Identification of host biomarkers from dried blood spots for monitoring treatment response in extrapulmonary tuberculosis.

There is a lack of objective tools for monitoring treatment response in extrapulmonary tuberculosis (EPTB). This study aimed to explore the utility of inflammatory biomarkers from the dry blood spots (DBS) as a tool for monitoring treatment response in EPTB. In a prospective cohort study, 40 inflammatory biomarkers were investigated in DBS samples from 105 EPTB cases using a Luminex platform. The samples were taken before, and, at the end of the 2nd and 6th months of treatment. A total of 11 inflammatory host biomarkers changed significantly with treatment in all EPTB patients. CXCL9/MIG, CCL20, CCL23, CXCL10/IP-10, CXCL1, CXCL2, and CXCL8 significantly declined in our cohort of EPTB (48 TB pleuritis and 57 TB lymphadenitis) patients at both time points. A biosignature consisting of MIG, CCL23, and CXCL2, corresponded with the treatment response in 81% of patients in the 2nd month and 79% of patients at the end of treatment. MIG, CCL23, IP-10, and CXCL2 changed significantly with treatment in all patients including those showing partial clinical response at the 2nd month of treatment. The changes in the levels of inflammatory biomarkers in the DBS correspond with the treatment success and can be developed as a routine test in low-resource settings.

Tissue-Resident Memory-Like CD8+ T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion.

Tissue-resident memory T (TRM) cells are well known to play critical roles in peripheral tissues during virus infection and tumor immunology. Our previous studies indicated that CD69+CD4+ and CD69+CD8+ T cells in tuberculous pleural effusion (TPE) were antigen-specific memory T cells. However, the phenotypical and functional characteristics of CD8+ TRM cells in tuberculosis remain unknown. We found that CD103+CD8+ T cells were the predominant subset of CD103+ lymphocytes in TPE; both CD103 and CD69 expressed on memory CD8+ T cells from TPE were significantly increased compared with those from paired peripheral blood. Phenotypically, CD103+CD69+ and CD103+CD69-CD8+ T cells expressed higher levels of CD45RO than CD103-CD69+CD8+ T cells did; CD103+CD69-CD8+ T cells highly expressed CD27, CD127, and CD62L and some chemokine receptors. We further compared the functional differences among the four distinct CD45RO+CD8+ T subsets identified by CD103 and CD69 expression. In consist with our published results, CD69+CD8+ T cells, but not CD103+CD8+, produced high levels of IFN-γ after treatment with BCG in the presence of BFA. Nevertheless, CD103-CD69+ and CD103+CD69+ memory CD8+ T cells expressed higher levels of Granzyme B, while CD103+CD69- memory CD8+ T cells were characterized as a possibly immunosuppressive subset by highly expressing CTLA-4, CD25, and FoxP3. Furthermore, TGF-ß extremely increased CD103 expression but not CD69 in vitro. Together, CD103+CD8+ T cells form the predominant subset of CD103+ lymphocytes in TPE; CD103 and CD69 expression defines distinct CD8+ TRM-like subsets exhibiting phenotypical and functional heterogeneity. Our findings provide an important theoretical basis to optimize and evaluate new tuberculosis vaccines.

Development and validation of a novel scoring system developed from a nomogram to identify malignant pleural effusion.

