OBJECTIVE: To study the clinical value of detecting carcinoembryonic antigen levels in pleural effusion (PCEA) and serum (SCEA) and their ratio (P/S) in the differential diagnosis of pleural effusions resulting from tuberculosis and lung cancer. METHODS: This retrospectively study was conducted among 82 patients with pleural effusion caused by pulmonary tuberculous (TB; control group) and 120 patients with pleural effusion resulting from lung cancer in our hospital between April, 2016 and March, 2018. PCEA, SCEA and P/S were compared between the two groups and among the subgroups of lung cancer patients with squamous cell carcinoma (SqCa), adenocarcinoma (ACA), small cell carcinoma (SCLC). The receiveroperating characteristic curve (ROC) analysis was used to confirm the optimal critical value to evaluate the diagnostic efficiency of different combinations of PCEA, SCEA and P/S. RESULTS: PCEA, SCEA and P/S were significantly higher in the overall cancer patients and in all the 3 subgroups of cancer patients than in the patients with TB ( < 0.05). The areas under the ROC curve of PCEA, SCEA and P/S were 0.925, 0.866 and 0.796, respectively; PCEA had the highest diagnostic value, whose diagnostic sensitivity, specificity, accurate rate, and diagnostic threshold were 83.33%, 96.34, 88.61%, and 3.26 ng/ml, respectively; SCEA had the lowest diagnostic performance; the diagnostic performance of P/S was between that of SCEA and PCEA, but its combination with SCEA greatly improved the diagnostic performance and reduced the rates of misdiagnosis and missed diagnosis. Parallel tests showed that the 3 indexes combined had significantly higher diagnostic sensitivity than each or any two of the single indexes ( < 0.05), but the diagnostic specificity did not differ significantly. The area under the ROC curve of combined detections of the 3 indexes was 0.941 for diagnosis of lung cancer-related pleural effusion, higher than those of any other combinations of the indexes. CONCLUSIONS The combined detection of PCEA, SCEA and P/S has a high sensitivity for diagnosis of lung cancer-related pleural effusion and provides important information for rapid and accurate diagnosis of suspected cases.
Carcinoembryonic Antigen ; analysis ; blood ; Case-Control Studies ; Diagnosis, Differential ; Humans ; Lung Neoplasms ; blood ; complications ; Pleural Effusion ; blood ; diagnosis ; immunology ; Pleural Effusion, Malignant ; blood ; chemistry ; diagnosis ; ROC Curve ; Retrospective Studies ; Sensitivity and Specificity ; Tuberculosis, Pulmonary ; complications

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

BACKGROUND/AIMS: The goal of this study was to monitor tuberculosis (TB)-specific T-cell responses in cerebrospinal fluid-mononuclear cells (CSF-MCs) and peripheral blood mononuclear cells (PBMCs) in patients with tuberculous meningitis (TBM) over the course of anti-TB therapy. METHODS: Adult patients (> or = 16 years) with TBM admitted to Asan Medical Center, Seoul, South Korea, were prospectively enrolled between April 2008 and April 2011. Serial blood or CSF samples were collected over the course of the anti-TB therapy, and analyzed using an enzyme-linked immunosorbent spot (ELISPOT) assay. RESULTS: Serial ELISPOT assays were performed on PBMCs from 17 patients (seven definite, four probable, and six possible TBM) and CSF-MC from nine patients (all definite TBM). The median number of interferon-gamma (IFN-gamma)-producing T-cells steadily increased during the first 6 months after commencement of anti-TB therapy in PBMCs. Serial CSF-MC ELISPOT assays revealed significant variability in immune responses during the first 6 weeks of anti-TB therapy, though early increases in CSF-MC ELISPOT results were associated with treatment failure or paradoxical response. CONCLUSIONS: Serial analysis of PBMCs by ELISPOT during the course of treatment was ineffective for predicting clinical response. However, increases in TB-specific IFN-gamma-producing T-cells in CSF-MC during the early phase of anti-TB therapy may be predictive of clinical failure.
Adult ; Antitubercular Agents/therapeutic use ; Biological Markers/blood/cerebrospinal fluid ; *Enzyme-Linked Immunospot Assay ; Female ; Humans ; Interferon-gamma/blood/cerebrospinal fluid ; *Interferon-gamma Release Tests ; Kinetics ; Male ; Middle Aged ; Predictive Value of Tests ; Prospective Studies ; Republic of Korea ; T-Lymphocytes/drug effects/*immunology/metabolism/microbiology ; Treatment Outcome ; Tuberculosis, Meningeal/blood/cerebrospinal fluid/*diagnosis/drug therapy/immunology/microbiology

