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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.

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.

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.

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.

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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.

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.

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.

Pleural effusion is one of the commonest presentations of tuberculosis, the clinical manifestations being typically abrupt resembling bacterial pneumonia. Since delayed hypersensitivity is the underlying immune response, bacterial load is very low. Owing to these facts, tuberculous pleurisy as an extra-pulmonary disease poses a diagnostic dilemma. The conventional bacteriological methods rarely detect Mycobacterium tuberculosis in pleural fluid and are of limited use in diagnosis of tuberculous pleurisy. We evaluated the efficacy of polymerase chain reaction (PCR) in the diagnosis of tuberculous pleurisy by targeting the gene segment coding for MPB64 protein specific forMycobacterium tuberculosis. Based on the clinical criteria, 82 patients with lymphocytic exudative pleural effusion were included in the study. Patients were analyzed in two groups; one group consisting of 48 patients of tubercular pleural effusion confimed by various diagnostic procedures and another group of 34 patients comprising of non-tubercular pleural effusion. There were no false positive results by PCR and the specificity worked out to be 100%. Twenty two patients tested positive for Mantoux with a sensitivity of 45%. ZN-staining for AFB was found in samples from 15 patients (20% sensitivity). ADA was positive for 28 patients with a sensitivity of 53%. PCR was positive for 32/48 patients (67% sensitivity). Thus, PCR was found to be more sensitive than any other conventional method in diagnosis of clinically suspected tubercular pleurisy.

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.

Background

The study was designed to investigate the clinical usefulness of Amplified Mycobacterium Tuberculosis Direct (AMTD) tests for diagnosing TB pleurisy.

Methods

One hundred and fifty-two patients for whom the exclusion of tuberculous pleural effusion was necessary were retrospectively analyzed.

Results

The sensitivity of AMTD in diagnosing pleural TB was 36.4% (20 of 55). Combining sputum and pleural effusion AFB smear, pleural biopsy, and AMTD test of pleural effusion increased sensitivity to 82.5% (33/40). There were significantly higher percentages of neutrophils in the pleural effusion in the positive than in the negative AMTD group (38.0±6.7% vs. 11.1±3.7%, p<0.001). Patients with symptom duration <18 days prior to pleural effusion studies had more positive AMTD tests than those with symptom >18 days (70% vs. 31.4%; OR 5.09; 95% CI 1.54–16.79; p = 0.011).

Conclusions

Combining AMTD tests with conventional diagnostic methods offer good sensitivity for pleural TB diagnosis. Patients in the early course of the disease are better candidates for AMTD tests.

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.

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.

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.

BACKGROUND

The prevalence of hypercalcemia has not previously been determined in newly diagnosed tuberculosis (TB) patients in Nigeria.

OBJECTIVE

To determine the incidence of hypercalcemia in Nigerian patients with newly diagnosed TB before the commencement of anti-TB treatment.

METHODS

The present study is a prospective examination of consecutive patients with newly diagnosed TB confirmed by bacteriological and/or histological methods at the National Hospital (Abuja, Nigeria) from January 2004 to December 2004.

RESULTS

Of 120 patients (70 males and 50 females), 70 had pulmonary TB, 10 had pulmonary and pleural TB, 20 had pleural TB without radiographic evidence of lung involvement, 18 had various other forms of extrapulmonary TB and two had disseminated TB. The mean age of the patients was 38.3±12.0 years. The mean albumin-adjusted serum calcium concentration was 2.53±0.22 mmol/L. Hypercalcemia was present in 27.5% of the patients, but only 12% of these patients showed symptoms of hypercalcemia. The type of TB and, in the case of pulmonary TB, the extent of lung involvement, had no effect on the serum calcium concentration.

CONCLUSION

Hypercalcemia is not uncommon among Nigerian patients with newly diagnosed TB, but it is rarely symptomatic.

