Objective The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains is exacerbating the global burden of tuberculosis (TB), highlighting the urgent need for new treatment strategies for TB. Methods The recombinant adenovirus vaccine expressing cyclic di-adenosine monophosphate (c-di-AMP) phosphodiesterase B (CnpB) (rAd-CnpB), was administered to normal mice via mucosal immunization, either alone or in combination with drug therapy, to treat Mtb respiratory infections in mice.Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of antibodies in serum and bronchoalveolar lavage fluid (BALF). Real-time quantitative PCR was performed to assess the transcription levels of cytokines interferon γ(IFN-γ) and interleukin 10(IL-10) in mouse lungs. Flow cytometry was used to determine the proportions of CD4⁺ and CD8⁺ T cell subsets in the lungs and spleens. ELISA was employed to measure the levels of cytokines IFN-γ, IL-2, IL-10, inflammatory factors IL-6, and tumor necrosis factor α (TNF-α) secreted by spleen cells following antigen stimulation. The bacteria loads in the lungs and spleens of Mtb-infected mice were enumerated by plate counting methods. Resluts Intranasal immunization with rAd-CnpB induced high titers of IgG in mouse serum and the production of IgG and IgA in BALF, along with alterations in T lymphocyte subsets in the lungs and spleens. Administration of rAd-CnpB, either alone or in combination with drugs, to Mtb-infected mice significantly increased serum IgG levels as well as IgA and IgG levels in BALF. rAd-CnpB immunization promoted the secretion of CnpB-specific cytokines and inflammatory factors by splenocytes in Mtb-infected mice. However, rAd-CnpB immunotherapy, either alone or combined with drugs, did not significantly affect the bacterial loads in the lungs and spleens of mice with Mtb respiratory infections. Conclusion Mucosal immunization with rAd-CnpB induced significant mucosal, humoral and cellular immune responses in mice, and significantly enhanced CnpB-specific cellular immune responses in Mtb-infected mice.
Animals ; Adenoviridae/immunology* ; Mycobacterium tuberculosis/genetics* ; Mice ; Female ; Phosphoric Diester Hydrolases/genetics* ; Tuberculosis Vaccines/administration & dosage* ; Tuberculosis/prevention & control* ; Mice, Inbred BALB C ; Cytokines ; Lung/microbiology* ; Immunization ; Bronchoalveolar Lavage Fluid/immunology* ; Immunity, Mucosal

Objectives: To evaluate the immunogenicity of Methods: Protein extracts from Results: Immunization with Conclusion This is the advanced study to investigate the immunogenicity of
Animals ; Antibodies, Bacterial/immunology* ; Antigens, Bacterial/immunology* ; Bacterial Proteins/immunology* ; Cross Reactions ; Cytokines/immunology* ; Female ; Genome, Bacterial ; Immunoglobulin G/immunology* ; Immunoglobulin M/immunology* ; Macrophages/immunology* ; Mice, Inbred BALB C ; Mycobacterium avium Complex/immunology* ; Mycobacterium tuberculosis/immunology* ; Tuberculosis Vaccines/administration & dosage* ; Whole Genome Sequencing

Objective The aim of this study was to evaluate the diagnostic performance of T-SPOT.TB for tuberculous lymphadenitis. Methods Suspected tuberculous lymphadenitis patients between September 2010 and September 2018 who had both peripheral blood T-SPOT.TB test and lymph node biopsy were retrospectively enrolled in this study. The cutoff value of T-SPOT.TB test for peripheral blood was set as 24 spot forming cell (SFC)/10 ⁶ periphreral blood monocyte cell (PBMC) according to the instruction of testing kits. The gold standard for diagnosis of TBL was the combination of microbiology results, histopathology results and patient's response to anti-TB treatment. Diagnostic efficacy of T-SPOT.TB was evaluated, including sensitivity, specificity, accuracy, predictive values, and likelihood ratio. Results Among 91 patients who met the inclusion criteria, we excluded 8 cases with incomplete clinical information and 6 cases who lost to follow-up. According to the gold standard, there were 37 cases of true TBL (9 confirmed TBL and 28 probable TBL), 30 cases of non-TBL, and 10 cases of clinically indeterminate diagnosis who were excluded from the final analyses. The T-SPOT.TB tests yielded 43 cases of positive response and 24 cases of negative response. The sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR) and negative likelihood ratio (NLR) of peripheral blood T-SPOT.TB for diagnosing TBL were 89.2%, 66.7%, 79.1%, 76.7%, 83.3%, 2.68 and 0.16, respectively. The number of SFCs of T-SPOT.TB in TBL patients [432(134-1264)/10 ⁶ PBMCs] was higher than that in non-TBL patients [0 (0-30) /10 ⁶PBMCs] with a significant difference (Z=-5.306, P <0.001). Conclusion T-SPOT.TB is a rapid and simple diagnostic test for TBL with a high sensitivity and negative predictive value.
Adolescent ; Adult ; Aged ; Female ; Humans ; Interferon-gamma Release Tests ; Male ; Middle Aged ; Mycobacterium tuberculosis/physiology* ; T-Lymphocytes/immunology* ; Tuberculosis, Lymph Node/diagnosis* ; Young Adult

