OBJECTIVETo construct a recombinant bacillus Calmette-Guérin vaccine (rBCG) secreting human interferon-alpha 2b (IFN alpha-2b).

METHODSBCG Ag85B signal sequence and IFN alpha-2b gene were amplified from the genome of BCG and of human peripheral blood by polymerase chain reaction (PCR), respectively. IFN alpha-2b gene was cloned in E. coli-BCG shuttle-vector pMV261 to get pMV261-IFN alpha-2b. A new recombinant plasmid pMV261-IFN alpha-2b was constructed by inserting BCG Ag85B signal sequence into pMV261-Ag85B-IFN alpha-2b. Then, BCG was transformed with this recombinant plasmid by electroporation, and designated as rBCG-IFN alpha-2b. The DNA and protein expressions of IFN alpha-2b gene in rBCG were determined by PCR and Western blot respectively. Also the quantity of IFN alpha-2b protein secreted by rBCG in culture supernatants was determined by enzyme linked immunosorbent assay (ELISA).

RESULTSBy partial nucleotide sequencing, the DNA sequences of human IFN alpha-2b and BCG Ag85B were consistent with that in the Gene Bank, and were correctly inserted into the shuttle expression vector pMV261 to construct recombinant plasmid pMV261-Ag85B-IFN alpha-2b. BCG was successfully transformed with this recombinant plasmid by electroporation and the recombinant BCG (rBCG-IFN alpha-2b) was capable of synthesizing and secreting cytokine IFN alpha-2b. The concentration of IFN alpha-2b in culture supernatants was quantified by ELISA and calculated to be approximately 301.45 pg/ml.

CONCLUSIONSRecombinant BCG secreting human IFN alpha-2b (rBCG-IFN alpha-2b) was constructed successfully and the specific IFN alpha-2b protein can be expressed highly and steadily by rBCG vaccine.

BCG Vaccine ; genetics ; immunology ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Interferon-alpha ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; Transformation, Bacterial

OBJECTIVEThe impact of dendritic cells (DCs) and regulatory T cells (Tr) on the pathogenesis of asthma have been investigated over the past decades. As the professional antigen presenting cells, DCs not only prime immune response directing Th0 cells toward different T subtypes but also induce immune tolerance. As an immunoregulator, Bacillus Calmette-Guerin (BCG) has potential to be applied in allergic diseases such as asthma for prevention. Previous study showed that neonatal BCG vaccination could induce Th1/Tr1 development in mice in vivo. To further identify the mechanism of neonatal BCG vaccination on T cell subsets differentiation, the present study was designed to investigate the impact of BCG vaccination on splenic DCs development in neonatal mice.

METHODSNeonatal specific pathogen free (SPF) BALB/c mice (2-3 days) were divided into intraperitoneal BCG-treated group, subcutaneous BCG-treated group and control group; simultaneously adult SPF BALB/c mice (6-8 weeks) were divided into intraperitoneal BCG-treated and control group. The BCG-treated mice were inoculated with 1 x 10(5) CFU BCG, the mice in control group were not inoculated with any vaccine. Four weeks post BCG vaccination, spleen cells were isolated. With flow cytometry, subtype and maturity of splenic DCs were analysed. Moreover, cells were further separated into mononuclear cell by Ficoll solution. The mononuclear cells were stimulated by 1 microg/ml lipopolysaccharide (LPS) for 18 or 10(5) CFU /ml BCG for 48 hours at 37 degrees C in a humidified atmosphere containing 5% CO2 and cytokines concentration was detected by ELISA.