BACKGROUND: Lymphocytes have a central role in human defences against mycobacteria. A study was designed to assess the relation between lymphocyte responses and clinical pattern of disease, nutrition and recovery during treatment in patients with tuberculosis. METHODS: Lymphocyte numbers and subsets (on the basis of CD3, CD4, and CD8 monoclonal antibodies) were measured in peripheral blood and, where appropriate, bronchoalveolar lavage or pleural fluid of patients with different forms of pulmonary tuberculosis. Eleven had localised pulmonary tuberculosis, 18 miliary tuberculosis and seven a tuberculous pleural effusion. RESULTS: CD4 lymphocytes were found in greatly increased numbers in pleural fluid and were relatively depleted in the blood. Lymphocyte numbers in bronchoalveolar lavage fluid varied widely in localised pulmonary and miliary tuberculosis but were highest in lavage fluid from patients with miliary tuberculosis. This was due to an increase in CD8 lymphocytes, which were also increased in the blood. Lymphocyte numbers bore no relation to nutrition, symptom duration, or radiographic profusion scores. In miliary tuberculosis the time taken for the chest radiograph to clear (mean (SD) 17.6 (7.8) weeks) correlated with lymphocyte numbers in lavage fluid, especially CD8 cells (r = 0.74), but not with the patients' age or nutrition. After 8 weeks' treatment, total and CD4 lymphocyte numbers in lavage fluid showed a substantial increase. CONCLUSION: The association of CD8 cells with delayed recovery is compatible with suppression of the antimycobacterial action of macrophages. The switch to predominance of CD4 cells in lavage fluid during successful treatment supports the view that they may have a role in eliminating mycobacteria.

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BACKGROUND--Pleural biopsy is usually considered important for the diagnosis of pleural effusions, especially for distinguishing between tuberculosis and neoplasia, even though tuberculous pleural fluid contains sensitive biochemical markers. In regions with a high prevalence of tuberculosis, and in patient groups with a low risk of other causes of pleurisy, the positive predictive value of these markers is increased. The criteria for performing a pleural biopsy under these circumstances have been investigated, using adenosine deaminase (ADA) as a pleural fluid marker for tuberculosis. METHODS--One hundred and twenty nine patients with a pleural effusion aged < or = 35 years (mean (SD) 25.2 (4.9) years) were studied. Seventy three were men. Eighty one effusions (62.8%) were tuberculous, 12 (9.3%) parapneumonic, and 10 (7.7%) neoplastic, five were caused by pulmonary thromboembolism, four by systemic lupus erythematosus, seven by empyema, three following surgery, one was the result of asbestosis, and one of nephrotic syndrome. In five cases no definitive diagnosis was reached. ADA levels were determined by the method of Galanti and Giusti. RESULTS--The diagnostic yield of procedures not involving biopsy was 94.5% (122/129). Pleural biopsy provided a diagnosis in a further two cases, but not in the remaining five. All tuberculous cases had pleural fluid levels of ADA of > 47 U/l (mean (SD) 111.1 (36.6) U/l). The only other cases in which ADA exceeded this level were six of the seven patients with empyema. Cytological examination of the pleural fluid diagnosed eight of the 10 neoplastic cases, compared with six diagnosed by pleural biopsy. CONCLUSIONS--In a region with a high prevalence of tuberculosis procedures not involving pleural biopsy have a very high diagnostic yield in patients with a pleural effusion aged < or = 35 years, making biopsy necessary only in cases in which pleural levels of ADA are below 47 U/l, pleural fluid cytology is negative and, in the absence of a positive basis for some other diagnosis, neoplasia is suspected.

Background

The pathogenesis of primary tuberculous pleurisy is a delayed-type hypersensitivity immunogenic reaction to a few mycobacterial antigens entering the pleural space rather than direct tissue destruction by mycobacterial proliferation. Although it has been shown that pulmonary tuberculosis induces 18-fluorodeoxyglucose (FDG) uptake in active lesions, little is known about the application of FDG positron emission/computed tomography (FDG PET/CT) to the management of primary tuberculous pleurisy.

Case presentation

We report a case of asymptomatic primary tuberculous pleurisy presenting with diffuse nodular pleural thickening without distinct pleural effusion and parenchymal lung lesions mimicking malignant mesothelioma. An initial FDG PET/CT scan demonstrated multiple lesions of intense FDG uptake in the right pleura and thoracoscopic biopsy of pleural tissue revealed caseous granulomatous inflammation. The patient received antituberculous therapy for 6 months, with clearly decreased positive signals on a repeated FDG PET/CT scan.

