patients were significantly enhanced and the maximum LT productivity was also seen after the third or fifth BCG administration. TNF-alpha productivities by peritoneal macrophages of guinea pigs were significantly enhanced and the maximum TNF-alpha productivity was seen after the second or third BCG administration. Our data might suggest that six consecutive intravesical BCG administrations could induce the increased productions of TNF-alpha and LT, which might play an important role in the antitumor activity in superficial bladder cancer.Although an immune response to bacillus Calmette Guerin (BCG) has often been associated with antitumor activity, the action mechanism(s) of intravesical BCG therapy for prophylaxis and treatment of superficial bladder cancer is not clearly understood. In an attempt to evaluate the roles of tumor necrosis factor (TNF)-alpha and lymphotoxin (LT) in the antitumor activity, TNF-alpha productivities by peripheral blood monocytes, serum levels of TNF-alpha, and LT productivities by peripheral blood lymphocytes were studied in superficial bladder cancer patients after six intravesical administrations of BCG. TNF-alpha productivities by peritoneal macrophages of guinea pigs were also studied after six intravesical administrations of BCG. The maximum TNF-alpha productivities by peripheral blood monocytes of superficial bladder cancer patients were seen after the fourth week of administration of BCG, and the serum TNF-alpha levels were also slightly increased after intravesical BCG administration in the superficial bladder cancer patients. LT productivities by peripheral blood lymphocytes of superficial bladder cancer patients were significantly enhanced and the maximum LT productivity was also seen after the third or fifth BCG administration. TNF-alpha productivities by peritoneal macrophages of guinea pigs were significantly enhanced and the maximum TNF-alpha productivity was seen after the second or third BCG administration. Our data might suggest that six consecutive intravesical BCG administrations could induce the increased productions of TNF-alpha and LT, which might play an important role in the antitumor activity in superficial bladder cancer.
Administration, Intravesical ; Animal ; Bladder Neoplasms/*metabolism/*therapy ; Female ; Guinea Pigs ; Human ; Mycobacterium bovis/*physiology ; Prospective Studies ; Support, Non-U.S. Gov't ; Tumor Necrosis Factor/*biosynthesis

BCG has been one of the vehicles for multi-recombinant vaccine. However, low transformation efficiency of BCG with plasmid DNA hampered studies involving expression of foreign antigens in BCG. In an effort to determine the optimal conditions, this study was initiated to investigate factors involved in the transformation of BCG with a Mycobacterium-Escherichia coli shuttle vector, pYUB18, by electroporation. Mycobacterium bovis BCG (strain 1173P2) was grown in Middlebrook (M) 7H9 broth containing albumin-dextrose-catalase and 0.05% tween 80, and transformed BCG was grown in M7H10 agar containing kanamycin for counting viable cells. Pretreatment of BCG with 10 mM CaCl2 improved the transformation efficiency, but overnight incubation of BCG with 1% glycine did not. The transformation efficiency in BCG also varied depending on voltage, resistance, and DNA concentration. The maximum transformation efficiency was obtained when the infinity resistance, 12.5 Kv/cm, and 100 ng of DNA were used, and reached 1.4 x 10(5) CFU/microgram of plasmid DNA, which is about 3-100 times greater than those from previous reports. The transformation conditions described in this study, therefore, will give us a better position for employing BCG as a vehicle for developing multi-recombinant vaccines.
Calcium Chloride/pharmacology ; Comparative Study ; DNA/metabolism ; Electrophysiology ; Electroporation* ; Escherichia coli/genetics* ; Genetic Vectors* ; Glycine/pharmacology ; Mycobacterium/genetics* ; Mycobacterium bovis/genetics* ; Osmolar Concentration ; Transformation, Bacterial/physiology* ; Transformation, Bacterial/drug effects

