Reanalysis and validation of the transcriptional pleural fluid signature in pleural tuberculosis.

Introduction: Pleural tuberculosis (PlTB), the most common site of extrapulmonary TB, is characterized by a paucibacillary nature and a compartmentalized inflammatory response in the pleural cavity, both of which make diagnosis and management extremely challenging. Although transcriptional signatures for pulmonary TB have already been described, data obtained by using this approach for extrapulmonary tuberculosis and, specifically, for pleural tuberculosis are scarce and heterogeneous. In the present study, a set of candidate genes previously described in pulmonary TB was evaluated to identify and validate a transcriptional signature in clinical samples from a Brazilian cohort of PlTB patients and those with other exudative causes of pleural effusion. Methods: As a first step, target genes were selected by a random forest algorithm with recursive feature elimination (RFE) from public microarray datasets. Then, peripheral blood (PB) and pleural fluid (PF) samples from recruited patients presenting exudative pleural effusion were collected during the thoracentesis procedure. Transcriptional analysis of the selected top 10 genes was performed by quantitative RT-PCR (RT-qPCR). Results: Reanalysis of the public datasets identified a set of candidate genes (CARD17, BHLHE40, FCGR1A, BATF2, STAT1, BTN3A1, ANKRD22, C1QB, GBP2, and SEPTIN4) that demonstrated a global accuracy of 89.5% in discriminating pulmonary TB cases from other respiratory diseases. Our validation cohort consisted of PlTB (n = 35) patients and non-TB (n = 34) ones. The gene expressions of CARD17, GBP2, and C1QB in PF at diagnosis were significantly different between the two (PlTB and non-TB) groups (p < 0.0001). It was observed that the gene expressions of CARD17 and GBP2 were higher in PlTB PF than in non-TB patients. C1QB showed the opposite behavior, being higher in the non-TB PF. After anti-TB therapy, however, GBP2 gene expression was significantly reduced in PlTB patients (p < 0.001). Finally, the accuracy of the three above-cited highlighted genes in the PF was analyzed, showing AUCs of 91%, 90%, and 85%, respectively. GBP2 was above 80% (sensitivity = 0.89/specificity = 0.81), and CARD17 showed significant specificity (Se = 0.69/Sp = 0.95) in its capacity to discriminate the groups. Conclusion: CARD17, GBP2, and C1QB showed promise in discriminating PlTB from other causes of exudative pleural effusion by providing accurate diagnoses, thus accelerating the initiation of anti-TB therapy.

Adenosine Deaminase in Pleural Effusion and Its Relationship with Clinical Parameters in Patients with Malignant Pleural Mesothelioma.

Pleural effusion adenosine deaminase (ADA) levels are elevated in various diseases. We investigated whether pleural effusion ADA levels differ among patients with malignant pleural mesothelioma (MPM), lung cancer (LC), and benign diseases, including tuberculous pleurisy. We examined 329 patients from February 2002 to July 2013. There were 131 MPM cases with ADA levels of 32.29 IU/L; 117 LC cases with ADA levels of 21.12 IU/L; 54 benign disease cases with ADA levels of 20.98 IU/L. A significant difference existed in pleural effusion ADA levels between MPM and benign disease patients. Pleural effusion ADA levels were significantly higher in MPM patients.

Macrophage mannose receptor, CD206, predict prognosis in patients with pulmonary tuberculosis.

Tuberculosis (TB) remains a leading cause of fatal infectious disease. Accumulations of macrophages are found in infected sites; thus, we hypothesized that a marker of activated macrophages may be related to prognosis of pulmonary TB (PTB). This study investigated serum soluble macrophage mannose receptor, sCD206, in PTB and examined its clinical significance. First, the concentration of sCD206 was measured in the sera of 96 patients with PTB (Tenryu cohort), and in pleural effusions from 29 patients with TB pleurisy. These were verified in another independent cohort (Shizuoka cohort). We found increased concentrations of sCD206 in sera, but not in pleural effusions of PTB patients. Notably, PTB patients with poor prognosis showed significantly higher levels of serum sCD206. At a cut-off value of 1,600 ng/mL in the Tenryu cohort, sCD206 predicted prognosis of PTB with area under the curve 0.847, sensitivity 77.3%, and specificity 86.5%. These results were validated in the Shizuoka cohort. Pathological analyses showed concordance of enhanced CD206 expression in lung and pleural tissues with caseating granuloma in TB. Serum sCD206 increased in PTB and was associated with prognosis. sCD206 is a potential biomarker for PTB.