BACKGROUND: This study aimed to establish and validate a novel scoring system based on a nomogram for the differential diagnosis of malignant pleural effusion (MPE) and benign pleural effusion (BPE). METHODS: Patients with PE and confirmed aetiology who underwent diagnostic thoracentesis were included in this study. One retrospective set (N = 1261) was used to develop and internally validate the predictive model. The clinical, radiological and laboratory features were collected and subjected to logistic regression analyses. The primary predictive model was displayed as a nomogram and then modified into a novel scoring system, which was externally validated in an independent set (N = 172). FINDINGS: The novel scoring system was composed of fever (3 points), erythrocyte sedimentation rate (4 points), effusion adenosine deaminase (7 points), serum carcinoembryonic antigen (CEA) (4 points), effusion CEA (10 points) and effusion/serum CEA (8 points). With a cutoff value of 15 points, the area under the curve, specificity and sensitivity for identifying MPE were 0.913, 89.10%, and 82.63%, respectively, in the training set, 0.922, 93.48%, 81.51%, respectively, in the internal validation set and 0.912, 87.61%, 81.36%, respectively, in the external validation set. Moreover, this scoring system was exclusively applied to distinguish lung cancer with PE from tuberculous pleurisy and showed a favourable diagnostic performance in the training and validation sets. INTERPRETATION: This novel scoring system was developed from a retrospective study and externally validated in an independent set based on six easily accessible clinical variables, and it exhibited good diagnostic performance for identifying MPE. FUNDING: NFSC grants (no. 81572942, no. 81800094).

The comparison of pleural fluid TNF-α levels in tuberculous and nontuberculous pleural effusion.

BACKGROUND: Tuberculous pleural effusion is the manifestation of Mycobacterium tuberculosis infection in pleura. With existing means, it is difficult to establish the diagnosis of tuberculosis (TB) and non-TB pleural effusions; thus, establishing the diagnosis of TB pleural effusion and non-TB pleural effusion is still a clinical problem. Tumour necrosis factor alpha (TNFα) is a potent inflammatory cytokine that plays an important role in immunity to Mycobacterium tuberculosis infections, this level of cytokine increases in pleural effusion due to tuberculosis. OBJECTIVE: To compare the TNF-α level of pleural fluid in TB and non-TB pleural effusion. METHODS: The samples in this study that fulfilled the inclusion criteria were patients with non-TB pleural tuberculosis effusion in the inpatient ward in Pulmonology Unit Dr. Soetomo General Hospital Surabaya, male and female, aged between 15 and 60 years. The data is divided into two: primary data and secondary data of patients who fulfilled inclusion and exclusion criteria. The data with normal distribution was analyzed using independent t2 test and if the data distribution is abnormal, it was analyzed using Fisher's exact test. RESULTS: There were 22 subjects divided into 2 groups that were 11 patients with TB pleural effusion and 11 patients with non-TB pleural effusion. The TNF-α level of pleural fluid in TB pleural effusion was 25.43±13.55pg/mL. The TNF-α level of pleural fluid in non-TB was 5.98±1.89pg/mL. The serum TNF-α level in TB pleural effusion was 83.22±88.15pg/mL. The serum TNF-α level in non-TB was 68.54±57.88pg/mL. There was higher level of TNF-α pleural fluid in TB pleural effusion than in non-TB pleural effusion (25.43±13.55pg/mL vs 5.98±1.89pg/mL, p value <0.05). The serum TNF-α level in patients with TB pleural effusion was higher than TNF-α serum level of non-TB pleural effusion. There was no significant difference between TNF-α level of pleural fluid and serum TNF-α levels in the TB pleural effusion group (p value >0.05). CONCLUSION: The TNF-α level of pleural fluid in TB pleural effusion was higher than non-TB pleural effusions and there was no significant difference between serum TNF-α levels in the TB pleural effusion group and in the non-TB pleural effusion group.

Differential diagnosis of tuberculous and malignant pleural effusions: comparison of the Th1/Th2 cytokine panel, tumor marker panel and chemistry panel.

