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1.  Different Subsets of Macrophages in Patients with New Onset Tuberculous Pleural Effusion 
PLoS ONE  2014;9(2):e88343.
Objective
Macrophages are the infiltrate components of tuberculous pleural effusion (TPE). This study is aimed at examining the role of different subsets of macrophages in pleural fluid (PF) and peripheral blood (PB) from patients with new onset TPE.
Methods
The numbers of PB and PF CD163+, CD206+ and CD115+ macrophages in 25 patients with new onset TPE and 17 healthy controls (HC) were determined by flow cytometry. The concentrations of serum and PF cytokines were determined by cytometric bead array (CBA) and enzyme-linked immunosorbentassay (ELISA). The potential association between the numbers of different subsets of macrophages and the values of clinical measures in TPE patients were analyzed.
Results
The numbers of PB CD14+CD163− M1-like and CD14+CD163− interleukin (IL)-12+ M1 macrophages were significantly higher than that in the HC, but lower than PF, and the numbers of PF CD14+CD163+, CD14+CD163+CD206+, CD14+CD163+CDll5+ M2-like, and CD14+CD163+IL-10+ M2 macrophages were less than PB in the TPE patients. The levels of serum IL-1, IL-6, IL-8, IL-12, tumor growth factor (TGF)-β1, and tumor necrosis factor (TNF)-α in the TPE patients were significantly higher than that in the HC, but lower than that in the PF. The levels of PF IL-10 were significantly higher than that in the PB of patients and HC. In addition, the levels of serum IL-12 and TNF-α were correlated positively with the values of erythrocyte sedimentation rate (ESR) and the numbers of ESAT-6- and culture filtrate protein 10 (CFP-10)-specific IFN-γ-secreting T cells, and the levels of PF TNF-α were correlated positively with the levels of PF adenosine deaminase (ADA) and lactate dehydrogenase (LDH) in those patients.
Conclusion
Our data indicate that Mycobacterium tuberculosis (M. tb) infection induces M1 predominant pro-inflammatory responses, contributing to the development of TPE in humans.
doi:10.1371/journal.pone.0088343
PMCID: PMC3919770  PMID: 24520370
2.  IL-33 levels differentiate tuberculous pleurisy from malignant pleural effusions 
Oncology Letters  2014;8(1):449-453.
Tuberculous pleural effusions (TPEs) and malignant pleural effusions (MPEs) are difficult to differentiate between in certain clinical situations. Interleukin (IL)-33 is a cytokine that participates in inflammatory responses and may have a role in pleural effusions. The present study aimed to investigate the concentrations and potential differential significance of IL-33 in patients with TPE and MPE. IL-33 levels in pleural effusion and serum samples were detected using sandwich enzyme-linked immunosorbent assay in 23 patients with TPE and 21 patients with MPE. The concentration of IL-33 (mean ± standard deviation) in the TPE patients (22.962±0.976 ng/l) was significantly higher than that in the MPE patients (12.603±5.153 ng/l; P<0.001; z=−4.572); however, there was no significant difference in the serum level of IL-33 in the patients with TPE compared with those with MPE (P>0.05). The concentration of IL-33 in the pleural effusions was positively correlated with that in the serum samples in each group (TPE: r=0.563, P=0.05; MPE: r=0.535, P<0.05). The cut-off value of pleural IL-33 for TPE was 19.86 ng/l, which yielded a sensitivity of 0.869, a specificity of 0.905 and an area under the corresponding receiver operating characteristic curve of 0.903. The present study identified that the level of pleural IL-33 is significantly increased in TPEs and may serve as a novel biomarker to differentiate between patients with TPE and MPE.
doi:10.3892/ol.2014.2109
PMCID: PMC4063658  PMID: 24959294
interleukin-33; pleural effusion; peripheral blood; tuberculous pleurisy; malignant pleural effusion
3.  Association between Elevated Pleural Interleukin-33 Levels and Tuberculous Pleurisy 
Annals of Laboratory Medicine  2012;33(1):45-51.
Background
Interferon-γ (IFN-γ) plays a crucial role in Mycobacterium tuberculosis induced pleural responses. Interleukin (IL)-33 up-regulates the production of IFN-γ. We aimed to identify whether an association between pleural IL-33 levels and tuberculous pleurisy exists and determine its diagnostic value.
Methods
Pleural IL-33, ST2 (a receptor of IL-33), adenosine deaminase (ADA), and IFN-γ, as well as serum IL-33 and ST2 were measured in 220 patients with pleural effusions (PEs). Patients with malignant (MPEs), parapneumonic (PPEs), tuberculous (TPEs), and cardiogenic (CPEs) pleural effusions were included.
Results
Pleural and serum IL-33 levels were highest or tended to be higher in patients with TPEs than in those with other types of PEs. The median pleural fluid-to-serum IL-33 ratio was higher in TPE cases (≥ 0.91) than in other PE cases (≤ 0.56). Pleural IL-33 levels correlated with those of pleural ADA and IFN-γ. However, the diagnostic accuracies of pleural IL-33 (0.74) and pleural fluid-to-serum IL-33 ratio (0.75) were lower than that of ADA (0.95) or IFN-γ (0.97). Pleural ST2 levels in patients with MPEs were higher than in patients with TPEs. Serum ST2 levels did not differ among the groups.
Conclusions
We identified an association between elevated pleural IL-33 levels and tuberculous pleurisy. However, we recommend conventional pleural markers (ADA or IFN-γ) as diagnostic markers of TPE.
doi:10.3343/alm.2013.33.1.45
PMCID: PMC3535196  PMID: 23301222
Interleukin-33; ST2; Tuberculosis; Pleural effusion
4.  Localized Eosinophil Degranulation Mediates Disease in Tropical Pulmonary Eosinophilia  
Infection and Immunity  2003;71(3):1337-1342.