RESULTS(1) CD11c(+) CD8alpha(+) and CD11c(+) CD8alpha(-) DCs were found in spleen cells of the BALB/c mice. In comparison with the control group, the percent of CD11c(+) CD8alpha(-) DCs in intraperitoneal BCG group significantly declined (P < 0.01) and that of CD11c(+) CD8alpha(+) DCs significantly increased (P < 0.01), there were no significant difference in DC subtypes between intraperitoneal and subcutaneous BCG-vaccinated mice. In contrast, the percent of CD11c(+) CD8alpha(-)DCs markedly increased (P < 0.01) and that of CD11c(+) CD8alpha(+)DCs noticeably reduced (P < 0.01) in adult BCG-vaccinated mice. The percent of CD11c(+)CD8alpha(-)DC was significantly higher and that of CD11c(+) CD8alpha(+)DC was significantly lower in adult-vaccinated BALB/c mice than that of neonatal-vaccinated ones. (2) The expression of costimulatory molecules CD40 on CD11c(+) CD8alpha(-) DCs and CD86 on CD11c(+) CD8alpha(+) DCs in neonatal BCG-treated BALB/c mice was higher than the controls. There were no significant difference in expression of costimulatory molecules on DC between neonatal BCG-vaccinated mice. Compared with the control group, expression of CD40 and MHC-II molecules on CD11c(+) CD8alpha(-) and CD11c(+) CD8alpha(+)DC was significantly higher and that of CD86 was significantly lower in adult BCG-vaccinated mice. The expression of costimulatory molecules on DC had no significant difference between neonatal and adult BCG vaccinated BALB/c mice. (3) As compared with the control mice, concentration of IL-12p70 induced by LPS and IL-10 induced by BCG in vitro from spleen cells culture supernatant was noticeably elevated (P < 0.05) in neonatal BCG-treated BALB/c mice, but that of IL-6 did not change by LPS or BCG stimulation.

CONCLUSION(1) By up-regulating splenic CD8alpha(+)DCs and inducing IL-12p70 and IL-10 production in BALB/c mice, neonatal BCG vaccination promoted Th1/Tr1 development. (2) The effect of BCG vaccination on DC was different between neonates and adult BALB/c mice.

Animals ; Animals, Newborn ; BCG Vaccine ; immunology ; Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; metabolism ; Immunophenotyping ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; immunology ; metabolism

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4⁺ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4⁺ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.
Cattle ; Animals ; Cytokines ; BCG Vaccine/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-2 ; Flow Cytometry/methods* ; Chemokine CXCL10/metabolism* ; Leukocytes, Mononuclear ; CD4-Positive T-Lymphocytes/metabolism* ; Tuberculosis ; Antibodies, Monoclonal/metabolism*

PURPOSE: Diagnosis of tuberculosis (TB) in children is more challenging than in adults. This study aimed to describe demographical, clinical and laboratory findings of children diagnosed with tuberculosis in Turkey, including the issues of contact tracing, culture positivity and forms of the disease. MATERIALS AND METHODS: Clinical and laboratory data of 51 children with a mean age of 8.0+/-4.6 years who were diagnosed with TB were retrospectively reviewed. Main diagnostic tools included tuberculin skin test, chest X-ray, sputum/gastric aspirate culture with sensitivity testing, and direct microscopy for acid-fast bacilli on available samples. Clinical characteristics and outcomes of the patients were examined. RESULTS: Thirty-six (70.6%) children were diagnosed with intra-thoracic and 15 (29.4%) with extra-thoracic tuberculosis. Twenty-eight of the patients had a positive Bacillus Calmette-Guerin vaccine scar (28/51, 54.9%) and 23/51 (45.1%) had a positive tuberculin skin test. An adult TB contact was identified in 27 (52.9%) of the cases. On direct microscopy, acid-fast bacilli were found in nine (17.6%) patients and positive culture for Mycobacterium tuberculosis was found in 19 (37.3%). Drug resistance to isoniazid was detected in four (7.8%). One patient with nephrotic syndrome and miliary tuberculosis died during follow-up. All other patients responded well to the treatment. CONCLUSION: Focusing on active contact tracing among all household contacts of tuberculous cases may be helpful in early identification and controlling childhood disease, even in regions with low disease prevalence. Adopting a suspicious and proactive approach in this particular age group is warranted.
Adolescent ; BCG Vaccine/metabolism ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Isoniazid/therapeutic use ; Male ; Mycobacterium tuberculosis/pathogenicity ; Retrospective Studies ; Risk Factors ; Tuberculin/metabolism ; Tuberculin Test ; Tuberculosis/*diagnosis/drug therapy/metabolism ; Tuberculosis, Pulmonary/diagnosis/drug therapy/metabolism ; Turkey