The differentiation between tuberculous plural effusion (TPE) and malignant plural effusion (MPE) remains a major clinical challenge in the diagnosis and management of pleural effusions, especially in developing countries with a high incidence of tuberculosis. We aimed to evaluate the diagnostic value of cytokines, tumor markers and biochemical markers in the differentiation of TPE and MPE. Two hundred and forty-two patients were included, of whom 134 were diagnosed with MPE and 108 were diagnosed with TPE. In total, 12 markers were tested in pleural effusion samples from all subjects: Interleukin-2 (IL-2), Tumor necrosis factor alpha (TNF-α), Interferon (IFN)-γ, interleukins-4, 6, 10 (IL-4,6,10), cytokeratin-19 fragment (CYFRA 21-1), carcinoembryonic antigen (CEA), neuron specific enolase (NSE), adenosine deaminase (ADA), lactate dehydrogenase (LDH) and high sensitivity C- reactive protein (Hs-CRP). The diagnostic value of each marker was evaluated and compared by receiver operating characteristic (ROC) curves. In the 12 markers evaluated, TNF-α, IFN-γ, IL-6, CYFRA 21-1, CEA, ADA and Hs-CRP were significantly different between the TPE and MPE groups, and the areas under the ROC curves were 0.624, 0.942, 0.619, 0.808, 0.903, 0.842 and 0.917, respectively. IFN-γ showed a better diagnostic performance than the other markers. With a cut-off value of >2.45 pg/mL, the sensitivity and specificity of IFN-γ were 91.11 and 91.94%, respectively. TNF-α, IFN-γ, IL-6, CYFRA 21-1, CEA, ADA and Hs-CRP were useful in the differentiation between the TPE and MPE groups. IFN-γ showed a better diagnostic performance than the multitude of other markers evaluated in this study, which is satisfactory for the discrimination of TPE and MPE.

Serial anti-tuberculous immune responses during the follow-up of patients with tuberculous pleurisy.

Little is known about the decay kinetics of interferon (IFN)-γ response and its influencing factors in tuberculous pleurisy. We enrolled thirty-two patients with tuberculous pleurisy prospectively and followed up at month 0, 6, and 9, at which time peripheral venous blood was drawn for interferon gamma release assay (IGRA) by means of QuantiFERON-TB Gold In-Tube (QFT-GIT). Demographic and clinical data were captured. To identify significant predictive factors influencing the IFN-γ response, multiple linear regression analyses were performed. Percentage of CD4+, CD8+, Vγ2Vδ2 T cells and Treg cells were measured by flow cytometry. The percentage of QFT-GIT-positive patients at baseline, month 6 and month 9 were 96.9% (30/32), 90.6% (29/32) and 84.4% (27/32), respectively. Quantitative IFN-γ response at baseline were significantly correlated with symptom duration (P = .003, R = 0.261) and age (P = .041, R = 0.132). Besides, the decreases of the IFN-γ response at month 6 and month 9 were positively correlated with the IFN-γ level at baseline. The dynamic tendency of the percentages of Treg cells was similar to the IFN-γ responses at each time-point. Quantitative IFN-γ response could be influenced by host immune status, instead of disease burden and anti-tuberculosis treatment. IGRA is probably not a useful biomarker of treatment efficacy in tuberculous pleurisy.

Evidence that changes in antimicrobial peptides during tuberculosis are related to disease severity, clinical presentation, specific therapy and levels of immune-endocrine mediators.

Given the role of host defense peptides (HDPs) in the defensive response against mycobacteria, we analyzed the circulating levels of LL-37, ß-defensin-2 and -3 in newly diagnosed patients with pulmonary (PTB) or pleural tuberculosis (PLTB) in whom measurements of pleural fluids were also performed. Severe PTB patients displayed higher circulating amounts of ß-defensin-3, statistically different from controls, further decreasing upon antimycobacterial treatment. LL-37 concentrations appeared within the normal range at diagnosis, but tended to increase during treatment, becoming statistically upon its completion in moderate cases. PLTB patients revealed decreased levels of ß-defensin-2 in presence of increased amounts of ß-defensin-3 and LL-37; in their plasma or pleural fluids. Considering the immune-endocrine dysregulation of tuberculosis, we also performed correlation analysis detecting positive associations between levels of cortisol, IL-6 and ß-defensin-3 in plasma from untreated severe patients as did their dehydroepiandrosterone and LL-37 values. Increased presence of ß-defensins, may represent an attempt to improve defensive mechanisms; which also take part in the inflammatory reaction accompanying TB, reinforced by the association with immune-endocrine mediators. The divergent profile of PLTB patients, decreased ß-defensin-2 but increased ß-defensin-3 and LL-37 levels, suggests a differential role of these HDPs in a situation characterized for its better protective response.

