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Objective: To detect the Th1 and Th2 cell percentage in pleural effusion mononuclear cells (PEMCs) stimulated by early secretory antigenic target protein-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) fusion protein (E/C) with flow cytometry (FCM), and therefore to explore the local antigen specific Th1 and Th2 response and its diagnostic value in tuberculous pleuritis., Methods: Forty patients with tuberculous pleural effusion and 30 patients with malignant pleural effusion were included in this study from Sep.2008 to Mar.2009. PEMCs were isolated and cryopreserved. After resuscitation, the cells were cultured with E/C (simultaneously with positive control and negative control), and antigen-specific Th1 and Th2 cells were detected with intracellular cytokine staining of FCM. Normal distribution data using t test, abnormal distribution data using Wilcoxon test., Results: In the TB group,the medians (quartile range) of Th1 cells and Th1/Th2 ratio among PEMCs stimulated by ESAT-6/CFP-10 fusion protein were 3.06% (1.59%-6.92%) and 17 (7.38-35.53), significantly higher than those of the negative control [0.38% (0.02%-1.80%) and 3.59 (0.49-25.09)], the differences being statistically significant (Z = -5.345 and 3.314, P < 0.01). The percentage of Th2 cells [(0.22 ± 0.19)%] was also increased compared with that of the negative control [(0.10 ± 0.08)%], the difference being statistically significant (t = 4.108, P < 0.01). In the malignant effusion group, the medians (quartile range) of Th1 percentage and Th1/Th2 ratio were 0.12% (0.05%-0.39%) and 1.05 (0.25-2.52), which were significantly different as compared with those of the TB group (Z = -6.624 and -5.536, P < 0.01). The Th2 percentage in the 2 groups were (0.22 ± 0.19)% and (0.15 ± 0.02)%, respectively (t = 1.954, P > 0.05). The receiver operating characteristic curve indicated that the area under the curve (AUC), sensitivity, and specificity were 0.937, 85.4%, and 90.6% respectively for Th1 to diagnose tuberculous pleurisy. For Th1/Th2, the AUC, sensitivity, and specificity were 0.883, 81.5%, and 90.6% respectively., Conclusions: The feature of ESAT-6/CFP-10 fusion protein-specific Th1 and Th2 response in tuberculous pleurisy was a mixed reaction of Th1 and Th2 with Th1 predominance. Th1 percentage and Th1/Th2 ratio could be diagnostic indexes for identifying tuberculous from malignant pleural effusions.

Objective: To investigate the effects of different cytokines (IL-22, IL-17, IFN-γ) on proliferation and apoptosis of human pleural mesothelial cells (PMC) during Mycobacterium tuberculosis infection., Methods: The expressions of IL-22R, IL-17R and IFN-γR1 on PMC purified from tuberculous pleural effusion (TPE) were determined by flow cytometry. The effects of one or more of IL-22, IL-17, and IFN-γ on Ki-67 expression and apoptosis of PMC were explored. Ki-67 expression of PMC under the conditions of the absence or presence of exogenous TPE and with a combination of control IgG, or anti-IL-22, -IL-17 or -IFN-γ mAbs were determined., Results: (1) During Mycobacterium tuberculosis infection, IL-22R, IL-17R and IFN-γR1 were highly expressed on the surface of PMC [(86.2 ± 2.9)%, (41.5 ± 4.4)% and (64.9 ± 5.8)% respectively]. (2) In group IL-22 + IL-17, the percentage of Ki-67(+)PMC was (31.5 ± 2.0) % and (26.1 ± 2.4) % respectively, which were both higher than that in the medium group [ (14.6 ± 0.7)%] (q = 6.8 and 4.9, respectively, both P < 0.05). In group IFN-γ, the percentage of Ki-67(+)PMC was (5.2 ± 1.2) %, which was lower than that in the medium group (q = 5.0, P < 0.05). In group IFN-γ+IL-22 and IFN-γ+IL-17, the percentages of Ki-67(+)PMC were (23.4 ± 1.7)% and (21.8 ± 3.8)% respectively, which were both higher than that in group IFN-γ (q = 7.3 and 6.7, respectively, both P < 0.05). In group TPE and TPE+IgG, the percentages of Ki-67(+)PMC were (63 ± 9) % and (63 ± 11) % respectively, which were both higher than that in the medium group (q = 19.6 and 19.7, respectively, both P < 0.05). In group TPE+ anti-IFN-γ mAb, the percentage of Ki-67(+)PMC was (82 ± 4) %, which was even higher than that in group TPE+IgG (q = 7.5,P < 0.05) . In group TPE + anti-IL-22 mAb, the percentage of Ki-67(+)PMC was (34 ± 3) %, which was lower than that in group TPE+IgG (q = 11.8, P < 0.05). In group TPE + anti-IL-17 mAb, the percentage of Ki-67(+)PMC was (58 ± 5) %, which showed no significant difference compared to that in group TPE+IgG (q = 2.1, P > 0.05). (3) The percentage of apoptotic PMC in group IFN-γ was (19.3 ± 1.1)%, which was higher than that in the medium group[ (4.3 ± 0.6)%] (q = 33.4,P < 0.05) . The percentage of apoptotic PMC in group IL-22 + IL-17 was (3.8 ± 0.6)% and (5.7 ± 0.8)% respectively, which had no significant difference compared to that in the medium group (q = 1.3 and 3.0, respectively, both P > 0.05). The percentage of apoptotic PMC in group IFN-γ + IL-22 and IFN-γ+ IL-17 were (6.5 ± 0.7) % and (8.7 ± 1.7)% respectively, which were both lower than that in group IFN-γ (q = 28.5 and 23.6, respectively, both P < 0.05)., Conclusion: During Mycobacterium tuberculosis infection, IFN-γ inhibited PMC proliferation and contributed to apoptosis, while IL-22 and IL-17 promoted PMC proliferation without influencing PMC apoptosis and succeeded in reversing the effect induced by IFN-γ.