OBJECTIVETo construct a Mycobacterium tuberculosis DNA vaccine pVAX1/ESAT-6 plasmid and investigate its immunoreactivity.

METHODSThe ESAT-6 gene fragment amplified from Mycobacterium tuberculosis genome was inserted into pVAX1 vector to construct the recombinant plasmid pVAX1/ESAT-6, which was identified by restriction enzyme digestion and sequencing. The recombinant plasmid was transformed into Hela cells using Sofast® Transfection reagent, and the cellular expressions of ESAT-6 mRNA and protein were analyzed by RT-PCR and immunofluorescence assay, respectively. The recombinant plasmid pVAX1/ESAT-6 was also transfected into mouse by electronic pulse method, and the mouse serum IFN-γ level and anti-ESAT-6 IgG antibody level were detected by ELISA, mouse lymphocyte proliferation assessed with flow cytometry, and IFN-γ-secreting lymphocytes counted using ELISPOT.

RESULTSDouble restriction-enzyme digestion and sequencing showed that the inserted fragment in the recombinant plasmid pVAX1/ESAT-6 was identical to ESAT-6 gene with an inframe insertion. RT-PCR yielded the target band as expected on agarose gel, and immunofluorescence assay of the transfected cells showed specific green fluorescence signals. The mice transfected with the recombinant plasmid showed significantly elevated serum level of anti-ESAT-6 IgG antibody and increased serum IFN-γ level, spleen cell proliferation, and number of IFN-γ-secreting lymphocytes.

CONCLUSIONThe Mycobacterium tuberculosis DNA vaccine pVAX1/ESAT-6 plasmid we constructed can induce high levels of cellular and humoral immunoreactivity in mice.

Animals ; Antibodies, Bacterial ; blood ; Antibody Formation ; Antigens, Bacterial ; immunology ; Bacterial Proteins ; immunology ; Female ; Genetic Vectors ; HeLa Cells ; Humans ; Immunity, Cellular ; Immunity, Humoral ; Immunoglobulin G ; blood ; Interferon-gamma ; blood ; Mice ; Mice, Inbred BALB C ; Mycobacterium tuberculosis ; immunology ; Plasmids ; immunology ; Tuberculosis Vaccines ; genetics ; immunology ; Vaccines, DNA ; genetics ; 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

OBJECTIVETo screen the specific antibody binding peptides of tuberculosis from phage-displayed random phage display (Ph.D.)-7 peptide libraries with purified IgG from tuberculosis serum, and to provide the basis for the development of serological detection reagent of tuberculosis.

METHODSPurified IgG of tuberculosis serum was used as solid ligand to screen the binding peptide from the Ph.D.-7 peptide library, according to the biopanning process of absorption, elution and amplification. Purified IgG of health people serum was used as the molecule of counter selection during the second and third selection. Phages were enriched after 3 rounds of screening, then 20 single phages separately eluted by IgG of tuberculosis and health people were randomly selected on each direction of the determination plates and amplified. The single chains DNA were extracted as template for sequencing. The combination abilities of selected clones to IgG of tuberculosis and health people were tested by indirect enzyme linked immunosorbent assay (ELISA), and the positive clone was identified. Serum samples, from 47 patients with tuberculosis and 37 healthy people vaccinated with BCG, were verified by positive phage clones using phage-ELISA. The verifying results of 24 serum samples used for panning were separately analyzed statistically.