Conclusion

FDG PET/CT imaging may be useful for evaluating disease activity in tuberculous pleurisy patients with an unknown time of onset.

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.

Background:

Sputum for acid fast bacilli (AFB) is seldom looked for in the etiological diagnosis of tuberculous pleural effusion usually due to the absence of any parenchymal lesion radiologically, but presence of tubercle bacilli in sputum may have important epidemiological and therapeutic implication.

Aims:

This study aims to evaluate the role of sputum examination for AFB in the patients of tuberculous pleural effusion with no apparent lung parenchymal lesion radiologically.

Settings and Design:

Forty-five consecutive indoor patients of suspected tuberculous pleural effusion having no apparent lung parenchymal lesion on chest radiography were selected for our study. It was a prospective and observational study conducted over a period of 1 year.

Materials and Methods:

After confirming the etiology of pleural effusion as tuberculous by biochemical, cytological, histopahtological, and microbiological tests, emphasis was given on sputum examination for AFB by smear examination and culture for Mycobacterium tuberculosis.

Results:

Sputum was bacteriologically (smear and /or culture) positive for tuberculosis in 10 out of 30 cases (33.33%) in which tuberculous etiology was confirmed by histology and /or bacteriology (definite tuberculosis). No sputum AFB (smear and culture) was found in 15 cases of probable tuberculosis where tuberculous etiology was established by indirect methods like Adenosine de aminase level more than 40 unit/l and other relevant investigations. Over all, sputum was bacteriologically smear and/or culture positive in 10 out of 45 cases (22.22%).

Conclusion:

Careful and thorough sputum examination in cases of tuberculous pleural effusion may help as a diagnostic tool and it has therapeutic and epidemiological implications.

Summary

The inflammatory response to Mycobacterium tuberculosis (M.tb) at the site of disease is Th1 driven. Whether the Th17 cytokines, IL-17 and IL-22, contribute to this response in humans is unknown. We hypothesized that IL-17 and IL-22 contribute to the inflammatory response in pleural and pericardial disease sites of human tuberculosis (TB).

We studied pleural and pericardial effusions, established TB disease sites, from HIV-uninfected TB patients. Levels of soluble cytokines were measured by ELISA and MMP-9 by luminex. Bronchoalveolar lavage or pericardial mycobacteria-specific T cell cytokine expression was analyzed by intracellular cytokine staining.

IL-17 was not abundant in pleural or pericardial fluid. IL-17 expression by mycobacteria-specific disease site T cells was not detected in healthy, M.tb-infected persons, or patients with TB pericarditis. These data do not support a major role for IL-17 at established TB disease sites in humans.

IL-22 was readily detected in fluid from both disease sites. These IL-22 levels exceeded matching peripheral blood levels. Further, IL-22 levels in pericardial fluid correlated positively with MMP-9, an enzyme known to degrade the pulmonary extracellular matrix. We propose that our findings support a role for IL-22 in TB-induced pathology or the resulting repair process.

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.

Important advances have been made in the immunodiagnosis of tuberculosis (TB) based on the detection of Mycobacterium tuberculosis (MTB)-specific T cells. However, the sensitivity and specificity of the immunological approach are relatively low because there are no specific markers for antigen-specific Th cells, and some of the Th cells that do not produce cytokines can be overlooked using this approach. In this study, we found that MTB-specific peptides of ESAT-6/CFP-10 can stimulate the expression of CD40L specifically in CD4+ T cells but not other cells from pleural fluid cells (PFCs) in patients with tuberculous pleurisy (TBP). CD4+CD40L+ but not CD4+CD40L− T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22 after stimulation with MTB-specific peptides. In addition, CD4+CD40L+ T cells were found to be mostly polyfunctional T cells that simultaneously produce IFN-γ, IL-2 and TNF-α and display an effector or effector memory phenotype (CD45RA−CD45RO+CCR7−CD62L−ICOS−). To determine the specificity of CD4+CD40L+ T cells, we incubated PFCs with ESTA-6/CFP-10 peptides and sorted live CD4+CD40L+ and CD4+CD40L− T cells by flow cytometry. We further demonstrated that sorted CD4+CD40L+, but not CD4+CD40L− fractions, principally produced IFN-γ, IL-2, TNF-α, IL-17 and IL-22 following restimulation with ESTA-6/CFP-10 peptides. Taken together, our data indicate that the expression of CD40L on MTB-specific CD4+ T cells could be a good marker for the evaluation and isolation of MTB-specific Th cells and might also be useful in the diagnosis of TB.