To explore the mechanisms underlying the eosinophil-mediated inflammation of tropical pulmonary eosinophilia (TPE), bronchoalveolar lavage (BAL) fluid, serum, and supernatants from pulmonary and blood leukocytes (WBC) from patients with acute TPE (n = 6) were compared with those obtained from healthy uninfected individuals (n = 4) and from patients with asthma (n = 4) or elephantiasis (n = 5). Although there were no significant differences in the levels of interleukin-4 (IL-4), IL-5, IL-13, eotaxin, granulocyte-macrophage colony-stimulating factor, RANTES, or eosinophil cationic protein, there was a marked increase in eosinophil-derived neurotoxin (EDN) both systemically and in the lungs of individuals with TPE compared to each of the control groups (P < 0.02). Moreover, there was a compartmentalization of this response, with EDN levels being higher in the BAL fluid than in the serum (P < 0.02). Supernatants from WBC from either whole blood or BAL cells were examined for chemokines, cytokines, eosinophil degranulation products, and arachidonic acid metabolites. Of the many mediators examined—particularly those associated with eosinophil trafficking—only EDN (in BAL fluid and WBC) and MIP-1α (in WBC) levels were higher for TPE patients than for the non-TPE control groups (P < 0.02). These data suggest it is the eosinophilic granular protein EDN, an RNase capable of damaging the lung epithelium, that plays the most important role in the pathogenesis of TPE.
doi:10.1128/IAI.71.3.1337-1342.2003
PMCID: PMC148813  PMID: 12595450
5.  Factors affecting pleural fluid adenosine deaminase level and the implication on the diagnosis of tuberculous pleural effusion: a retrospective cohort study 
BMC Infectious Diseases  2013;13:546.
Background
Adenosine deaminase (ADA) is useful in the diagnosis of tuberculous pleural effusion (TPE). This study aims to determine the factors affecting pleural fluid ADA levels and to establish the optimal ADA levels for diagnosis of TPE for different age groups.
Methods
This was a retrospective study from January 2007 to October 2011. One hundred and sixty patients who had pleural fluid ADA performed for investigation of pleural effusion were analyzed. Variables examined included demographics, pleural fluid characteristics and peripheral blood counts. The ADA cut-offs according to age were selected using the receiver operating characteristic (ROC) curve.
Results
The mean pleural fluid ADA was significantly higher in the TPE group (100 ± 35 IU/L) compared to non TPE patients (30 ± 37 IU/L). There was significant correlation between pleural fluid ADA and age, pleural fluid protein, LDH, and fluid absolute lymphocyte count. The strongest correlation was seen with age (r = −0.621). For patients ≤ 55 years old the ROC for ADA had area under curve (AUC) of 0.887. A pleural fluid ADA of 72 IU/L had sensitivity of 95.1%, specificity of 87.5%, positive predictive value (PPV) of 95.1% and negative predictive value (NPV) of 87.5% for the diagnosis of TPE. For patients > 55 years old the AUC is 0.959. ADA of 26 IU/L had a sensitivity of 94.7%, specificity of 80.4%, PPV of 62% and NPV of 97.8%.
Conclusions
There is a significant negative correlation between pleural fluid ADA and age. For older patients, a lower ADA cut-off should be used to exclude TPE.
doi:10.1186/1471-2334-13-546
PMCID: PMC3835552  PMID: 24238276
Adenosine deaminase; Tuberculosis; Pleural effusion; Biological markers
6.  Diagnostic value of interleukin 22 and carcinoembryonic antigen in tuberculous and malignant pleural effusions 
The aim of this study was to investigate the diagnostic value of interleukin 22 (IL-22) and carcinoembryonic antigen (CEA) in tuberculous pleural effusions (TPEs) and malignant pleural effusions (MPEs). Pleural effusion samples from 56 patients were classified on the basis of diagnosis as TPE (n=28) and MPE (n=28). The concentration of IL-22 was determined by ELISA. Lactate dehydrogenase (LDH), adenosine dehydrogenase (ADA) and CEA levels were also determined in all patients. A significant difference was observed in the levels of ADA and CEA (P<0.01), but not in the levels of LDH (P>0.05) between TPE and MPE. The concentration of IL-22 in TPE was significantly higher compared to MPE (P<0.01). With a threshold value of 49 pg/ml, IL-22 had a sensitivity of 82.14% (23/28) and a specificity of 96.43% (27/28) for differential diagnosis. The combined detection of IL-22 and CEA had a sensitivity of 100% (28/28) and a specificity of 96.43% (27/28) to distinguish TPE from MPE. TPEs showed significantly higher levels of IL-22 compared to MPEs. The combined detection of IL-22 and CEA may be more valuable in the differential diagnosis between TPE and MPE.
doi:10.3892/etm.2011.344
PMCID: PMC3440797  PMID: 22977645
interleukin 22; carcinoembryonic antigen; pleural effusion; differential diagnosis
7.  Reciprocal Immunomodulatory Effects of Gamma Interferon and Interleukin-4 on Filaria-Induced Airway Hyperresponsiveness 
Infection and Immunity  2001;69(3):1463-1468.
Tropical pulmonary eosinophilia (TPE) is a severe asthmatic syndrome of lymphatic filariasis, in which an allergic response is induced to microfilariae (Mf) in the lungs. Previously, in a murine model for TPE, we have demonstrated that recombinant interleukin-12 (IL-12) suppresses pulmonary eosinophilia and airway hyperresponsiveness (AHR) by modulating the T helper (Th) response in the lungs from Th2- to Th1-like, with elevated gamma-interferon (IFN-γ) production and decreased IL-4 and IL-5 production. The present study examined the immunomodulatory roles of IL-4 and IFN-γ in filaria-induced AHR and pulmonary inflammation using mice genetically deficient in these cytokines. C57BL/6, IL-4 gene knockout (IL-4−/−), and IFN-γ−/− mice were first immunized with soluble Brugia malayi antigens and then inoculated intravenously with 200,000 live Mf. Compared with C57BL/6 mice, IL-4−/− mice exhibited significantly reduced AHR, whereas IFN-γ−/− mice had increased AHR. Histopathologically, each mouse strain showed increased cellular infiltration into the lung parenchyma and bronchoalveolar space compared with naïve animals. However, consistent with changes in AHR, IL-4−/− mice had less inflammation than C57BL/6 mice, whereas IFN-γ−/− mice had exacerbated pulmonary inflammation with the loss of pulmonary architecture. Systemically, IL-4−/− mice produced significantly higher IFN-γ levels compared with C57BL/6 mice, whereas IFN-γ−/− mice produced significantly higher IL-4 levels. These data indicate that IL-4 is required for the induction of filaria-induced AHR, whereas IFN-γ suppresses AHR.
doi:10.1128/IAI.69.3.1463-1468.2001
PMCID: PMC98042  PMID: 11179313
8.  Pulmonary inflammation induced by a recombinant Brugia malayi gamma-glutamyl transpeptidase homolog: involvement of humoral autoimmune responses. 