High-throughput autoantibody analysis in malignant pleural effusion and tuberculosis pleural effusion.

BACKGROUND: Malignant pleural effusion (MPE) and tuberculosis pleural effusion (TPE) are 2 kinds of common pleural diseases. Finding efficient and accurate biomarkers to distinguish the 2 is of benefit to basic and clinical research. In the present study, we carried out the first high-throughput autoantibody chip to screen the beneficial biomarker with samples of MPE and TPE and the corresponding serum. METHODS: We collected pleural effusion and serum of patients with MPE (n = 10) and TPE (n = 10) who had been in Beijing Chao-Yang hospital from June 2013 to August 2014. Using RayBio Human Protein Array-G2 to measure the concentration of 487 defined autoantibodies. RESULTS: Fold changes of Bcl-2-like protein 11 (BIM) autoantibody in MPE-serum/TPE-serum and MPE/TPE groups were 10 (P = .019) and 6 (P = .001); for decorin autoantibody, MPE-serum/TPE-serum ratio was 0.6 (P = .029), and MPE/TPE ratio was 0.3 (P < .001). CONCLUSION: BIM autoantibody is a promising MPE biomarker by high-throughput autoantibody analysis in MPE and TPE.

IgA and IgG antibody detection of mycobacterial antigens in pleural fluid and serum from pleural tuberculous patients.

BACKGROUND: A previous study demonstrated pleural fluid (PF) IgA immunodominance for the fused MT10.3:MPT64 protein in pleural tuberculosis (PLTB) cases. However, no clue on the role of IgA and IgG against this and other antigens in PF and serum concerning improved diagnosis is available. Thus, the aim of the present study was to validate PF IgA-MT10.3:MPT64 and evaluate PF and serum IgA and IgG reactivity against this protein, its peptides (F2) and single MPT64, MT10.3 and the PPE59 mycobacterial specific antigens. IgA and IgG ELISA were measured against the antigen in PLTB (n = 29) and other non-TB pleurisy (n = 39) patient samples. RESULTS: The immunodominance of PF IgA-MT10.3:MPT64 was confirmed in PLTB (86.2%) followed by PPE59 (62%), while serum IgA-F2 exhibited 51.7% sensitivity. PF and serum IgG-MT10.3:MPT64 led to 65.5 and 51.7% sensitivity, respectively. However, MT10.3 and MPT64 displayed overall lower sensitivity (≤34.5) for both antibodies. All results at 95% fixed specificity. Combinatory results indicated 93.1% sensitivity for PF IgA-MT10.3:MPT64/-PPE59 and IgA/IgG-MT10.3:MPT64 at 92.3% specificity, followed by IgA-MT10.3:MPT64/-MPT64 or /-F2 (89.6%) without jeopardizing specificity (94.9%). The combinatory results of the PF adenosine deaminase test (ADA) and IgA-MT10.3:MPT64/-F2 demonstrated the highest sensitivity (96.6%), with a specificity of 92.3%. CONCLUSIONS: The PF IgA-MT10:MPT64 immune dominance was validated in PLTB, and its combinatory results with PPE59 or MPT64 or F2 antigens as well as with IgG, are reported herein for the first time, improving their potential to assist diagnosis. Combining PF-ADA and IgA-MT10.3:MPT64/-F2 results achieved better accuracy. Moreover, serum IgG, although less accurate, displays potential beyond microbiological tests.

Diagnostic accuracy of interleukin-22 and adenosine deaminase for tuberculous pleural effusions.