To assess an open technique of pleural biopsy as an aid to diagnosis in pleural disease 107 African patients with radiological evidence of pleural effusion underwent biopsy. In 87 there was radiological evidence of an effusion but not of underlying lung disease; 73 patients (84%) in this group were ultimately diagnosed as suffering from tuberculosis and of these 56 (77%) had a positive pleural biopsy. There was a heavy male predominance of tuberculous infection (male:female ratio approximately 5:1) and half of the patients were aged 21 to 30 years. In the 20 patients with radiological changes in the lung a diagnosis was established by biopsy in 13 cases. Four of these were tuberculous and a further two cases of tuberculosis were established on clinical grounds.

Immune factors influencing progression to active tuberculosis (TB) remain poorly defined. In this study, we investigated the expression of immunoregulatory cytokines and receptors by using lung bronchoalveolar lavage cells obtained from patients with pulmonary TB, patients with other lung diseases (OLD patients), and healthy volunteers (VOL) by using reverse transcriptase PCR, a transforming growth factor β (TGF-β) bioactivity assay, and an enzyme immunoassay. TB patients were significantly more likely than OLD patients to coexpress TGF-β receptor I (RI) and RII mRNA, as well as interleukin-10 (IL-10) mRNA (thereby indicating the state of active gene transcription in the alveolar cells at harvest). In contrast, gamma interferon (IFN-γ) and IL-2 mRNA was seen in both TB and OLD patients. Likewise, significantly elevated pulmonary steady-state protein levels of IL-10, IFN-γ, and bioactive TGF-β were found in TB patients versus those in OLD patients and VOL. These data suggest that the combined production of the immunosuppressants IL-10 and TGF-β, as well as coexpression of TGF-β RI and RII (required for cellular response to TGF-β), may act to down-modulate host anti-Mycobacterium tuberculosis immunity and thereby allow uncontrolled bacterial replication and overt disease. Delineating the underlying mechanisms of M. tuberculosis-triggered expression of these immune elements may provide a molecular-level understanding of TB immunopathogenesis.

We have used the murine model of aerosol-induced experimental tuberculosis to assess the effects of four clinical isolates and a reference strain of Mycobacterium tuberculosis on resistant C57BL/6 mice and susceptible DBA/2 mice. Histological studies and detection of 25 cytokines potentially involved in the infection were carried out. DBA/2 mice showed higher concentrations of bacilli in bronchoalveolar lavage fluid and lung tissue. Furthermore, these mice evidenced a larger granulomatous infiltration in the parenchyma due to an increased rate of emigration of infected foamy macrophages from the granulomas to the neighboring pulmonary alveolar spaces. The better control of bacillary concentrations and pulmonary infiltration observed in C57BL/6 mice from week 3 postinfection could result from their higher RANTES, ICAM-1, and gamma interferon (IFN-γ) mRNA levels. On the other hand, the higher MIP-2 and MCP-3 mRNA levels seen in DBA/2 mice would result in stronger lung recruitment of macrophages and neutrophils. Additionally, DBA/2 mice showed increased inducible nitric oxide synthase expression, induced by the larger number of foamy macrophages, at weeks 18 and 22. This increment was a consequence of phagocytosed bacillary debris, was independent of IFN-γ expression, and could exert only a bacteriostatic effect. The results of the study suggest that DBA/2 mice are more susceptible than C57BL/6 mice to M. tuberculosis infection due to a higher bronchial dissemination of bacilli inside poorly activated foamy macrophages.