Molecular Medicine  2001;7(5):344-354.
BACKGROUND: A major allergen from the lymphatic filarial parasite Brugia malayi implicated in the pathogenesis of tropical pulmonary eosinophilia (TPE) has recently been cloned and identified as the homolog of the membrane-bound mammalian enzyme gamma-glutamyl transpeptidase (gamma-GT). Patients with acute TPE show autoreactive antibodies against endogenous gamma-GT from the pulmonary epithelium. MATERIALS AND METHODS: Recombinant B. malayi gamma-GT, alone or adsorbed to aluminium hydroxide (AL), was used in a BALB/c mouse model to analyze its antigenic/allergenic potential, its potential to induce pulmonary inflammation, and its capacity to induce autoreacting antibodies. RESULTS: Mice immunized with B. malayi gamma-GT showed significant levels of gamma-GT-specific IgG1, IgG2a, IgG3, IgA, IgE antibodies, and mild blood eosinophilia, even in the absence of adjuvant. Intranasal challenge with B. malayi gamma-GT induced peribronchial and perivascular inflammation characterized by a mixed infiltrate of lymphocytes, neutrophils, eosinophils, and macrophages. Both IL-4 and IFN-gamma were detected in the peripheral blood and in the bronchoalveolar lavage fluid of immunized and intranasally challenged mice. Histological analysis of murine lungs using affinity-purified antibodies from mice immunized with the parasite's gamma-GT revealed the presence of autoimmune antibodies against pulmonary epithelium. Western blot analysis identified the 55 kDa heavy chain subunit of the murine gamma-GT as the target of autoreactive/crossreacting antibodies. CONCLUSION: Our data from the in vivo mouse model demonstrate the potent allergenicity/antigenicity of B. malayi gamma-GT, and its capacity to induce pulmonary inflammation upon intranasal challenge. This leads to breakdown of tolerance against endogenous murine gamma-GT. Thus, humoral autoimmunity against the airways epithelium may contribute to the pathogenesis of TPE.
PMCID: PMC1950044  PMID: 11474580
9.  Pleural tuberculosis: is radiological evidence of pulmonary-associated disease related to the exacerbation of the inflammatory response? 
Clinics  2012;67(11):1259-1263.
OBJECTIVE:
Pleural tuberculosis is the most frequently occurring form of extra pulmonary disease in adults. In up to 40% of cases, the lung parenchyma is concomitantly involved, which can have an epidemiological impact. This study aims to evaluate the pleural and systemic inflammatory response of patients with pleural or pleuropulmonary tuberculosis.
METHODS:
A prospective study of 39 patients with confirmed pleural tuberculosis. After thoracentesis, a high resolution chest tomography was performed to evaluate the pulmonary involvement. Of the 39 patients, 20 exhibited only pleural effusion, and high resolution chest tomography revealed active associated-pulmonary disease in 19 patients. The total protein, lactic dehydrogenase, adenosine deaminase, vascular endothelial growth factor, interleukin-8, tumor necrosis factor-α, and transforming growth factor-β1 levels were quantified in the patient serum and pleural fluid.
RESULTS:
All of the effusions were exudates with high levels of adenosine deaminase. The levels of vascular endothelial growth factor and transforming growth factor-β1 were increased in the blood and pleural fluid of all of the patients with pleural tuberculosis, with no differences between the two forms of tuberculosis. The tumor necrosis factor-α levels were significantly higher in the pleural fluid of the patients with the pleuropulmonary form of tuberculosis. The interleukin-8 levels were high in the pleural fluid of all of the patients, without any differences between the forms of tuberculosis.
CONCLUSION:
Tumor necrosis factor-α was the single cytokine that significantly increased in the pleural fluid of the patients with pulmonary involvement. However, an overlap in the results does not permit us to suggest that cytokine is a biological marker of concomitant parenchymal involvement. Although high resolution chest tomography can be useful in identifying these patients, the investigation of fast acid bacilli and cultures for M. tuberculosis in the sputum is recommended for all patients who are diagnosed with pleural tuberculosis.
doi:10.6061/clinics/2012(11)06
PMCID: PMC3488982  PMID: 23184200
Cytokines; Inflammation; Pleural Diseases; Tuberculosis
10.  Acute tropical pulmonary eosinophilia. Characterization of the lower respiratory tract inflammation and its response to therapy. 
Journal of Clinical Investigation  1987;80(1):216-225.
Although acute tropical pulmonary eosinophilia (TPE) is well recognized as a manifestation of filarial infection, the processes that mediate the abnormalities of the lung in TPE are unknown. To evaluate the hypothesis that the derangements of the lower respiratory tract in this disorder are mediated by inflammatory cells in the local milieu, we utilized bronchoalveolar lavage to evaluate affected individuals before and after therapy. Inflammatory cells recovered from the lower respiratory tract of individuals with acute, untreated TPE (n = 8) revealed a striking eosinophilic alveolitis, with marked elevations in both the proportion of eosinophils (TPE 54 +/- 5%; normal 2 +/- 5%; P less than 0.001) and the concentration of eosinophils in the recovered epithelial lining fluid (ELF) (TPE 63 +/- 20 X 10(3)/microliter; normal 0.3 +/- 0.1 X 10(3)/microliter; P less than 0.01). Importantly, when individuals (n = 5) with acute TPE were treated with diethylcarbamazine (DEC), there was a marked decrease of the lung eosinophils and concomitant increase in lung function. These observations are consistent with the concept that at least some of the abnormalities found in the lung in acute TPE are mediated by an eosinophil-dominated inflammatory process in the lower respiratory tract.