OBJECTIVE: To investigate the role of tumor necrosis factor receptor-associated factor 6 (TRAF6) in regulating Bacillus Calmette-Guérin (BCG)-induced macrophage apoptosis. METHODS: The expression of TRAF6 in peripheral blood samples of 50 patients with active tuberculosis (TB) and 50 healthy individuals were detected using quantitative real-time PCR (qPCR). RAW264.7 macrophages were infected with BCG at different MOI and for different lengths of time, and the changes in expressions of Caspase 3 and TRAF6 were detected with Western blotting and qPCR. In a RAW264.7 cell model of BCG infection with TRAF6 knockdown established using RNA interference technique, the bacterial load was measured and cell apoptotic rate and mitochondrial membrane potential (MMP) were determined with flow cytometry. The expression levels of TRAF6, Caspase 3, PARP, BAX and Bcl-2 in the cells were detected using Western blotting, and the expressions of TRAF6 and Caspase 3 were also examined with immunofluorescence assay. RESULTS: The expression of TRAF6 was significantly upregulated in the peripheral blood of patients with active TB as compared with healthy subjects (P < 0.001). In RAW264.7 cells, BCG infection significantly increased the expressions of Caspase 3 and TRAF6, which were the highest in cells infected for 18 h and at the MOI of 15. TRAF6 knockdown caused a significant increase of bacterial load in BCG-infected macrophages (P=0.05), lowered the cell apoptotic rate (P < 0.001) and reduced the expressions of Caspase 3 (P=0.002) and PARP (P < 0.001). BCG-infected RAW264.7 cells showed a significantly increased MMP (P < 0.001), which was lowered by TRAF6 knockdown (P < 0.001); the cells with both TRAF6 knockdown and BCG infection showed a lowered BAX expression (P=0.005) and an increased expression of Bcl-2 (P=0.04). CONCLUSION TRAF6 promotes BCG-induced macrophage apoptosis by regulating the intrinsic apoptosis pathway.
Apoptosis ; BCG Vaccine ; Caspase 3/metabolism* ; Humans ; Intracellular Signaling Peptides and Proteins ; Macrophages ; Mycobacterium bovis/metabolism* ; Poly(ADP-ribose) Polymerase Inhibitors ; TNF Receptor-Associated Factor 6/metabolism* ; bcl-2-Associated X Protein/metabolism*

AIMTo observe the stability of BCG-induced insulin resistance model.

METHODSThe glucose tolerance, serum glucose, FFA, insulin, triglycerides, cholesterol, TNF-alpha and ALT level were measured. The change of GDR was measured by euglycemic clamp in model rats after given i.v. BCG 2, 4 and 8 weeks.

RESULTSAfter 2, 4 and 8 weeks, the GIR and glucose tolerance of the animals deceased significantly. After 2, 4 and 8 weeks, BCG infusion resulted in a pronounced reduction in glucose tolerance and insulin-stimulated glucose disposal rate [GDR = GDR: (29 +/- 6) vs (13 +/- 7) mg.kg-1.min-1 2 weeks; (29 +/- 6) vs (11 +/- 7) mg.kg-1.min-1 4 weeks and (23 +/- 3) vs (16 +/- 3) mg.kg-1.min-1 8 weeks, respectively, P < 0.01]. BCG infusion resulted in a pronounced increase in the weights of the liver [(6.2 +/- 0.9) vs (8.2 +/- 1.3) g, P < 0.05] and spleens [(0.51 +/- 0.11) vs (1.4 +/- 0.4) g, P < 0.01]. The histo-pathological results showed that BCG infusion resulted severe inflammation in the livers and spleens and the ratio of beta/alpha in pancreas increased. The serum levels of triglyceride, FFA and glucose were unchanged, but the level of serum TNF-alpha [543 +/- 60) vs (759 +/- 137) pg.mL-1, P < 0.05] and insulin [(31 +/- 5) vs (36 +/- 5) mu.L-1, P > 0.05] increased.

CONCLUSIONThis novel model of immune insulin resistance is completely and constantly established.

Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus ; metabolism ; Glucose Clamp Technique ; Glucose Tolerance Test ; Injections, Intravenous ; Insulin ; blood ; Insulin Resistance ; immunology ; Male ; Mycobacterium bovis ; Random Allocation ; Rats ; Rats, Wistar ; Spleen ; pathology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo study the protective effect of atractylenolide I on immunological liver injury induced by BCG and LPS.

METHODKunming mice were randomly divided into 6 groups: the normal group, the model group, positive control biphenyl group, the atractylenolide I high does group, the atractylenolide I middle dose group and the atractylenolide I low dose group (60, 120, 240 mg x kg(-1)), with 12 mice in each group. Immunological liver injury in mice was induced by BCG and LPS to compared liver index and spleen index and detect content of serum ALT, AST, MDA and GSH-px in serum and NO, iNOS, TNF-alpha in serum and liver homogenate. Liver pathological changes were observed by HE staining.