OBJECTIVE: Reliable markers for accurately diagnosing tuberculous pleural effusions (TPE) are needed. This study sought to investigate the diagnostic potential of pleural interleukin-22 (IL-22) and compare it with the performance of adenosine deaminase (ADA). METHOD: This prospective study involved 49 patients with TPE and 60 patients with pleural effusion of other causes. Pleural levels of IL-22 and ADA were determined, respectively, using ELISA or an enzymatic method. A receiver operating characteristic curve was constructed and the area under the curve (AUC) was calculated to summarize the diagnostic accuracy of single markers or marker combinations. RESULTS: Levels of IL-22 in pleural effusion were significantly higher in TPE patients than in other patients (322.36 ± 406.65 vs. 83.13 ± 22.15 pg/ml, P < 0.05). With a cut-off value of 97.82 pg/ml, the diagnostic sensitivity of IL-22 for TPE was 71.42%, specificity was 81.67%, and the area under the curve (AUC) was 0.83. ADA levels were also increased in TPE, and its AUC for diagnosing TPE was 0.90. The combination of IL-22 and ADA enhanced diagnostic accuracy, offering sensitivity of 83.67%, specificity of 91.67%, and an AUC of 0.93. CONCLUSION: IL-22 may be useful for diagnosing TPE, and combining it with ADA may further enhance diagnostic accuracy. Our results justify more rigorous studies with larger samples to confirm the diagnostic potential of IL-22 for TPE.

Vitamin D3 Status and the Association with Human Cathelicidin Expression in Patients with Different Clinical Forms of Active Tuberculosis.

Low vitamin D (vitD3) is one of the most common nutritional deficiencies in the world known to be associated with numerous medical conditions including infections such as tuberculosis (TB). In this study, vitD3 status and its association with the antimicrobial peptide, human cathelicidin (LL-37), was investigated in Ethiopian patients with different clinical forms of TB. Patients with active TB (n = 77) and non-TB controls (n = 78) were enrolled in Ethiopia, while another group of non-TB controls (n = 62) was from Sweden. Active TB included pulmonary TB (n = 32), pleural TB (n = 20), and lymph node TB (n = 25). Concentrations of 25-hydroxyvitamin D3 (25(OH)D3) were assessed in plasma, while LL-37 mRNA was measured in peripheral blood and in samples obtained from the site of infection. Median 25(OH)D3 plasma levels in active TB patients were similar to Ethiopian non-TB controls (38.5 versus 35.0 nmol/L) and vitD3 deficiency (.

The neuro-endocrine-immune relationship in pulmonary and pleural tuberculosis: a better local profile in pleural fluid.

BACKGROUND: Tuberculosis (TB) is a major health problem worldwide. In TB, the immune and central nervous systems modulate each other. The two main components of this network are the hypothalamic-pituitary-adrenal axis (HPA) and autonomic nervous system (ANS). OBJECTIVE: To elucidate neuro-endocrine-immune (NEI) interactions in pulmonary (PTB) or pleural (PLTB) TB, we analysed the relationship among compounds from these systems. METHODS: We quantified levels of catecholamines, hormones and cytokines in plasma from patients with PTB (n = 46) or PLTB (n = 12) and controls (n = 32), and in the pleural fluid from PLTB patients. Transcript expression for genes involved in glucocorticoid-related function (quantitative real-time polymerase chain reaction) was also analysed in mononuclear cells (MCs) from peripheral blood (PBMC) or pleural effusion (PEMC) compartments. RESULTS: Both patient groups had increased plasma levels of pro- and anti-inflammatory cytokines, cortisol, growth hormone (GH) and dopamine, whereas insulin-like growth factor 1 (IGF-1) and dehydroepiandrosterone levels were decreased. The pleural fluid contained increased levels of pro-inflammatory cytokines, GH and IGF-1 and reduced levels of steroid hormones compared with their plasma counterparts. PBMCs from PTB patients had increased expression of transcripts for 11ß-hydroxysteroid dehydrogenase (11ßHSD1) and a decreased glucocorticoid receptor (GR) ratio (GRα/GRß). In PLTB cases, expression of 11ßHSD1 and GRα transcripts was higher in PEMCs. CONCLUSION: PTB patients seem to display adverse NEI dysregulation. Changes in pleural fluid are compatible with a more effective NEI reaction.

The expressions and roles of different forms of IL-22 in Mycobacterium tuberculosis infection.