Images
PMCID: PMC442221  PMID: 3298321
11.  Abnormal Immune Responses in Persons with Previous Extrapulmonary Tuberculosis in an In Vitro Model That Simulates In Vivo Infection with Mycobacterium tuberculosis 
Persons with previous extrapulmonary tuberculosis have reduced peripheral blood mononuclear cell cytokine production and CD4+ lymphocytes compared to persons with previous pulmonary tuberculosis or latent tuberculosis infection, but specific defects related to Mycobacterium tuberculosis infection of macrophages have not been characterized. The objective of this study was to further characterize the in vitro immune responses to M. tuberculosis infection in HIV-seronegative persons with previous extrapulmonary tuberculosis. Peripheral blood mononuclear cells were isolated from HIV-seronegative persons with previous extrapulmonary tuberculosis (n = 11), previous pulmonary tuberculosis (n = 21), latent M. tuberculosis infection (n = 19), and uninfected tuberculosis contacts (n = 20). Experimental conditions included M. tuberculosis-infected macrophages cultured with and without monocyte-depleted peripheral blood mononuclear cells. Concentrations of interleukin 1β (IL-1β), IL-4, IL-6, CXCL8 (IL-8), IL-10, IL-12p70, IL-17, CCL2 (monocyte chemoattractant protein 1), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) were measured by multiplex cytokine array. When M. tuberculosis-infected macrophages were cocultured with monocyte-depleted peripheral blood mononuclear cells, IFN-γ (P = 0.01), TNF-α (P = 0.04), IL-10 (P < 0.001), and IL-6 (P = 0.03) exhibited similar continua of responses, with uninfected persons producing the lowest levels, followed by extrapulmonary tuberculosis cases, pulmonary tuberculosis controls, and persons with latent M. tuberculosis infection. A similar pattern was observed with CXCL8 (P = 0.04), IL-10 (P = 0.02), and CCL2 (P = 0.03) when monocyte-depleted peripheral blood mononuclear cells from the four groups were cultured alone. Persons with previous extrapulmonary tuberculosis had decreased production of several cytokines, both at rest and after stimulation with M. tuberculosis. Our results suggest that persons who develop extrapulmonary tuberculosis have a subtle global immune defect that affects their response to M. tuberculosis infection.
doi:10.1128/CVI.00221-12
PMCID: PMC3416084  PMID: 22675156
12.  Factors Influencing Residual Pleural Opacity in Tuberculous Pleural Effusion 
Journal of Korean Medical Science  2008;23(4):616-620.
Tuberculous pleural effusion (TPE) leads to residual pleural opacity (RPO) in a significant proportion of cases. The aim of this study was to investigate which TPE patients would have RPO following the treatment. This study was performed prospectively for a total of 60 TPE patients, who underwent pleural fluid analysis on the initial visit and chest radiographs and computed tomography (CT) scans before and after the administration of antituberculous medication. At the end of antituberculous medication, the incidence of RPO was 68.3% (41/60) on CT with a range of 2-50 mm. Compared with the non-RPO group, the RPO group had a longer symptom duration and lower pleural fluid glucose level. On initial CT, loculation, extrapleural fat proliferation, increased attenuation of extrapleural fat, and pleura-adjacent atelectasis were more frequent, and parietal pleura was thicker in the RPO group compared with the non-RPO group. By multivariate analysis, extrapleural fat proliferation, loculated effusion, and symptom duration were found to be predictors of RPO in TPE. In conclusion, RPO in TPE may be predicted by the clinico-radiologic parameters related to the chronicity of the effusion, such as symptom duration and extrapleural fat proliferation and loculated effusion on CT.
doi:10.3346/jkms.2008.23.4.616
PMCID: PMC2526418  PMID: 18756047
Computed Tomography; Pleural Effusion; Residual Thickening; Tuberculosis
13.  Clonal Immune Responses of Mycobacterium-Specific γδ T Cells in Tuberculous and Non-Tuberculous Tissues during M. tuberculosis Infection 
PLoS ONE  2012;7(2):e30631.
Background
We previously demonstrated that unvaccinated macaques infected with large-dose M.tuberculosis(Mtb) exhibited delays for pulmonary trafficking of Ag-specific αβ and γδ T effector cells, and developed severe lung tuberculosis(TB) and “secondary” Mtb infection in remote organs such as liver and kidney. Despite delays in lungs, local immunity in remote organs may accumulate since progressive immune activation after pulmonary Mtb infection may allow IFNγ-producing γδ T cells to adequately develop and traffic to lately-infected remote organs. As initial efforts to test this hypothesis, we comparatively examined TCR repertoire/clonality, tissue trafficking and effector function of Vγ2Vδ2 T cells in lung with severe TB and in liver/kidney without apparent TB.
Methodology/Principal Findings
We utilized conventional infection-immunity approaches in macaque TB model, and employed our decades-long expertise for TCR repertoire analyses. TCR repertoires in Vγ2Vδ2 T-cell subpopulation were broad during primary Mtb infection as most TCR clones found in lymphoid system, lung, kidney and liver were distinct. Polyclonally-expanded Vγ2Vδ2 T-cell clones from lymphoid tissues appeared to distribute and localize in lung TB granuloms at the endpoint after Mtb infection by aerosol. Interestingly, some TCR clones appeared to be more predominant than others in lymphocytes from liver or kidney without apparent TB lesions. TCR CDR3 spetratyping revealed such clonal dominance, and the clonal dominance of expanded Vγ2Vδ2 T cells in kidney/liver tissues was associated with undetectable or low-level TB burdens. Furthermore, Vγ2Vδ2 T cells from tissue compartments could mount effector function for producing anti-mycobacterium cytokine.
