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Objective:To evaluate the expressions of three biomarkers combination of CD27, CD38 and human leucocyte antigen (HLA)-DR in the application of discrminating active tuberculosis (ATB) and latent tuberculosis infection (LTBI).Methods:Sixty cases of ATB and 44 cases of LTBI were enrolled from March 2021 to February 2022 in Huashan Hospital, Fudan University and Wuxi Fifth People′s Hospital. Freshly isolated peripheral blood mononuclear cells (PBMC) from patients were stimulated with 6 kDa early secretory antigenic target/culture filtrate protein 10 peptide pools. The expressions of CD27, CD38 and HLA-DR on Mycobacterium tuberculosis-specific CD4 + T lymphocytes were evaluated by polychromatic flow cytometry. Mann-Whitney U test was used for statistical analysis. The area under the receiver operator characteristic curve (AUROC) was used to evaluate the diagnostic value of biomarkers in discriminating ATB and LTBI. Results:The frequencies of CD27 -, CD38 +, HLA-DR +, CD27 -CD38 +, CD27 -HLA-DR + and CD38 + HLA-DR + in ATB group were all higher than those in LTBI group, and the differences were all statistically significant ( U=26.00, 451.00, 384.00, 8.00, 7.00 and 184.00, respectively, all P<0.001). The AUROC of CD27 -CD4 + interferon-γ(IFN-γ) + T lymphocytes was 0.71 with a cut-off value of 52.31%, with the sensitivity of 50.00% and specificity of 87.20%. The AUROC of CD38 + CD4 + IFN-γ + T lymphocytes was 0.82 with a cut-off value of 30.25%, with the sensitivity of 73.40% and specificity of 89.70%. The AUROC of HLA-DR + CD4 + IFN-γ + T lymphocytes was 0.85 with a cut-off value of 36.60%, with the sensitivity of 66.00% and specificity of 94.90%. The AUROC of CD27 -CD38 + CD4 + IFN-γ + T lymphocytes was 0.80 with a cut-off value of 8.82%, with the sensitivity of 90.60% and specificity of 61.50%. The AUROC of CD27 -HLA-DR + CD4 + IFN-γ + T lymphocytes was 0.83 with a cut-off value of 18.62%, with the sensitivity of 75.00% and specificity of 79.50%. The AUROC of CD38 + HLA-DR + CD4 + IFN-γ + T lymphocytes was 0.93 with a cut-off value of 22.35%, with the sensitivity of 79.70% and specificity of 100.00%. Conclusions:The expressions of CD27 -, CD38 + and HLA-DR + in Mycobacterium tuberculosis-specific CD4 + T lymphocytes are higher in ATB group compared to LTBI group. ATB and LTBI could be well discriminated by detecting the expressions of CD27, CD38 and HLA-DR on CD4 + IFN-γ + T lymphocytes with flow cytometry.
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Objective:To investigate the role of glycoprotein A repetitions predominant (GARP) in the pathogenesis of tuberculosis through regulatory T cell (Treg), in order to provide new targets for the treatment of tuberculosis.Methods:Sixty patients with active pulmonary tuberculosis (ATB) admitted to Huashan Hospital, Fudan University and Wuxi Fifth People′s Hospital from January to September 2021 were included. And six individuals with latent tuberculosis infection (LTBI), and 16 healthy controls (HC) were recruited during the same period. Flow cytometry was performed to detect the proportion of Treg in the peripheral blood, and the expressions of GARP and transforming growth factor-β1 (TGF-β1) on Treg in different groups. Mann-Whitney U test was used for statistical analysis. Results:Among the 60 patients with ATB, 23 patients did not receive anti-tuberculosis drug therapy, 17 patients were treated for less than three months, ten patients were treated for three to less than six months, and ten patients were treated for greater than or equal to six months. The percentage of CD4 + CD25 + forkhead box protein 3 (Foxp3) + Treg in untreated ATB patients was 7.50%(5.67%, 9.00%), which was higher than that in HC (5.57%(5.03%, 6.09%)), and the difference was statistically significant ( U=95.00, P=0.010). The percentage of GARP expressing in CD4 + CD25 + Foxp3 + Treg in untreated ATB patients was 10.37%(7.79%, 12.90%), which was higher than that in LTBI (7.02%(5.15%, 8.81%)) and HC (5.33%(4.26%, 6.67%)), respectively, and the differences were both statistically significant ( U=31.00, P=0.040; U=36.00, P<0.001, respectively), while there was no significant difference between LTBI and HC ( U=25.00, P=0.095). The percentage of CD4 + CD25 + Foxp3 + Treg expressing TGF-β1 in untreated ATB patients was 7.13%(4.25%, 8.89%), which was higher than that in HC (3.59%(2.10%, 5.17%)), and the difference was statistically significant ( U=71.00, P=0.001). The expressions of GARP in CD4 + CD8 -CD25 + Foxp3 + Treg in patients with ATB treated for less than three months group, three to less than six months group and greater than or equal to six months group were 7.82%(3.94%, 13.17%), 6.92%(5.61%, 9.47%) and 7.26%(5.82%, 9.64%), respectively. The expressions of TGF-β1 in CD4 + CD8 -CD25 + Foxp3 + Treg in the above three treatment groups were 11.16%(7.91%, 15.23%), 8.66%(5.43%, 12.54%) and 7.82%(6.01%, 9.53%), respectively, and the expression of TGF-β1 in CD4 + CD8 -CD25 + Foxp3 + Treg in the patients with ATB treated for less than three months group was higher than that in the greater than or equal to six months group, the difference was statistically significant ( U=37.50, P=0.024). Conclusions:Foxp3/GARP/TGF-β1 pathway may be involved in the immune mechanism of Treg regulating the pathogenesis of tuberculosis, and GARP may be a new target for anti-tuberculosis therapy.