Despite evidence suggesting an anti-Mycobacterium tuberculosis effector function of CD4+ T cells that produce and retain IL-22 in macaques, the general role of IL-22 in tuberculosis infection is still poorly characterized. To explore the immune mechanism in the pathogenesis of tuberculosis in humans, here we evaluated different forms of IL-22 in populations with different tuberculosis infection statuses. We enrolled 156 subjects including 49 patients with pulmonary tuberculosis, 27 patients with tuberculous pleurisy (TPE), 38 individuals with latent tuberculous infection (LTBI) and 42 healthy controls (HC). We found significantly higher IL-22 levels at the tuberculosis infection site than in the peripheral blood as well as higher antigen-specific IL-22 levels in the culture supernatant for patients with active tuberculosis than in healthy controls. The proportions of IL-22 + CD4+ T and IL-22 + CD8+ T cells in patients with active tuberculosis were significantly higher than those in the latent tuberculosis infection group and the healthy control group, based on intracellular cytokine staining. However, surprisingly, we found membrane-bound IL-22+ T cells, including CD4+ T cells and CD8+ T cells, by surface staining, especially in patients with active tuberculosis. Furthermore, the expression of membrane-bound IL-22 significantly decreased after drug therapy. In conclusion, our results suggest that IL-22 has various roles in tuberculosis immune responses. In particular, membrane-bound IL-22+ T cells may play important roles in the human immune response to Mycobacterium.

Diagnostic Significance of Measuring Vascular Endothelial Growth Factor for the Differentiation between Malignant and Tuberculous Pleural Effusion.

Malignancy and tuberculosis are common causes of lymphocytic exudative pleural effusion. However, it is occasionally difficult to differentiate malignant pleural effusion from tuberculous pleural effusion. Vascular endothelial growth factor (VEGF) is a critical cytokine in the pathogenesis of malignant pleural effusion. Endocan is a dermatan sulfate proteoglycan that is secreted by endothelial cells. Importantly, endocan mediates the vascular growth-promoting action of VEGF. The aim of this study was to evaluate the diagnostic significance of VEGF and endocan in pleural effusion. We thus measured the levels of VEGF and endocan in the pleural effusion and serum samples of patients with lung cancer (n = 59) and those with tuberculosis (n = 32) by enzyme-linked immunosorbent assay. Lung cancer included 40 cases of adenocarcinoma, 13 of squamous cell carcinoma, and 6 of small cell carcinoma. Pleural effusion VEGF levels were significantly higher in the malignant group than in the tuberculosis group (2,091.47 ± 1,624.80 pg/mL vs. 1,291.05 ± 1,100.53 pg/mL, P < 0.05), whereas pleural effusion endocan levels were similar between the two groups (1.22 ± 0.74 ng/mL vs. 0.87 ± 0.53 ng/mL). The areas under the curve of VEGF and endocan were 0.73 and 0.52, respectively. Notably, the VEGF levels were similar in malignant pleural effusion, irrespective of the histological type of lung cancer. Moreover, no significant difference was found in the serum VEGF and endocan levels between patients with lung cancer and those with tuberculosis. In conclusion, high VEGF levels in pleural effusion are suggestive of malignant pleural effusion.

Utility of Macrophage-activated Marker CD163 for Diagnosis and Prognosis in Pulmonary Tuberculosis.