RESULTBoth of atractylenolide I and biphenyl remarkably decrease the increased live index and spleen index (P < 0.05), improve the histopathological changes in liver and pathological grades of liver tissues and relieve the inflammatory reaction induced by BCG and LPS. They showed a notable effect in improving MDA and GSH-px in serum.

CONCLUSIONAtractylenolide I can obviously protect immunological injury liver a dose-dependent manner within the range of test doses. Its mechanism may be related to release or over expression of inhibitory inflammatory medium such as NO, iNOS and TNF-alpha.

Animals ; Chemical and Drug Induced Liver Injury ; immunology ; metabolism ; pathology ; prevention & control ; Lactones ; pharmacology ; Lipopolysaccharides ; adverse effects ; Liver ; drug effects ; enzymology ; metabolism ; pathology ; Male ; Mice ; Mycobacterium bovis ; immunology ; Oxidative Stress ; drug effects ; immunology ; Sesquiterpenes ; pharmacology

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

OBJECTIVETo investigate the pathophysiological role of CXCL16 in immunological liver injury induced by Bacille de Calmette et Guerin (BCG) and lipopolysaccharides (LPS).

METHODSImmunological liver injury was induced by BCG and LPS in mice, and the expression of CXCL16 was detected in the liver tissues by real-time quantitative PCR and immunohistochemical examination. The relationship of the expression of CXCL16 and the extent of hepatic necrosis was investigated histopathologically and immunohistochemically. Mononuclear cells were isolated from the liver tissues and their numbers were counted; T lymphocytes populations in the liver tissue were also analyzed with FACS.

RESULTSThe immunological liver injury model was successfully created. Up-regulation of CXCL16 in injured livers correlated with the extent of liver injury and the amountmononuclear cell infiltrations.

CONCLUSIONThese findings suggest that up-regulation of CXCL16 was closely correlated with liver injury extent during the immunological liver injury induced by BCG-LPS in mice, and intrahepatic recruitment of specific lymphocytes might be an important mechanism of liver injury.

Animals ; Chemical and Drug Induced Liver Injury ; Chemokine CXCL16 ; Chemokine CXCL6 ; Chemokines, CXC ; biosynthesis ; genetics ; Lipopolysaccharides ; Liver Diseases ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mycobacterium bovis ; Receptors, Scavenger ; biosynthesis ; genetics

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

OBJECTIVETo observe the effect of polysaccharide ATPS-2 from Armillariella tabescens on the immunological liver injury in mice induced by BCG plus LPS.

METHODBCG and LPS were adopted to establish BCG plus LPS liver injury model in mice. The content of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and NO, the activity of superoxide dismutase (SOD) and malondiadehyde (MDA) content of liver homogenate in mice were measured by colorimetric method. The content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), on serum were measured by enzyme linked immunosorbent assay (ELISA) , and T- and B-lymphocyte proliferation were measured by MTT. Index of liver, spleen and thymus were calculated after treatment.

RESULTPolysaccharide ATPS-2 from A. tabescens (25, 50, 100 mg x kg(-1)) could obviously reduce the high level of ALT, AST, NO and TNF-alpha, IL-1 on serum, inhibit the high level of MDA, increase the low activity of SOD in liver homogenate and enhance T-and B-lymphocyte proliferation, elevate the spleen, thymic index and decrease liver index of the mice to different extent.

CONCLUSIONPolysaccharide ATPS-2 from A. tabescens had apparently protective effects in the immunological liver injury mice induced by BCG plus LPS.

Agaricales ; chemistry ; Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; B-Lymphocytes ; cytology ; Cell Proliferation ; drug effects ; Chemical and Drug Induced Liver Injury ; Interleukin-1 ; blood ; Lipopolysaccharides ; Liver ; metabolism ; pathology ; Liver Diseases ; immunology ; metabolism ; pathology ; Male ; Malondialdehyde ; metabolism ; Mice ; Mycobacterium bovis ; Nitric Oxide ; blood ; Polysaccharides ; isolation & purification ; pharmacology ; Protective Agents ; isolation & purification ; pharmacology ; Random Allocation ; Superoxide Dismutase ; metabolism ; T-Lymphocytes ; cytology ; Tumor Necrosis Factor-alpha ; blood