Conclusion
We were the first to demonstrate clonal immune responses of mycobacterium-specific Vγ2Vδ2 T cells in the lymphoid system, heavily-infected lungs and lately subtly-infected kidneys or livers during primary Mtb infection. While clonally-expanded Vγ2Vδ2 T cells accumulated in lately-infected kidneys/livers without apparent TB lesions, TB burdens or lesions appeared to impact TCR repertoires and tissue trafficking patterns of activated Vγ2Vδ2 T cells.
doi:10.1371/journal.pone.0030631
PMCID: PMC3271047  PMID: 22319574
14.  Effect of Neutralizing Transforming Growth Factor β1 on the Immune Response against Mycobacterium tuberculosis in Guinea Pigs  
Infection and Immunity  2004;72(3):1358-1363.
Transforming growth factor β (TGF-β) is a cytokine which has been shown to suppress the antimycobacterial immune responses of humans and experimental animals. In this study, the contributions of TGF-β to cytokine production in vivo were investigated by using the established guinea pig model of tuberculous pleurisy. Mycobacterium bovis BCG-vaccinated guinea pigs were injected intrapleurally with heat-killed virulent Mycobacterium tuberculosis. Eight days following induction of an antigen-specific pleural effusion, guinea pigs were injected intrapleurally with anti-TGF-β1 or isotype control antibody. The following day, pleural exudates were removed, and the fluid volume and characteristics of the infiltrating cells were determined. Pleural fluid was analyzed for total interferon (IFN) and tumor necrosis factor (TNF) protein levels by using appropriate bioassays. RNA from pleural effusion cells was examined to determine TGF-β1, TNF-α, IFN-γ, and interleukin-8 mRNA levels by using real-time PCR. Proliferative responses of pleural effusion lymphocytes were examined in response to concanavalin A and purified protein derivative (PPD) in vitro. Treatment with anti-TGF-β1 resulted in decreased pleural fluid volume and decreased cell numbers in the pleural space along with an increased percentage of lymphocytes and a decreased percentage of neutrophils. The bioactive TNF protein levels in pleural fluid were increased in guinea pigs treated with anti-TGF-β1, while the bioactive IFN protein concentrations were not altered. Expression of TGF-β1 and TNF-α mRNA was significantly increased following TGF-β1 neutralization. Finally, PPD-induced proliferative responses of pleural cells from anti-TGF-β1-treated animals were significantly enhanced. Thus, TGF-β1 may be involved in the resolution of this local, mycobacterial antigen-specific inflammatory response.
doi:10.1128/IAI.72.3.1358-1363.2004
PMCID: PMC356004  PMID: 14977939
15.  Imbalance of Th17 Cells and Regulatory T Cells in Tuberculous Pleural Effusion ▿ 
Clinical and Vaccine Immunology : CVI  2011;18(10):1608-1615.
Both T helper interleukin 17 (IL-17)-producing cells (Th17 cells) and regulatory T cells (Tregs) have been found to be increased in human tuberculous pleural effusion (TPE); however, the possible interaction between Th17 cells and Tregs in TPE remains to be elucidated. The objective of the present study was to investigate the distribution of Th17 cells in relation to Tregs, as well as the mechanism of Tregs in regulating generation and differentiation of Th17 cells in TPE. In the present study, the numbers of Th17 cells and Tregs in TPE and blood were determined by flow cytometry. The regulation and mechanism of CD39+ Tregs on generation and differentiation of Th17 cells were explored. Our data demonstrated that the numbers of Th17 cells and CD39+ Tregs were both increased in TPE compared with blood. Th17 cell numbers were correlated negatively with Tregs in TPE but not in blood. When naïve CD4+ T cells were cultured with CD39+ Tregs, Th17 cell numbers decreased as CD39+ Treg numbers increased, and the addition of the anti-latency-associated peptide monoclonal antibody to the coculture reversed the inhibitory effect exerted by CD39+ Tregs. This study shows that Th17/Treg imbalance exists in TPE and that pleural CD39+ Tregs inhibit generation and differentiation of Th17 cells via a latency-associated peptide-dependent mechanism.
doi:10.1128/CVI.05214-11
PMCID: PMC3187020  PMID: 21813663
16.  Different Patterns of Cytokines and Chemokines Combined with IFN-γ Production Reflect Mycobacterium tuberculosis Infection and Disease 
PLoS ONE  2012;7(9):e44944.
Background
IFN-γ is presently the only soluble immunological marker used to help diagnose latent Mycobacterium tuberculosis (M.tb) infection. However, IFN-γ is not available to distinguish latent from active TB infection. Moreover, extrapulmonary tuberculosis, such as tuberculous pleurisy, cannot be properly diagnosed by IFN-γ release assay. As a result, other disease- or infection-related immunological biomarkers that would be more effective need to be screened and identified.
Methodology
A panel of 41 soluble immunological molecules (17 cytokines and 24 chemokines) was tested using Luminex liquid array-based multiplexed immunoassays. Samples, including plasma and pleural effusions, from healthy donors (HD, n = 12) or patients with latent tuberculosis infection (LTBI, n = 20), pulmonary tuberculosis (TB, n = 12), tuberculous pleurisy (TP, n = 15) or lung cancer (LC, n = 15) were collected and screened for soluble markers. Peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) were also isolated to investigate antigen-specific immune factors.
Principal Findings
For the 41 examined factors, our results indicated that three patterns were closely associated with infection and disease. (1) Significantly elevated plasma levels of IL-2, IP-10, CXCL11 and CXCL12 were present in both patients with tuberculosis and in a sub-group participant with latent tuberculosis infection who showed a higher level of IFN-γ producing cells by ELISPOT assay compared with other latently infected individuals. (2) IL-6 and IL-9 were only significantly increased in plasma from active TB patients, and the two factors were consistently highly secreted after M.tb antigen stimulation. (3) When patients developed tuberculous pleurisy, CCL1, CCL21 and IL-6 were specifically increased in the pleural effusions. In particular, these three factors were consistently highly secreted by pleural fluid mononuclear cells following M.tb-specific antigen stimulation. In conclusion, our data imply that the specific secretion of soluble immunological factors, in addition to IFN-γ, may be used to evaluate M.tb infection and tuberculosis disease.
doi:10.1371/journal.pone.0044944
PMCID: PMC3441719  PMID: 23028695
17.  Regulation of CD4+ T Cells by Pleural Mesothelial Cells via Adhesion Molecule-Dependent Mechanisms in Tuberculous Pleurisy 
PLoS ONE  2013;8(9):e74624.