RATIONALE: Among infectious diseases, tuberculosis (TB) is a leading cause of death worldwide. Accumulated knowledge has revealed that macrophages are deeply involved in the progression and pathogenesis of TB. We hypothesized that the evaluation of a macrophage activation marker may be useful in the diagnosis and assessment of pulmonary TB. OBJECTIVES: To examine the utility of the macrophage activation marker soluble CD163 (sCD163) as a diagnostic tool and measure of disease severity for pulmonary TB and tuberculous pleurisy. METHODS: We compared the concentration of sCD163 in serum samples of 180 patients with active pulmonary TB with concentrations in serum samples of 45 age- and sex-matched control subjects. We also measured sCD163 in pleural fluid samples of 100 patients with pleural disease, including 31 patients with tuberculous pleurisy. MEASUREMENTS AND MAIN RESULTS: We found increased serum concentrations of sCD163 in patients with active pulmonary TB compared with those of control subjects (1,643 ± 1,737 ng/ml vs. 533.9 ± 49.3 ng/ml; P < 0.0001). sCD163 levels were also higher in pleural fluid samples of patients with pulmonary TB than in those of patients with non-TB pleurisy (5,239 ± 2,436 ng/ml vs. 2,877 ± 1,191 ng/ml; P < 0.0001). The levels of sCD163 in pleural effusions were significantly higher than serum levels obtained simultaneously from the same patients, particularly for patients with tuberculous pleurisy. Patients with a serum level of sCD163 above 1,300 ng/ml, had a mortality rate that was five times higher than that of patients with a lower sCD163 level (44.6% vs. 6.6%; P < 0.0001 by log-rank test). Microscopic examination of lung and pleural tissue samples showed concordance of enhanced CD163 expression with the presence of caseating granulomas in tissue obtained from patients with TB. CONCLUSIONS: The macrophage activation marker CD163 was increased in patients with active pulmonary TB compared with age- and sex-matched control subjects. Increased levels of sCD163 were associated with increased mortality in patients with pulmonary TB. sCD163 also showed promise as a diagnostic tool for tuberculous pleurisy. These results warrant further study of sCD163 as a potentially useful biomarker for the diagnosis and assessment of pulmonary TB. Clinical trial registered with www.umin.ac.jp/ctr/index-j.htm (UMIN000003400).

[Interleukin-20 and interleukin-22 in pleural effusion].

OBJECTIVE: To investigate the concentrations and clinical significance of interleukin (IL)-20 and IL-22 in pleural effusion with various etiologies. METHODS: Pleural effusion (PE) and corresponding serum samples were obtained from 88 patients from Wuhan Tuberculosis Prevention and Control Institute from June 2011 to June 2013. There were 27 cases with malignant pleural effusion, 24 with tuberculous pleural effusion, 17 with bacterial pleural effusion and 20 with transudativeeffusion. The pleural and serum levels of IL-20 and IL-22 were determined by sandwich enzyme-linked immunosorbent assays (ELISA). RESULTS: (1) Except for transudativeeffusion, the concentration of IL-20 in malignant pleural effusion (36.8±5.1) ng/L, tuberculous pleural effusion (34.8±6) ng/L, bacterial pleural effusion (41.7±20.2) ng/L, were significantly higher than that of the corresponding serum concentration (29.7±5.97) ng/L, (27.3 ±6.7) ng/L, (25.6±4.7) ng/L (t=5.044, 3.804, 3.452, P<0.05). However, the concentration of IL-20 in pleural effusions of different causes showed no significant difference; malignant (36.8±5.1) ng/L, tuberculous(34.8±6.0) ng/L, bacterial (41.7±20.2) ng/L, transudate (34.1±7.3) ng/L (P>0.05). The concentration of IL-22 (median, quartiles) in tuberculouseffusion was 146.1 (39.8) ng/L and bacterial effusion 59.6 (484.3) ng/L was significantly higher than those in the corresponding serum concentrations 18.7 (9.8) ng/L, 15.7 (17.2) ng/L (Z value respectively -3.971, -3.290, P<0.05). The concentration of IL-22 in tuberculous pleural effusion, bacterial pleural effusion, transudative pleural effusion was significant higher than those in malignant pleural effusion respectively (all P<0.001). (2)The concentrations of IL-22 in malignant pleural effusion was correlated positively with those in serum (r=0.729, P<0.001). (3) With a cut-off value of 19.7 ng/L, pleural IL-22 exhibited a high sensitivity and specificity of 95.1% (39/41) and 88.9%(24/27) respectively, when used for distinguishing infectious pleural effusion (including tuberculous and bacterial effusion) from malignant pleural effusion (P<0.001). CONCLUSIONS: Higher levels of IL-22 in tuberculous and bacterial pleural effusion were found when compared with corresponding serum levels and might be involved in the pathogenesis of infectious pleural effusion. Pleural IL-22 measurement provided reliable diagnostic efficiency for distinguishing infectious from malignant pleural effusion.