RESULTSAfter 3 rounds of panning, remarkable enrichment of phages that could specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 12 sequences were obtained. 12 phage clones with different sequences were amplified and detected with indirect ELISA and single phage H12 showed higher affinity with IgG of tuberculosis (S/N ≥ 2.1) and was identified as the positive clone. It was found that, in indirect ELISA with single Phage H12, the A(450) value of tuberculosis patients (0.105 ± 0.010) was significantly higher than that of healthy individuals (0.070 ± 0.005), and the t value was 2.912 (P < 0.0001). The A(450) value of 12 serum samples of tuberculosis patients and 12 samples of health individuals used for panning were 0.144 ± 0.016 and 0.052 ± 0.004, and the t value was 5.69 (P < 0.0001).

CONCLUSIONBy using phage-displayed random peptide libraries, we obtained the specific antibody binding peptides of tuberculosis, which showed specific binding activity with IgG of tuberculosis. It can provide a basis for the establishment of a new serological detection method of tuberculosis with peptide.

Adult ; Case-Control Studies ; Female ; Humans ; Immunoglobulin G ; blood ; Male ; Middle Aged ; Molecular Sequence Data ; Mycobacterium tuberculosis ; immunology ; Peptide Library ; Peptides ; immunology ; Tuberculosis ; diagnosis ; immunology ; microbiology ; Young Adult

PURPOSE: Bacillus Calmette-Guerin (BCG) vaccine has widely been used to immunize against tuberculosis, but its protective efficacy is variable in adult pulmonary tuberculosis, while it is not efficiently protective against progressive infection of virulent Mycobacterium tuberculosis strains. In this study, the protective effects of plasmid DNA vaccine constructs encoding IL-12 or IL-18 with the BCG vaccine were evaluated against progressive infection of M. tuberculosis, using mouse aerosol challenge model. MATERIALS AND METHODS: Plasmid DNA vaccine constructs encoding IL-12 or IL-18 were constructed and mice were immunized with the BCG vaccine or with IL-12 DNA or IL-18 DNA vaccine constructs together with the BCG vaccine. RESULTS: The BCG vaccine induced high level of interferon gamma (IFN-gamma) but co-immunization of IL-12 or IL-18 DNA vaccine constructs with the BCG vaccine induced significantly higher level of IFN-gamma than a single BCG vaccine. The BCG vaccine was highly protective at early stage of M. tuberculosis infection, but its protective efficacy was reduced at later stage of infection. The co-immunization of IL-12 DNA vaccine constructs with the BCG vaccine was slightly more protective at early stage of infection and was significantly more protective at later stage infection than a single BCG vaccine. CONCLUSION: Co-immunization of IL-12 DNA vaccine with the BCG vaccine induced more protective immunity and was more effective for protection against progressive infection of M. tuberculosis.
Animals ; BCG Vaccine/*immunology ; Female ; Immunoenzyme Techniques ; Interferon-gamma/blood ; Interleukin-12/*genetics ; Interleukin-18/*genetics ; Mice ; Mice, Inbred C57BL ; Plasmids/genetics ; Tuberculosis/blood/*immunology/prevention & control ; Vaccines, DNA/*genetics/*immunology

OBJECTIVETo establish a recombinant plasmid of CFP32 of Mycobacterium tuberculosis in E. coli, and to analyze its antigenicity.

METHODSRv0577 gene was amplified by polymerase chain reaction from genome of Mycobacterium tuberculosis, and then cloned into vector pMD18-T followed by the subclone into the expression vector pET21a. Recombinant CFP32 was expressed and purified. The antigenicity of the recombinant protein was analyzed by using Western-blot. The purified recombinant CFP32 protein was used as an antigen to screen the sera of 7 pulmonary TB patients (n = 97), as well as the other pulmonary disease patients (n = 25), and the clinically healthy controls (n = 38) by ELISA.

RESULTSRecombinant plasmid of CFP32 was established, and be expressed efficiently in E. coli BL21 (DE3). The relative molecular mass of the protein was about 300,000 by SDS-PAGE analysis. The protein purified by Ni-NTA was in a purity over 90%, which was confirmed by Western-blot analysis. ELISA analysis showed its sensitivity and specificity were 63.9% (62/97) and 96.8% (2/63) respectively.