Objective: To develop a rat model of tuberculous pleurisy and to explore the mechanism of intrapleural inflammatory and immunological responses., Methods: Fifty Wistar rats were injected intrapleurally with 0.03 mg of standard human mycobacterium tuberculous bacilli H37Rv each. The rats were killed in group on days 1, 2, 3, 5, 7, 10, 15, 20, 30 and 60 after the day of intrapleural injection. The thorax was opened and the amount of pleural effusion was recorded, and histopathology of pleural tissues and lung tissues were observed. The white blood cell (WBC) count and differentials, levels of total protein (TP), glucose (GLU) and lactic dehydrogenase (LDH) of pleural effusions were determined. Pleural fluid was analyzed for the levels of soluble intercellular adhesion molecule-1 (sICAM-1), transforming growth factor beta1 (TGF-beta1) and interferon gamma (IFN-gamma) by using appropriate bioassays. Ten rats were intrapleurally received 2 ml of normal saline and another 10 rats received 2 ml of undiluted PPD solution each as control., Results: Bilateral pleural effusions appeared within 15 days in all rats intrapleurally received tuberculous bacilli. The peak amount of pleural fluid was on day 5 (6.7 +/- 0.5 ml). The neutrophils were the predominant cells for the first 24 hours, and then were followed by lymphocytes. In the pleural fluid, total protein concentration was between 51-55 g/L. The levels of glucose and LDH were 5.2 mmol/L and 18.1 micromol.s(-1).L(-1) on day 1 and changed to 2.8 mmol/L and 28.9 micromol.s(-1).L(-1) on day 15 respectively. The biochemistry parameters were in accordance with characteristics of tuberculous pleurisy. The sICAM-1 level increased early (21.9 ng/ml on day 1) and peaked on day 3 (38.0 ng/ml), then decreased over time (4.4 ng/ml on day 15). The level of IFN-gamma was 41.2 pg/ml on day 1 and increased and maintained at high levels over time. TGF-beta1 levels increased and peaked on day 7 (47.2 ng/ml), and then on day 15 decreased to a level lower than that of day 1. The ratio of IFN-gamma/TGF-beta1 increased from 1.32 on day 1 to 5.69 on day 15. Correlation analysis showed that sICAM-1 and IFN-gamma were closely related with WBC count and its differentials, as well as with LDH levels. Histopathological study revealed early pleural inflammation and late caseation., Conclusions: Wistar rats can be used as an experimental model for tuberculous pleurisy. Tuberculous inflammatory and immunological responses in acute tuberculous pleurisy is enhanced rather than suppressed.

IFN gamma levels and IFN gamma production capability were studied. The results showed that IFN gamma levels, IFN gamma production in the tuberculous pleurisy (n = 30) were significantly higher than those in the malignant pleural effusion (n = 23) and the pleural transudate. However, there was no detectable IFN gamma in the blood, IFN gamma production were lower in the blood than in the pleural effusion. The finding suggests that IFN gamma may play an important role in local anti-mycobacterial infection.

The levels of soluble interleukin 2 receptor alpha (sIL-2R alpha) were measured in pleural fluid from 38 patients including 16 cases with tuberculous pleurisy, 14 with malignant pleural effusions and 8 with transudative effusions by using ELISA technique. The level of sIL-2R alpha in tuberculous pleurisy is markedly higher than in malignant pleural effusions (P < 0.01). The levels of sIL-2R alpha in tuberculous pleurisy and in malignant pleural effusions are significantly higher than in transudative effusions (P < 0.01). The clinical significance is discussed.