Tuberculosis is a chronic infectious disease caused by Mycobacterium (M.) tuberculosis. Innate immunity plays an important role in the response to M. tuberculosis. Toll-like receptors (TLRs) are important pattern recognition receptors in innate immunity. TLRs serve as switches that play decisive roles in identifying pathogens-related components. Previous studies found that TLR1, TLR2, TLR4, TLR9 were essential to promote the development of innate immune responses. The SNPs of rs4833095, rs5743618, rs3923647 of TLR1, rs57473708, rs3804099 of TLR2 and rs352139, rs5743836 of TLR9 were closely related to the susceptibility of tuberculosis in some populations. And there appeared certain relationship between the polymorphisms of TLR3, TLR6, TLR7, TLR8, TLR10 and the susceptibility of tuberculosis. The normal function of TLRs ensures the body's normal immune response to M. tuberculosis. The diversity of TLRs genes allows different individuals to respond differently to the same pathogen. Studies targeting on the relationship between single nucleotide polymorphism in TLRs and susceptibility to tuberculosis can predict the susceptibility to tuberculosis in some populations, as well as discover new drugs targets.
Genetic Predisposition to Disease ; Humans ; Mycobacterium tuberculosis ; Polymorphism, Single Nucleotide ; Research/trends* ; Signal Transduction/immunology* ; Toll-Like Receptors/genetics* ; Tuberculosis/immunology*

Antigen Rv2628 of Mycobacterium tuberculosis is associated with latent tuberculosis infection. In this study, Rv2628 was prokaryotic expressed and purified, its immunological characteristics was evaluated with macrophage cell line RAW264.7 and BALB/c mice. The results show that Rv2628 was mainly expressed in form of inclusion body confirmed by SDS-PAGE, and could react with rabbit anti-H37Rv polyclonal antibody detected by Western blotting assay, indicating that the protein had an effective immunoreactivity. The interactions between Rv2628 and macrophage cell line RAW264.7 confirmed that it could effectively induce cells to produce pro-inflammatory cytokines, the relative expression level of IL-6 mRNA was higher than the control group in 1-12 h. BALB/c mice were subcutaneously immunized with Rv2628 protein, the production of IFN-gamma and IL-4 in the spleen cells was determined by Sandwich ELISA, in the Rv2628 immunized group, the level of IFN-gamma was significantly higher than that of IL-4 (P < 0.000 1). It indicated the protein induced Th1-tendency immune responses. At the same time, Rv2628(11-30) peptide used as coating antigen, the murine serum antibody titer detected by indirect-ELISA was 1:1 600, which demonstrated that Rv2628 could also induce humoral immune responses. In summary, Rv2628 could induce specific pro-inflammatory cytokines, affectively induce strongly Th1-tendency immune response and humoral response, it could be a potential target for developing subunit vaccine against TB. In addition, it laid foundation for probing the cross-talk between M. tb and host.
Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; immunology ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Interferon-gamma ; immunology ; Interleukin-4 ; immunology ; Interleukin-6 ; immunology ; Macrophages ; immunology ; Mice ; Mice, Inbred BALB C ; Mycobacterium tuberculosis ; Th1 Cells ; immunology ; Tuberculosis ; immunology

OBJECTIVETo investigate the mechanism of Mycobacterium tuberculosis invasion to mouse dendritic cells (DC).

METHODSMycobacterium tuberculosis strain H37Rv was co-cultured with mouse DC2.4 cells.The mRNA expression of Toll-like receptor 2/4(TLR2/4) in DC2.4 cells was detected by fluorescent quantitative real-time PCR and the protein expression of nuclear factor κB(NF-κB) was assessed by Western blotting.The extracellular concentration of tumor necrosis factor α(TNF-α) was measured by ELISA methods during Mycobacterium Tuberculosis invasion.Indirect immunofluorescent staining and flow cytometry assay were used to detect the expression of CD80 and CD86 on DC2.4 cells before and after invasion.