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*

OBJECTIVETo investigate the antitumor effect of recombinant IFN-alpha-2b-BCG on mouse bladder cancer MB49 cells in vitro, and to explore its antitumor mechanisms.

METHODSMB49 cells were co-cultured with recombinant BCG or wild BCG, and than were examined by light and transmission electron microscopy. The cell growth was assessed by MTT assay, and apoptosis rate and MHC-I of the MB49 cells was detected by flow cytometry using AO and Hoechst33258 fluorescence immunostaining.

RESULTSThe hIFN-alpha-2b-BCG-treated tumor cells showed slow growth, detachment of some cells, and various degree of degeneration. Light microscopy revealed organelle disorganization, chromatin aggregation, nuclear pyknosis, and cytolysis in some cells. Cellular membrane bulged and some bubbles were seen under fluorescence microscope using AO staining. Hoechst33258 assay also depicted frequent apoptosis in the tumor cells. The MTT assay showed that rBCG more actively than the wild BCG inhibited the proliferation of MB49 cells. The apoptosis rate of the recombinant BCG group was 19.7% and 46.6% at the time point of 24 h and 48 h, respectively, significantly higher than 10.8% and 20.9%, respectively, in the wild BCG group. The results of flow cytometry indicated that both types of BCG enhanced the expression of MHC-I in the MB49 cells, but more effective in the recombinant BCG group.

CONCLUSIONThe recombinant hIFN-alpha-2b-BCG has more strong immuno-modulatory properties, anti-tumor effect on MB49 cells and induces apparent cytotoxicity in the bladder cancer cells in vitro.

Adjuvants, Immunologic ; pharmacology ; Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; BCG Vaccine ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cytotoxicity, Immunologic ; Histocompatibility Antigens Class I ; metabolism ; Interferon-alpha ; pharmacology ; Mice ; Recombinant Proteins ; pharmacology ; Urinary Bladder Neoplasms ; metabolism ; pathology

This study was aimed to investigate the effects of xenogeneic antigen neu-Fc in combination with the recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and Bacillus Calmette-Guerin (BCG) on the regulation of Th1 and Th2 immune response in vitro. The rat neu L2-S2 domain was engineered as a chimeric protein with human IgG Fc. The eukaryotic expression vector was constructed. The recombinant protein was stably expressed in CHO cells and purified by rProtein A Sepharose Fast Flow column. The recombinant protein was identified by SDS-PAGE and Western blot. Peripheral blood mononuclear cells (PBMNCs) were obtained by means of standard Ficoll separation from the blood of healthy donors. Neu-Fc-induced PBMNC proliferation was tested by MTT. The production of IL-12 and IL-10 was measured by ELISA. The results showed that the level of IL-12 decreased and IL-10 increased after PBMNCs were incubated with MCF-7 cultural supernatant. 10 nmol/L neu-Fc strongly induced the cell proliferation. Compared with neu-Fc or GM-CSF or BCG treatment alone, neu-Fc in combination with GM-CSF and BCG significantly stimulated IL-12 production and inhibited IL-10 production (p < 0.01). It is concluded that the neu-Fc can stimulate the proliferation activity of PBMNCs. neu-Fc, GM-CSF and BCG costimulation efficiently induces Th1 immune response.
Animals ; BCG Vaccine ; immunology ; CHO Cells ; Cricetinae ; Cricetulus ; Granulocyte-Macrophage Colony-Stimulating Factor ; immunology ; Humans ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Rats ; Recombinant Proteins ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology

Recent studies show that the vector of recombinant Bacillus Calmette-Guérin (rBCG) has a series of advantages. With exogenous gene and vaccine in one inoculation, it can obtain strong and persistent immune response at one time so that BCG is considered as a kind of ideal vector for live recombinant vaccine. This review outlines the application of rBCG vaccine and its vector in infectious diseases caused by bacteria, viruses, other microorganisms and parasites.
AIDS Vaccines ; genetics ; immunology ; Antigens, Bacterial ; genetics ; immunology ; BCG Vaccine ; genetics ; immunology ; metabolism ; Communicable Disease Control ; methods ; Genetic Vectors ; genetics ; HIV Infections ; prevention & control ; HIV-1 ; Mycobacterium tuberculosis ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Tuberculosis ; prevention & control ; Vaccines, Synthetic ; immunology

OBJECTIVETo study the expression of signal transducer and activator of transcription 5b (STAT5b) mRNA and interleukin-4 (IL-4) mRNA in blood mononuclearcells in a rat model of asthma and the effect of montelukast (MK) and BCG-polysaccharide and nucleic acid injection (BCG-PSN) on STAT5b mRNA and IL-4 mRNA expression.

METHODSFifty-two male Sprague-Dawley rats (weight:140-200 g) were randomly divided into four groups: asthma, MK treated and BCG-PSN-treated and control groups. Rat model of asthma was prepared by ovalbumin (OVA) sensitization. The rats were sacrificed 24 hrs after the last sensitization. Blood eosinophils (EOS) were counted. Plasma contens of IL-4 and interferon-gamma (IFN-gamma) were measured using ELISA. Expression of STAT5b mRNA and IL-4 mRNA in blood mononuclearcells was detected with SYBR GREEN I fluorescent quantitation PCR method.

RESULTSBlood contents of STAT5b mRNA and IL-4 mRNA in the untreated asthma group were significantly higher than those in the other three groups (<0.01). Blood EOS count and plasma IL-4 contents in the untreated asthma group significantly increased, while plasma IFN-gamma contents significantly decreased compared with the other three groups (<0.01). There were no significant differences in the parameters measured among the MK-treated, the BCG-PSNjtreated and the control groups. STAT5b mRNA expression was positively correlated to IL-4 mRNA expression, IL-4 content and EOS count (r=0.730,0.650, 0.664, respectively; <0.01), but negatively correlated to IFN-gamma content (r=-0.798; <0.01).

CONCLUSIONSSTAT5b mRNA and IL-4 mRNA were strongly expressed in blood mononuclearcells in rats with asthma, and there was a positive correlation between them. MK and BCG-PSN had inhibitory effects on the expression of STAT5b mRNA and IL-4 mRNA, which might be contributed to suppression of airway inflammation in asthma.

Acetates ; pharmacology ; Animals ; Asthma ; blood ; drug therapy ; pathology ; BCG Vaccine ; pharmacology ; Interferon-gamma ; biosynthesis ; Interleukin-4 ; genetics ; Leukocytes, Mononuclear ; metabolism ; Male ; Quinolines ; pharmacology ; RNA, Messenger ; blood ; Rats ; Rats, Sprague-Dawley ; STAT5 Transcription Factor ; genetics

Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69% in total bacterial protein and 74.09% in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M. tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.
BCG Vaccine ; biosynthesis ; immunology ; Bacterial Proteins ; biosynthesis ; immunology ; Chaperonin 60 ; Chaperonins ; biosynthesis ; immunology ; Cloning, Molecular ; Escherichia coli ; metabolism ; Genetic Vectors ; Humans ; Mycobacterium tuberculosis ; genetics ; immunology ; Plasmids ; genetics ; Sequence Analysis, DNA ; Vaccines, Synthetic ; biosynthesis ; immunology