Background
Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) have been demonstrated to be expressed on pleural mesothelial cells (PMCs), and to mediate leukocyte adhesion and migration; however, little is known about whether adhesion molecule-dependent mechanisms are involved in the regulation of CD4+ T cells by PMCs in tuberculous pleural effusion (TPE).
Methods
Expressions of ICAM-1 and VCAM-1 on PMCs, as well as expressions of CD11a and CD29, the counter-receptors for ICAM-1 and VCAM-1, respectively, expressed on CD4+ T cells in TPE were determined using flow cytometry. The immune regulations on adhesion, proliferation, activation, selective expansion of CD4+ helper T cell subgroups exerted by PMCs via adhesion molecule-dependent mechanisms were explored.
Results
Percentages of ICAM-1-positive and VCAM-1‒positive PMCs in TPE were increased compared with PMC line. Interferon-γ enhanced fluorescence intensity of ICAM-1, while IL-4 promoted VCAM-1 expression on PMCs. Percentages of CD11ahighCD4+ and CD29highCD4+ T cells in TPE significantly increased as compared with peripheral blood. Prestimulation of PMCs with anti‒ICAM-1 or ‒VCAM-1 mAb significantly inhibited adhesion, activation, as well as effector regulatory T cell expansion induced by PMCs.
Conclusions
Our current data showed that adhesion molecule pathways on PMCs regulated adhesion and activation of CD4+ T cells, and selectively promoted the expansion of effector regulatory T cells.
doi:10.1371/journal.pone.0074624
PMCID: PMC3777994  PMID: 24069325
18.  Anergic Pulmonary Tuberculosis Is Associated with Contraction of the Vd2+T Cell Population, Apoptosis and Enhanced Inhibitory Cytokine Production 
PLoS ONE  2013;8(8):e71245.
Objective
To study the association of anergic pulmonary tuberculosis with Vδ2+ T cells and related cytokine levels.
Methods
82 pulmonary tuberculosis patients were divided into two groups according to their purified protein derivative tuberculin skin test (TST) results: 39 with TST-negative anergic pulmonary tuberculosis and 43 with TST-positive pulmonary tuberculosis, while 40 healthy volunteers were used as control. Based on chest X-ray results, the tuberculosis lesions were scored according to their severity, with a score of ≤ 2.5 ranking as mild, 2.5-6 as moderate and ≥ 6 as severe. The Vδ2+ T cell percentage and their expression levels of the apoptosis-related membrane surface molecule FasL in peripheral blood and bronchoalveolar lavage fluids (BALF) were analyzed by flow cytometry, while IL-2, IL-4, IL-6 and IL-10 cytokine and γ-interferon (γ-IFN) concentrations in peripheral blood were determined by ELISA.
Results
Most of the patients with chest X-ray lesion scores higher than 6 belonged to the anergic tuberculosis group (P<0.05). Anergic pulmonary tuberculosis patients displayed reduced peripheral blood Vδ2+ T cell counts (P<0.05) and higher FasL expression in peripheral blood Vδ2 + T cells (P <0.05). The Vδ2+ T cell percentages in the BALF of all tuberculosis patients were lower than in their peripheral blood (P <0.05), and IL-4 and IL-10 concentrations in peripheral blood of anergic tuberculosis patients were higher than in TST-positive tuberculosis patients and healthy controls (P <0.05).
Conclusion
Anergic pulmonary tuberculosis is accompanied by reduced Vδ2+ T cell percentage, and elevated Vδ2+ T cell FasL expression as well as enhanced IL-4 and IL-10 levels in peripheral blood.
doi:10.1371/journal.pone.0071245
PMCID: PMC3732239  PMID: 23936496
19.  Diagnostic Accuracy of Adenosine Deaminase and Lymphocyte Proportion in Pleural Fluid for Tuberculous Pleurisy in Different Prevalence Scenarios 
PLoS ONE  2012;7(6):e38729.
Background
Tuberculous pleural effusion (TPE) is a paucibacillary manifestation of tuberculosis, so isolation of Mycobacterium tuberculosis is difficult, biomarkers being an alternative for diagnosis. Adenosine deaminase (ADA) is the most cost-effective pleural fluid marker and is routinely used in high prevalence settings, whereas its value is questioned in areas with low prevalence. The lymphocyte proportion (LP) is known to increase the specificity of ADA for this diagnosis. We analyse the diagnostic usefulness of ADA alone and the combination of ADA ≥40 U/l (ADA40) and LP≥50% (LP50) in three different prevalence scenarios over 11 years in our area.
Materials and Methods
Biochemistry, cytology and microbiology studies from 472 consecutive pleural fluid samples were retrospectively analyzed. ADA and differential cell count were determined in all samples. We established three different prevalence periods, based on percentage of pleural effusion cases diagnosed as tuberculosis: 1998–2000 (31.3%), 2001–2004 (11.8%), and 2005–2008 (7.4%). ROC curves, dispersion diagrams and pre/post-test probability graphs were produced. TPE accounted for 73 episodes (mean prevalence: 15.5%). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for ADA40 were 89%, 92.7%, 69.2% and 97.9%, respectively. For ADA40+LP50 the specificity and PPV increased (98.3% and 90%) with hardly any decrease in the sensitivity or NPV (86.3% and 97.5%). No relevant differences were observed between the three study periods.
Conclusions/Significance
ADA remains useful for the diagnosis of TPE even in low-to-intermediate prevalence scenarios when combined with the lymphocyte proportion.
doi:10.1371/journal.pone.0038729
PMCID: PMC3377686  PMID: 22723878
20.  Elevated IP-10 and IL-6 from Bronchoalveolar Lavage Cells are Biomarkers of Non-Cavitary Tuberculosis 
SUMMARY
Objective
Tuberculosis (TB) causes nearly 1.5 million deaths annually worldwide. Active TB disease can destroy lung parenchyma leading to cavities. Immune responses that predispose or protect individuals from lung damage during tuberculosis are poorly defined.
