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*

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*

A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4+ and CD8+ cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.
Adjuvants, Immunologic/genetics/*metabolism ; Administration, Intranasal ; Animals ; Antigens, Protozoan/genetics/*immunology ; BCG Vaccine/administration & dosage/*genetics ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Chickens ; Coccidiosis/*prevention & control ; Disease Models, Animal ; Drug Carriers/administration & dosage ; Eimeria tenella/genetics/*immunology ; Genetic Vectors ; Injections, Subcutaneous ; Interleukin-2/genetics/*metabolism ; Protozoan Vaccines/administration & dosage/genetics/*immunology ; Spleen/immunology ; Vaccines, Synthetic/administration & dosage/genetics/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

To obtain a vaccine to defend from dormancy Mycobacterium tuberculosis, we constructed the recombinant Bacilli Calmette-Guérin (BCG) vaccine with Rv3133c encoding dormancy-correlated transcriptional regulatory protein DosR in Mycobacterium tuberculosis as a target gene, and evaluated its immunogenicity in BALB/c mice. In this study, we constructed the recombinant plasmids of rpMV361-Rv3133c using gene colon technology. We then transformed BCG strains with above-mentioned plasmids to obtain recombinant vaccine of rBCG-Rv3133c. We used the rBCG strains successfully constructed to vaccinate in BALB/c mice. 30d and 180d after immunization, the specific antibody titers were determined to investigate humoral responses induced by recombinant vaccine. We detected changes of splenocyte subsets of CD4+T, CD8+ T cells and cytokine of IFN-gamma secreted by splenocytes for evaluation of cellular immune responses. The results showed that the rBCG-Rv3133c was able to induce higher levels of antibody titer, stronger proliferative responses and higher IFN-gamma production comparing with BCG vaccine. The results also suggested that this recombinant vaccine was a more efficacious tuberculosis vaccine for further study.
Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; genetics ; immunology ; BCG Vaccine ; immunology ; Bacterial Proteins ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Interferon-gamma ; immunology ; Mice ; Mice, Inbred BALB C ; Mycobacterium tuberculosis ; immunology ; Protein Kinases ; genetics ; immunology ; Recombinant Proteins ; immunology ; T-Lymphocyte Subsets ; immunology ; Tuberculosis ; prevention & control ; Vaccination ; Vaccines, Synthetic ; immunology

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

OBJECTIVETo investigate the treatment and prognosis of the patients with oral mucosal melanoma (OMM).

METHODSThe clinicopathological and follow-up data of patients with OMM in Sun Yat-sen University Cancer Center from January 1976 to December 2005 were analyzed retrospectively.

RESULTSFifty-one cases were analyzed. The pathological lymph node metastasis rate was 61% (31/51) and the affected sites were confined to level I(b)-III (94%). The overall three year and five yearsurvival rates were 35% and 21% respectively. No significant difference of three year and five year survival rates were found between the group of incisional biopsy and the group of excisional biopsy. The prognosis was not affected by pigmentation. The survival rate of the patients receiving surgery combined with biotherapy or biochemotherapy was significantly higher than that of the patients treated by other modalities (P = 0.003).

CONCLUSIONSIn patients with OMM, lymph node metastasis was mostly confined to level I(b)-III. Incisional biopsy and pigmentation were not associated with an unfavorable prognosis. The prognosis of the patients with OMM was poor and the patients may get a better prognosis by receiving surgery combined with biotherapy or biochemotherapy.

Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; BCG Vaccine ; therapeutic use ; Combined Modality Therapy ; Female ; Follow-Up Studies ; Humans ; Interferon-gamma ; therapeutic use ; Interleukin-2 ; therapeutic use ; Lung Neoplasms ; secondary ; Lymph Node Excision ; Lymphatic Metastasis ; Male ; Melanoma ; drug therapy ; pathology ; surgery ; Melanoma-Specific Antigens ; metabolism ; Middle Aged ; Mouth Mucosa ; pathology ; surgery ; Mouth Neoplasms ; drug therapy ; pathology ; surgery ; Retrospective Studies ; S100 Proteins ; metabolism ; Survival Rate

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

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

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