RESULTSThe invasion of Mycobacterium tuberculosis in DC2.4 cells was observed after 2 h of co-incubation.The rates of invasion were (37.9±5.6)%,(51.2±7.6)%,(57.2±8.9)% and(63.9±6.8)% at 6,8,10 and 12 h after co-incubation,respectively.The mRNA expression level of TLR2 /4 was significantly increased at 6 h but decreased at 10 h after co-incubation.The expressions of NF-κB p65 and TNF-α were higher in DC2.4 cells after being invaded by 6,8,and 10 h and then gradually decreased.CD80 and CD86 expression were increased on DC2.4 at 6 h after co-incubation.

CONCLUSIONInvasion of Mycobacterium tuberculosis strain H37Rv to DC might enhance its antigen-presenting function through activation of TLR2/4-NF-kB signaling pathway.

Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; Mice ; Mycobacterium tuberculosis ; NF-kappa B ; metabolism ; Signal Transduction ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

PURPOSE: Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. MATERIALS AND METHODS: The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. RESULTS: The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7x10(4) CFU/mL and 2.0x10(6) CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). CONCLUSION: The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.
Antigens, Bacterial/*analysis/immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Mycobacterium tuberculosis/*immunology

OBJECTIVETo detect the expression of Mycobacterium tuberculosis secreted protein Ag85B in paraffin-embedded tissues by immunohistochemistry (IHC), and to evaluate its application in the pathological diagnosis of tuberculosis.

METHODSOne hundred and five tuberculosis specimens (54 pulmonary tuberculosis, 51 lymph nodal tuberculosis) and 51 specimens of other diseases (8 lung cancer, 10 pulmonary abscess, 10 bronchiectasis, 7 lymphoma, 5 necrotizing lymphadenitis, 4 reactive hyperplasia lymphoid, and 7 sarcoidosis) were collected from January 2012 to July 2013 from Beijing Chest Hospital, Capital Medical University. One-step IHC was performed on paraffin-embedded tissues using antibody directed against Ag85B.

RESULTSIHC and Ziehl-Neelsen (ZN) acid-fast staining showed that distribution and intensity of Ag85B expression were concordant with the distribution and number of acid-fast bacilli. IHC showed significantly higher sensitivity than ZN staining (50.5%, 53/105 vs. 31.4%, 33/105; χ² = 7.877, P = 0.005). The combined sensitivity of IHC and ZN staining was 59.0%. Moreover, oil immersion was not necessary for IHC, allowing more rapid diagnosis.

CONCLUSIONIHC detection of Ag85B is a simple method with higher sensitivity than ZN staining, and demonstrated good value in the pathological diagnosis of tuberculosis.

Acyltransferases ; metabolism ; Antigens, Bacterial ; metabolism ; Biomarkers ; metabolism ; Bronchiectasis ; diagnosis ; immunology ; Humans ; Immunohistochemistry ; Lymphadenitis ; diagnosis ; immunology ; Mycobacterium tuberculosis ; immunology ; Sarcoidosis ; diagnosis ; Staining and Labeling ; Tuberculosis, Lymph Node ; diagnosis ; immunology ; Tuberculosis, Pulmonary ; diagnosis ; immunology

As the first line of immune defense for Mycobacterium tuberculosis (Mtb), macrophages also provide a major habitat for Mtb to reside in the host for years. The battles between Mtb and macrophages have been constant since ancient times. Triggered upon Mtb infection, multiple cellular pathways in macrophages are activated to initiate a tailored immune response toward the invading pathogen and regulate the cellular fates of the host as well. Toll-like receptors (TLRs) expressed on macrophages can recognize pathogen-associated-molecular patterns (PAMPs) on Mtb and mediate the production of immune-regulatory cytokines such as tumor necrosis factor (TNF) and type I Interferons (IFNs). In addition, Vitamin D receptor (VDR) and Vitamin D-1-hydroxylase are up-regulated in Mtb-infected macrophages, by which Vitamin D participates in innate immune responses. The signaling pathways that involve TNF, type I IFNs and Vitamin D are inter-connected, which play critical roles in the regulation of necroptosis, apoptosis, and autophagy of the infected macrophages. This review article summarizes current knowledge about the interactions between Mtb and macrophages, focusing on cellular fates of the Mtb-infected macrophages and the regulatory molecules and cellular pathways involved in those processes.
Animals ; Apoptosis ; Autophagy ; Humans ; Interferon Type I ; metabolism ; Macrophages ; immunology ; metabolism ; Mycobacterium tuberculosis ; physiology ; Receptors, Calcitriol ; metabolism ; Steroid Hydroxylases ; metabolism ; Toll-Like Receptors ; metabolism ; Tuberculosis ; immunology ; metabolism ; pathology ; Tumor Necrosis Factors ; metabolism

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