Design
Enrolled subjects (N=73) had bilateral infiltrates and underwent bronchoalveolar lavage (BAL) to sample lung immune cells and assay BAL cell cytokine production.
Results
All had sputum culture demonstrating Mycobacterium tuberculosis and 22/73 (30%) had cavities on their chest radiograph. Those with cavities at presentation had higher percent PMN in BAL as well as lower IP-10 (p<0.01) and IL-6 (p=0.013) in BAL cell supernatants, compared to those without cavities. There was no correlation between cavities and other BAL or serum cytokines. IP-10 was negatively associated with BAL PMN. IP-10 and IL-6 expression above median decrease the odds of cavities by 79% and 78% in logistic regression models. IP-10 and IL-6 clustered with IFN-γ and TNF-α in a principal component analysis while IL-4 clustered with PMN.
Conclusion
Increasing IP-10 and IL-6 production by BAL cells is associated with non-cavitary tuberculosis in patients who present with radiographically advanced TB. IP-10 and IL-6 may reflect an effective Th-1 immune control pathway for TB, attenuating tuberculous lung destruction.
doi:10.5588/ijtld.12.0610
PMCID: PMC4050635  PMID: 23743311
biomarkers; cavitary tuberculosis; Th-1; innate immunity
21.  Serum biomarkers as potential predictors of antitumor activity of cetuximab-containing therapy for locally advanced head and neck cancer 
Oral Oncology  2011;47(10):961-966.
Objectives
We sought to identify biomarkers of antitumor activity in patients with locally advanced head and neck cancer treated with therapy containing cetuximab, an epidermal growth factor receptor (EGFR) inhibitor.
Materials and Methods
Patients with stage III-IVB head and neck cancer received cisplatin, docetaxel, and cetuximab (TPE) followed by radiotherapy, cisplatin, and cetuximab (XPE) and maintenance cetuximab in a phase II clinical trial. Serum and tissue biomarkers were examined for treatment-related changes and for association with clinical outcomes.
Results
Concentrations of 31 cytokines, chemokines and growth factors were measured before and after 3 cycles (9 weeks) of induction TPE using multi-analyte immunobead-based profiling (Luminex Corp., Austin, TX), with selected analytes validated by a single analyte enzyme-linked immunosorbent assay.Tumor biomarkers included phosphorylated signal transducer and activator of transcription-3 (pSTAT3), EGFR and human papillomavirus (HPV). Thirty-one patients had baseline biomarkers and 25 had paired samples, pre- and post-TPE. Adjusting for false discovery, 14 analytes including MCP1c, IP-10, Leptin, interleukin (IL)-5, Eotaxin, IL-6, G-CSF, CXCL5 changed significantly post TPE induction. Serum vascular endothelial growth factor (VEGF) and IL-6 levels were associated with tumor response as assessed by positron emission tomography and progression-free survival, however, the association was not significant after adjustment for false discovery. Analytes were not associated with toxicities, smoking history, HPV status, EGFR amplification, or pSTAT3 tumor protein levels.
Conclusions
Baseline serum biomarkers, in particular VEGF and IL-6, were identified as potentially useful prognostic markers of cetuximab-containing therapy. Validation is warranted in future studies specifically designed to detect biomarker associations.
doi:10.1016/j.oraloncology.2011.07.034
PMCID: PMC3402506  PMID: 21889392
head and neck cancer; cytokines; tumor markers; epidermal growth factor receptor; cetuximab; vascular endothelial growth factor; interleukin-6
22.  Identification of M.tuberculosis-Specific Th1 Cells Expressing CD69 Generated in vivo in Pleural Fluid Cells from Patients with Tuberculous Pleurisy 
PLoS ONE  2011;6(8):e23700.
Th1 cell-mediated immune responses at the site of active infection are important to restrict the growth of M.tuberculosis (MTB) and for the spontaneous resolution of patients with tuberculous pleurisy (TBP). In the present study, we found that without any stimulation, CD4+ T cells in pleural fluid cells (PFCs) from patients with TBP expressed significantly higher levels of CD69 than PBMCs from patients with tuberculosis (TB) or healthy donors. CD4+CD69+ T cells expressed T-bet and IL-12Rβ2. After stimulation with MTB-specific antigens, CD4+CD69+ T cells expressed significantly higher levels of IFN-γ, IL-2 and TNF-α than CD4+CD69− T cells, demonstrating that CD4+CD69+ T cells were MTB-specific Th1 cells. In addition, CD4+CD69+ T cells were mostly polyfunctional Th1 cells that simultaneously produced IFN-γ, IL-2, TNF-α and displayed an effector or effector memory phenotype (CD45RA−CCR7−CD62L−CD27−). Moreover, the percentages of CD4+CD69+ T cells were significantly and positively correlated with polyfunctional T cells. Interestingly, sorted CD4+CD69+ but not CD4+CD69− fractions by flow cytometry produced IFN-γ, IL-2 and TNF-α that were significantly regulated by CD4+CD25+ Treg cells. Taken together, based on the expression of CD69, we found a direct quantitative and qualitative method to detect and evaluate the in vivo generated MTB-specific polyfunctional CD4+ T cells in PFCs from patients with TBP. This method can be used for the potential diagnosis and enrichment or isolation of MTB-specific Th1 cells in the investigations.
doi:10.1371/journal.pone.0023700
PMCID: PMC3161751  PMID: 21887301
23.  Higher Frequency of T-Cell Response to M. tuberculosis Latency Antigen Rv2628 at the Site of Active Tuberculosis Disease than in Peripheral Blood 
PLoS ONE  2011;6(11):e27539.
Rationale
Due to the invasive nature of the procedures involved, most studies of Mycobacterium tuberculosis (Mtb)-specific immunity in humans have focused on the periphery rather than the site of active infection, the lung. Recently, antigens associated with Mtb-latency and -dormancy have been described using peripheral blood (PB) cells; however their response in the lung is unknown. The objective of this report was to evaluate, in patients prospectively enrolled with suspected active tuberculosis (TB), whether the latency antigen Rv2628 induces local-specific immune response in bronchoalveolar lavage (BAL) cells compared to PB cells.
