肌肉注射用M.Vaccae菌苗毒副反应的研究

对WHO推荐的皮内注射Vaccae菌苗，经改进研制出肌肉注射剂型，并进行动物模型的全身过敏性试验、局部肌肉刺激试验、急性毒性试验以及热原试验等毒副反应观察，实验结果证实，该菌苗均无异常反应。

Effects of Mycobacterium vaccae vaccine in a mouse model of tuberculosis: protective action and differentially expressed genes

Background:Tuberculosis is a leading cause of death worldwide.BCG is an effective vaccine,but not widely used in many parts of the world due to a variety of issues.Mycobacterium vaccae(M.vaccae)is another vaccine used in human subjects to prevent tuberculosis.In the current study,we investigated the potential mechanisms of M.vaccae vaccination by determining differentially expressed genes in mice infected with M.tuberculosis before and after M.vaccae vaccination.Methods:Three days after exposure to M.tuberculosis H37 Rv strain(5×10~5 CFU),adult BALB/c mice randomly received either M.vaccae vaccine(22.5μg)or vehicle via intramuscular injection(n=8).Booster immunization was conducted 14 and 28 days after the primary immunization.Differentially expressed genes were identified by microarray followed by standard bioinformatics analysis.Results:M.vaccae vaccination provided protection against M.tuberculosis infection(most prominent in the lungs).We identified 2,326 upregulated and 2,221 downregulated genes in vaccinated mice.These changes could be mapped to a total of 123 signaling pathways(68 upregulated and 55 downregulated).Further analysis pinpointed to the MyD88-dependent TLR signaling pathway and PI3 K-Akt signaling pathway as most likely to be functional.Conclusions:M.vaccae vaccine provided good protection in mice against M.tuberculosis infection,via a highly complex set of molecular changes.Our findings may provide clue to guide development of more effective vaccine against tuberculosis.

Nebulized Mycobacterium vaccae protects against asthma by attenuating the imbalance of IRF4/IRF8 expression in dendritic cells

Objective:To assess the effects of nebulized inhaled Mycobacterium vaccae on allergic airway inflammation,airway hyperresponsiveness,and Th1/Th2 cell imbalance in mice with ovalbumin(OVA)-induced asthma.Methods:Mice received OVA sensitization and challenge for establishment of the asthmatic model.For intervention,mice received Mycobacterium vaccae nebulization once every other day from the first day of sensitization to the day before challenge.After challenge,pulmonary histological analysis and airway responsiveness measurement were performed.In addition,Th1/Th2 cytokines and OVA-specific IgE levels in bronchoalveolar lavage fluid were measured by ELISA.Th1/Th2 subset ratios and the expression of interferon-regulatory factor 4(IRF4),IRF8 and Toll-like receptor 4(TLR4)in dendritic cells were evaluated by flow cytometry.Results:Severe inflammatory infiltration and airway hyperresponsiveness were observed in OVA-induced asthmatic mice.Asthmatic mice showed higher Th2 cytokine concentration and increased percentage of Th2 cells,along with lower Th1 cytokine concentration and reduced percentage of Th1 cells compared with the normal control.Moreover,an imbalance of IRF4^(+)and IRF8^(+)in dendritic cells was found in asthmatic mice.Nebulized inhaled Mycobacterium vaccae reduced airway hyperresponsiveness and inflammation in OVA-induced asthmatic mice.In addition,nebulized inhaled Mycobacterium vaccae enhanced TLR4 and IRF8 expression,and alleviated the imbalance of Th1/Th2 as well as IRF4^(+)and IRF8^(+)in dendritic cells.Conclusions:Nebulized inhaled Mycobacterium vaccae protects against asthma by alleviating the imbalance of Th1/Th2 and IRF4/IRF8 in OVA-induced asthmatic mice.

<i>Mycobacterium vaccae</i>Immunization to Pregnant BALB/c Mice Ameliorated Lung Histopathology and Bone Marrow Eosinophila in Ovalbumin Sensitized Offsprings

Context: One of the treatment strategies for atopic diseases is to skew immune response away from Th2 dominance by using Mycobacterial strains. Objective: We wanted to find out whether M. vaccae administration to pregnant mice had any preventive effect on the offsprings in the development of a murine asthma model. Materials and Methods: Pregnant BALB/c mice were divided into two groups;first group received heat-killed M. vaccae subcutaneously on 12th day of pregnancy and the latter group received PBS. After delivery, newborn mice of each group were further divided into two subgroups as M. vaccae/Ovalbumin (OVA), M. vaccae/control, PBS/OVA and PBS/ control. To establish experimental murine asthma model, mice were intraperitoneally sensitized and challenged intratracheally with Ovalbumin. We analysed airway histopathology, bone marrow eosinophil progenitors and splenic cell cytokine profiles of the offsprings. Results: Comparison of offsprings in M. vaccae/OVA group were not different than PBS controls with respect to thicknesses of airway epithelium, basement membrane, subepithelial smooth muscles and number of hyperplasic goblet cells as well as bronchial associated lymphoid tissue density and eosinophil progenitors in the bone marrow. Comparison of M. vaccae/OVA group to asthma model revealed significant differences and lower levels of OVA-induced IL-5. Conclusions: We propose that immunization of pregnant BALB/c with M. vaccae could prevent histopathological alterations in the airways related to the asthma model and down-regulates IL-5 secretion from splenocytes of offsprings.

Immunotherapeutical Potential of Mycobacterium Vaccae on M. Tuberculosis Infection in Mice

Tuberculosis remains the worldwide infectious disease. To identify the therapeutic potential of M. vaccae in treating tuberculosis, M. vaccae was injected into Mycobacterium tuberculosis （M. tuberculosis） infected mice. The optimal dose of M. vaccae （22.5 μg/mouse） treated mice showed lower pathological change index, spleen weight index, lung weight index and vital M. tuberculosis count than those of the untreated group. Treatment with M. vaccae enhanced the percentages of CD3^＋ and CD4^＋ T cells, IFN-γ^＋CD4^＋ T cells, innate immune cells including NK cells, NK1.1^＋ T cells and γδ T cells, and reduced the percentage of IL-4^＋CD4^＋ T cells. Therefore, M. vaccae could protect the mice from M. tuberculosis infection and improved mouse innate and adaptive cell-mediated immunity, suggesting that M. vaccae is a potential immunotherapeutic agent in pulmonary tuberculosis. Cellular ＆ Molecular Immunology.

Expression,purification and characterization of Mycobacterium tuberculosis RpfE protein

Resuscitation promoting factor E （RpfE） is one of the five Rpf-like proteins in Mycobacterium tuberculos＆ （M. tuberculosis）. These Rpf-like proteins are secretory, which make them candidates for recognition by the host immune system. In this study, the RpfE gene was amplified from M. tuberculosis, cloned into the expression vectors pDE22 and pPRO EXHT, and were expressed in Mycobacterium vaccae （M. vaccae） and Escherichia coli DHSa, respec- tively. Both recombinant RpfE proteins were purified by Ni-Sepharose affinity chromatography, and were given to C57BL/6 mice. The RpfE proteins elicited T cell proliferation, and stimulated the production of gamma interferon （IFN-y）, interleukin-10 （IL-10） and IL-12. Our results indicated that the RpfE protein expressed in M. vaccae could more efficiently stimulate cellular immune response, making it a promising candidate as a subunit vaccine.