Potential diagnostic value of serum/pleural fluid IL-31 levels for tuberculous pleural effusion.

The aim of this study was to explore the diagnostic value of IL-31 levels in the pleural fluid and plasma to differentially diagnose tuberculous and malignant pleural effusion. We enrolled 91 cases, including tuberculous pleural effusion (TPE, n = 50), malignant pleural effusion (MPE, n = 41), other cases including pneumonia with pleural fluid, pulmonary tuberculosis and healthy people as controls. Whole blood was stimulated with the M. tuberculosis-specific antigens and plasma was collected. The multiplex bead-based cytokine immunoassay was employed to measure the levels of various cytokines. IL-31 was found to be the most prominent cytokine (P < 0.0001), and with an optimal cut-off value of 67.5 pg/mL, the sensitivity and specificity for the diagnosis of TPE were 86% and 100%, respectively. Furthermore, the tuberculosis-specific IL-31 levels in the plasma of TPE patients were higher than that of MPE patients (P = 0.0002). At an optimal cut-off value of 23.9 pg/mL, the sensitivity and specificity for the diagnosis of TPE were 92.9% and 85.7%, respectively. Ultimately, the combination of pleural fluid with the plasma tuberculosis-specific IL-31 levels improved the sensitivity and specificity to 94.0% and 95.1%, respectively. Thus, we identified a novel biomarker for the diagnosis of TPE for clinical application.

Use of lateral flow assays to determine IP-10 and CCL4 levels in pleural effusions and whole blood for TB diagnosis.

One of the key problems in combating TB is the lack of fast and accurate diagnostic tests that are affordable and easy to use in resource-limited settings. We have used a field-friendly up-converting phosphor (UCP) reporter technology in a lateral flow (LF) based test for the diagnosis of respiratory infections. In this study we analysed samples obtained from patients presenting with symptoms suggestive of TB but prior to confirmation by microbiology in The Gambia. Following clinical and microbiological evaluation they were classified as either having TB or other respiratory disorder (ORD). Analysis of blood was performed for those with pulmonary TB and pleural fluid for those with pleural TB. UCP-LF test for detection and quantitation of IP-10 and CCL4 were used being the two chemokine markers that have been shown to increase in active TB disease. UCP-LF test accurately determined concentrations of both markers as compared to ELISA and multiplex cytokine array. However, only IP-10 could discriminate between TB and ORD, and this was significantly enhanced by analysing the site of infection (pleural fluid), which showed 92% correct classification. Future work will assess the use of multiple markers to increase diagnostic accuracy.

Short communication: circulating plasma HIV-1 viral protein R in dual HIV-1/tuberculosis infection.

Circulating free HIV-1 viral protein R (Vpr) is found in up to one third of subjects with HIV-1 infection. Free Vpr presumably shares some of the immunopathogenic effects of cell-associated Vpr. Here we assessed Vpr in plasma and pleural fluid from HIV/tuberculosis (TB) dually infected subjects with pleural TB and from plasma of patients with pulmonary HIV/TB. Vpr was assessed by western blot analysis. In plasma from HIV/TB subjects with pulmonary TB free Vpr could be detected in 47%. Only one subject, among 26 tested, with HIV monoinfection showed plasma Vpr activity. The majority (87.5%) of patients with pleural HIV/TB demonstrated free Vpr reactivity in their plasma. However, no Vpr activity was found in autologous pleural fluid samples from pleural HIV/TB patients. Standard (s) Vpr reactivity was reduced markedly by the addition of sVpr to pleural fluid from HIV-uninfected subjects. A high incidence of plasma Vpr reactivity in HIV/TB patients implies heightened processing and release of this HIV-1 accessory protein during HIV/TB coinfection. The contribution of free Vpr to HIV-1 immunopathogenesis during HIV/TB needs to be studied.

Different subsets of macrophages in patients with new onset tuberculous pleural effusion.

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)-ß1, 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.