Material/Methods
Among the 41 subjects enrolled, 20 resulted with active TB. Among the 21 without active disease, 9 were defined as subjects with latent TB-infection (LTBI) [Quantiferon TB Gold In-tube positive]. Cytokine responses to Rv2628 were evaluated by enzyme linked immunospot (ELISPOT) assay and flow cytometric (FACS) analysis. RD1-secreted antigen stimulation was used as control.
Results
There was a significantly higher frequency of Rv2628- and RD1-specific CD4+ T-cells in the BAL of active TB patients than in PB. However the trend of the response to Rv2628 in subjects with LTBI was higher than in active TB in both PB and BAL, although this difference was not significant. In active TB, Rv2628 and RD1 induced a cytokine-response profile mainly consisting of interferon (IFN)-γ-single-positive over double-IFN-γ/interleukin (IL)-2 T-cells in both PB and BAL. Finally, BAL-specific CD4+ T-cells were mostly effector memory (EM), while peripheral T-cell phenotypes were distributed among naïve, central memory and terminally differentiated effector memory T-cells.
Conclusions
In this observational study, we show that there is a high frequency of specific T-cells for Mtb-latency and RD1-secreted antigens (mostly IFN-γ-single-positive specific T-cells with an EM phenotype) in the BAL of active TB patients. These data may be important for better understanding the pathogenesis of TB in the lung.
doi:10.1371/journal.pone.0027539
PMCID: PMC3213161  PMID: 22102905
24.  Bronchoalveoloar lavage fluid cytokines and chemokines as markers and predictors for the outcome of interstitial lung disease in systemic sclerosis patients 
Arthritis Research & Therapy  2009;11(4):R111.
Introduction
Interstitial lung disease (ILD) is a frequent manifestation of systemic sclerosis (SSc), and cytokines can contribute to the disease pathology. The aim of the current study was to identify specific changes in cytokine levels that may serve as disease markers and possible targets for therapy.
Methods
Cytokines were measured with bioplex analysis in 38 bronchoalveolar fluids (BALFs) from 32 SSc patients (27 with alveolitis and 11 without alveolitis) and 26 control patients. In the case of SSc patients, cytokines were correlated with the respective bronchoalveolar lavage (BAL) cell differentiation, lung function, and thoracic HR-CT score. For 35 BALF samples derived from 29 SSc patients, follow-up investigations of clinical data, lung-function parameter, or thoracic HR-CT scans were available to evaluate the predictive capacity of BALF cytokines and chemokines.
Results
High IL-7 levels were characteristic of SSc-associated interstitial lung disease (ILD) and, in addition, when compared with ILD-negative SSc patients, ILD-positive SSc patients revealed higher IL-4, IL-6, IL-8, and CCL2 (MCP-1) BALF levels. High CCL2 and IL-8 BALF concentrations were associated with neutrophilic and mixed alveolitis. Cytokine levels of IL-4, IL-8, and CCL2 correlated negatively with lung-function parameters; CCL2 concentrations also correlated with HR-CT scores. High concentrations of several cytokines were associated with the progress of ILD and end-stage ILD. Univariate analyses revealed high IL-2 and tumor necrosis factor-alpha (TNF-α) levels as the best predictors for progressive disease, together with lung-function parameters, young age, and neutrophilic alveolitis. Multivariate analyses partially confirmed these results but did not sufficiently converge because of the limited number of patients.
Conclusions
The association of BALF cytokines with lung fibrosis and its progress suggests that cytokines contribute to the pathogenesis of ILD and hence could be regarded as potential therapeutic targets.
doi:10.1186/ar2766
PMCID: PMC2745793  PMID: 19615053
25.  251 Interferon-Gamma Release Assay—Useful Tool for the Diagnosis of Pleural Tuberculosis 
Background
Pleural tuberculosis represents a big problem to diagnose despite of many diagnostic tools. CD4+T-lymphocytes play an imprortant role in immune response against Mycobacterium tuberculosis. They are accumulated in pleural space affected with Mycobacterium tuberculosis and release an inflammatory cytokines (interferon-γ, interleukin-2). These cytokines interact with macrophages to stimulate effective antimycobacterial actions. The pleural fluid lymphocytes and interferon-γ level are elevated in many cases such as viral infections, malignancy or tuberculosis. We decided to evaluate a benefit of utilization of antigen specific Interferon-gamma release assay (IGRA) in pleural effusion (PE) to the diagnostics of pleural tuberculosis.
Methods
We present results of 29 patients with lymphocytic PE of unknown cause. The total leukocytes count, lymfocyte subsets by flow cytometry with monoclonal antibodies directed against the T-lymphocyte antigens (CD3, CD4, CD8) and HLA DR antigen as a marker of T-lymphocyte activation were determined in PE. IGRA test was used to determine a level of specific interferon-gamma in PE and in whole blood too. Furthermore we analysed results of tuberculin skin test and Mycobacterium tuberculosis cultivation in the sputum and in PE.
Results
Of the 29 lymphocytic PE was IGRA test positive in 3 cases (10,3%). These patients had the pleural effusion with activated CD4 + T-lymphocytes predominance, elevated CD4/CD8 ratio. Mycobacterium tuberculosis cultivation was negative in both sputum and pleural fluid except 1 patient with positive sputum culture. The tuberculin skin test was higher than 15 mm of all 3 patients. The clinical diagnosis of active pleural tuberculosis was confirmed in these patients. AntiTB treatment was successful of all them. Lymphocytic PE with negative IGRA test was in 26 patients (89,7%): malignancy in 7, heart failure effusion in 3, parapneumonic effusion in 8 and other pathological causes was in 8 patients.
Conclusions
Our results acknowledge that IGRA test applied to pleural fluid is very helpful to the diagnostics of pleural tuberculosis because the pleural fluid interferon-γ level is measured after stimulation of T-lymphocytes by tuberculous specific antigens. We conclude that another important benefit of interferon-γ release assays is the differential diagnostics of CD4+T-lymphocytic pleural effusions.
doi:10.1097/01.WOX.0000412008.84232.39
PMCID: PMC3512887

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