CONCLUSIONThe recombinant expression plasmid pET21a CFP32 has been constructed and CFP32 proteins has been successfully expressed and be purified in E. coli and, ELISA analysis has identified the recombinant CFP32 as a candidate antigen for TB serodiagnosis.

Antigens, Bacterial ; blood ; Bacterial Proteins ; genetics ; immunology ; Cloning, Molecular ; Escherichia coli ; Gene Expression ; Humans ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; Plasmids ; Recombinant Proteins ; Serologic Tests ; Tuberculosis, Pulmonary ; diagnosis ; microbiology

This study is to investigate the effects of paeoniflorin (Pae) on the levels of related serum antibodies and cAMP of splenocytes in rats with adjuvant arthritis. Complete Freund's adjuvant was used to induce AA in rats. The level of circulating immune complexes in serum was determined by PEG6000 assay, and the levels of anti-C II antibody and anti-TB antibody in serum were measured by enzyme-linked immunosorbent assay (ELISA), the level of cAMP in splenocytes was measured by radioimmunoassay, separately. Pae (25, 50, and 100 mg x kg(-1)) and GTW (40 mg x kg(-1)) were given by intragastric administration for 7 days from the 17th day after immunization. Pae (50 and 100 mg x kg(-1)) reduced the levels of circulating immune complexes, anti-C II antibody and anti-TB antibody in serum in rats with adjuvant arthritis. The inhibition ratios of Pae groups to AA model group were dosage-dependent; Pae (12.5, 62.5, and 312.5 mg x L(-1)) decreased the elevated levels of cAMP in splenocytes in vitro. Pae (ig) decreased the levels of related serum antibodies and elevated the level of cAMP in rats with adjuvant arthritis.
Animals ; Antibodies, Bacterial ; blood ; Antigen-Antibody Complex ; blood ; Arthritis, Experimental ; chemically induced ; immunology ; Benzoates ; isolation & purification ; pharmacology ; Bridged-Ring Compounds ; isolation & purification ; pharmacology ; Cyclic AMP ; metabolism ; Dose-Response Relationship, Drug ; Freund's Adjuvant ; Glucosides ; isolation & purification ; pharmacology ; Isoantibodies ; blood ; Monoterpenes ; Mycobacterium tuberculosis ; immunology ; Paeonia ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spleen ; cytology ; metabolism

Regulatory T cells, which stimulate or inhibit the effector functions of distinct T cell subsets, are critical in the control of the immune response. We investigated the effect of TGF-beta and IL-10 on T cell subsets according to the Th1/Th2 immune status. Sixty-two patients with asthma and 38 patients with pulmonary tuberculosis were included. Allergy skin tests, tuberculin tests, and chest radiography were performed. The levels of circulating IL-4, IFN-gamma, TGF-beta1, and IL-10 were measured using ELISA. The level of TGF-beta1 was higher in patients with asthma than in those with tuberculosis, but the IL-10 levels were the same between the asthma and tuberculosis groups. Atopy was unrelated to the tuberculin response. The IFN-gamma level was correlated with the IL-10 level, and the level of IL-4 was unrelated to the IL-10 or TGF-beta1 level. The level of IL-10 was higher in the negative tuberculin reactors than in the positive tuberculin reactors among patients with asthma, and TGF-beta1 was higher in the positive tuberculin reactors than in the negative tuberculin reactors among patients with tuberculosis. These results demonstrate that the regulatory effects of circulating TGF-beta and IL-10 on T cell cytokines may be different between Th2-type asthma and Th1 tuberculosis.
Adult ; Asthma/*blood/immunology ; Cytokines/*blood ; Female ; Humans ; Interferon Type II/blood ; Interleukin-10/blood ; Interleukin-4/blood ; Male ; Research Support, Non-U.S. Gov't ; Respiratory Function Tests ; Skin Tests ; Th1 Cells/*metabolism ; Th2 Cells/*metabolism ; Transforming Growth Factor beta/blood ; Tuberculin Test ; Tuberculosis/*blood/immunology