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Objective:To investigate the diagnostic values of interleukin-22 (IL-22), interferon-γ(IFN-γ)and macrophage migration inhibition factor (MIF) in pleural effusion for tuberculosis pleurisy.Methods:From April 2018 to May 2019, a total of 77 patients including 45 cases of tuberculous pleurisy, 19 cases of malignant pleurisy, 13 cases of parapneumonia and 13 cases of healthy control in Wuxi Fifth People′s Hospital were enrolled. The levels of IL-22, IFN-γ and MIF in plasma and pleural effusion were detected by enzyme linked immunosorbent assay (ELISA). Mann-Whitney U test was used for statistical analysis.The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic values of IL-22, IFN-γ and MIF for tuberculous pleurisy. Results:The median levels of IL-22, IFN-γ, MIF and adenosine deaminase in 45 cases with pleural effusion in tuberculosis pleurisy group were 396.8 ng/L, 2 200.0 ng/L, 241.3 μg/L and 70.8 U/L, respectively, which were all significantly higher than 32 cases with non-tuberculosis pleurisy group, including 19 cases with malignant pleurisy and 13 cases with parapneumonia (52.8 ng/L, 232.3 ng/L, 179.6 μg/L and 17.0 U/L, respectively). The differences were all statistically significant ( U=179.000, 118.500, 287.000, 162.000, respectively, all P<0.05). The median levels of IL-22 and IFN-γ in plasma of tuberculosis pleurisy group were 20.0 ng/L and 45.9 ng/L, respectively, which were both higher than healthy control group (14.3 ng/L and 33.4 ng/L, respectively). The level of MIF was 96.2 μg/L, which was lower than healthy control (159.5 μg/L). The differences were all statistically significant ( U=74.000, 13.000 and 73.000, respectively, all P<0.05). The areas under ROC curve (AUC) of IL-22, IFN-γ and MIF in pleural effusion for the diagnosis of tuberculosis pleurisy were 0.876, 0.917 and 0.682, respectively.The sensitivities were 93.75%, 100.00% and 63.64%, respectively; the specificities were 82.22%, 91.11% and 65.85%, respectively. The median levels of IL-22 and IFN-γ in plasma in tuberculosis pleurisy group at two months of follow-up after anti-tuberculosis therapy were 16.0 ng/L and 33.9 ng/L, respectively, which were both lower than baseline (20.0 ng/L and 44.7 ng/L, respectively). The differences were both statistically significant ( U=2.156 and 2.221, respectively, both P<0.05). Conclusion:IFN-γ and IL-22 in pleural effusion could be used as effective indicators to identify tuberculous pleurisy, and the dynamic monitoring of IL-22 in patients′plasma could be an important biomarker in evaluating the efficacy of anti-tuberculosis treatment.
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ObjectiveTo evaluate the application of a whole blood interferon-γ (IFN-γ) release assay QuantiFERON-TB Gold In Tube (QFT-GIT) in the diagnosis of tuberculous pleural effusion.Methods IFN-γ released by specific T cells stimulated by early secreted antigenic target 6 × 103protein (ESAT-6),culture filtrate protein 10 (CFP -10) and TB7.7 were measured by QFT-GIT test in 44 tuberculous pleural effusion patients and 16 non-tuberculous pleurisy controls.The IFN-γ release level between groups was compared by Mann-Whitmey test.ResultsThe positive rates of QFT-GIT in patients with tuberculous pleural effusion and non tuberculous pleurisy were 95.5％ and 12.5％,respectively.The sensitivity,specificity,positive predictive value and negative predictive value of QFT-GIT were 95.6％,87.5％,95.6％ and 87.5％,respectively.The antigen-specific IFN-γ release level in the patients with tuberculous pleural effusion was significantly higher than that in non-tuberculous pleurisy controls (P＜0.01).Conclusions The whole blood INF-γ release assay QFT-GIT is a sensitive and specific assay for detecting pleural tuberculosis infection.It could be a useful diagnostic tool for the diagnosis of tuberculous pleural effusion in China.
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To study the chemosensitivity and the mechanisms of recombinant adenovirus vector expressing ING4 and IL-24 (Ad-ING4-IL-24) on lung adenocarcinoma in vitro and in vivo, the expression of ING4 and IL-24 in A549 cells was detected by RT-PCR and Western blotting. The growth inhibition, apoptosis rate and apoptosis body were measured by MTT, flow cytometry and Hoechst staining respectively. For in vivo study, we first established the A549 tumor model by grafting A549 cells in athymic nude mice; and then injected Ad-ING4-IL-24 into the tumors. Two weeks after injection, we killed the mice, removed the tumors, weighted and calculated the ratios of tumor-suppression. We also detected the expressions of ING4, IL-24, bax, bcl-2, VEGF with immunohistochemistry. The results indicated that ING4 and IL-24 were proved successfully transcription and expression in A549 cells. More interestingly, the joint group inhibited the growth of A549 cells and induced apoptosis. The in vivo data showed that the joint group suppressed the tumor growth conspicuously through up-regulating the expression of bax, and down-regulating the expression of bcl-2, VEGF. The study proved that Ad-ING4-IL-24 significantly enhanced the chemosensitivity to anticancer drug DDP in lung adenocarcinoma, which may related with cell apoptosis and antiangiogenesis.