Formation of Foamy Macrophages by Tuberculous Pleural Effusions Is Triggered by the Interleukin-10/Signal Transducer and Activator of Transcription 3 Axis through ACAT Upregulation.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its human host relies on numerous immune evasion strategies, such as the deregulation of the lipid metabolism leading to the formation of foamy macrophages (FM). Yet, the specific host factors leading to the foamy phenotype of Mtb-infected macrophages remain unknown. Herein, we aimed to address whether host cytokines contribute to FM formation in the context of Mtb infection. Our approach is based on the use of an acellular fraction of tuberculous pleural effusions (TB-PE) as a physiological source of local factors released during Mtb infection. We found that TB-PE induced FM differentiation as observed by the increase in lipid bodies, intracellular cholesterol, and expression of the scavenger receptor CD36, as well as the enzyme acyl CoA:cholesterol acyl transferase (ACAT). Importantly, interleukin-10 (IL-10) depletion from TB-PE prevented the augmentation of all these parameters. Moreover, we observed a positive correlation between the levels of IL-10 and the number of lipid-laden CD14+ cells among the pleural cells in TB patients, demonstrating that FM differentiation occurs within the pleural environment. Downstream of IL-10 signaling, we noticed that the transcription factor signal transducer and activator of transcription 3 was activated by TB-PE, and its chemical inhibition prevented the accumulation of lipid bodies and ACAT expression in macrophages. In terms of the host immune response, TB-PE-treated macrophages displayed immunosuppressive properties and bore higher bacillary loads. Finally, we confirmed our results using bone marrow-derived macrophage from IL-10-/- mice demonstrating that IL-10 deficiency partially prevented foamy phenotype induction after Mtb lipids exposure. In conclusion, our results evidence a role of IL-10 in promoting the differentiation of FM in the context of Mtb infection, contributing to our understanding of how alterations of the host metabolic factors may favor pathogen persistence.

Distinct functions of CXCR3+ and CCR4+CD4+ T-cells accumulated in human tuberculosis pleural fluid.

BACKGROUND: Chemokine receptors and their ligands play a prominent role in regulating leucocyte migration. In the local milieu of inflammation, a high concentration of chemokines can recruit different chemokine receptor-expressing lymphocytes. OBJECTIVE: To understand the distinct immunological functions of CXC chemokine receptor 3 (CXCR3+) and CC chemokine receptor 4 (CCR4+) cluster of differentiation 4 (CD4+) T-cells accumulated in human tuberculosis (TB) pleural fluid after tuberculous antigen stimulation. METHODS: Mononuclear cells were isolated from the peripheral blood of healthy donors, cord blood and TB pleural fluid, and expression of CXCR3 and CC chemokine receptor 4 (CCR4), cytokines and cytolytic molecules by CD4+ T-cells with or without stimulation were analysed using fluorescence-activated cell sorting. RESULTS: CXCR3 and CCR4 expression on CD4+ T-cells from pleural fluid mononuclear cells (PFMCs) was significantly higher than in peripheral blood mononuclear cells (PBMCs). T-cell receptor signalling resulted in the upregulation of CXCR3 and CCR4 expression on CD4+ T-cells from cord blood mononuclear cells (CBMCs) and PBMCs in a time-dependent manner, but not from PFMCs. After stimulation with Mycobacterium tuberculosis antigens, CXCR3+CCR4-CD4+ T-cells were dominated by multifunctional T-helper 1 cells; however, CXCR3+CCR4+CD4+ T-cells exhibited cytotoxicity and degranulation by expressing granzyme B, perforin, CD107a/b and tumour necrosis factor-related apoptosis-inducing ligand. CONCLUSION: Our results indicated that CXCR3 or CCR4 expression on CD4+ T-cells had different biological activities against tuberculous infection, and could be a potential marker for the diagnosis of TB.

Detection of Mycobacterium tuberculosis from paraffin-embedded tissues by GeneXpert MTB/RIF.

GeneXpert MTB/RIF (Xpert) assay, a rapid and automated system based on real-time PCR and molecular beacon technology, proved to be a sensitive and specific tool capable of detecting Mycobacterium tuberculosis and rifampin resistance in clinical specimens. In this study we provide a Xpert-dedicated successful protocol for processing paraffin-embedded tissue and assess the feasibility of the Xpert assay-based tuberculosis (TB) diagnosis on these specimens, thus proving the Xpert assay as a valuable TB diagnostic tool in supporting conventional histopathological methods.

TB/HIV pleurisy reduces Th17 lymphocyte proportion independent of the cytokine microenvironment.

T-helper (Th) 17 cells are a pro-inflammatory subset of CD4(+) effector T-cells critical in mucosal immunity. Imbalances in Th17 cell proportion have been implicated in the pathogenesis of several diseases; however, this has not been adequately explored in tuberculosis (TB) and human immunodeficiency virus (HIV) co-infection. Since Th17 cells are predominantly mucosally associated, we assessed Th17 proportion and associated microenvironment in pleural effusions from patients co-infected with TB/HIV. Our results show that TB(+)HIV(+) pleurisy results in significantly reduced frequency of CD4(+)IL-17(+)RORC(+)STAT3(+) Th17 cells compared to TB(-)HIV(-)ex vivo (p = 0.0054) and was confirmed in conditioned media studies in vitro (p = 0.0001). This was not associated with alterations in Th17 polarising cytokines IL-6, IL-21 and IL-23 or changes in Th17 signature cytokines IL-17A and F. However, the mRNA expression of Th17 signalling molecules, IL-6 (p = 0.0022), IL-6R (p = 0.0247), IL-1ß (p = 0.0022) and signal transducer and activator (STAT) 3 (p = 0.0022) were significantly upregulated. Notably, TB(+)HIV(+) pleural fluid contained significantly higher concentrations of IL-1ß (p = 0.0008), IL-22 (p = 0.0115), IL-31 (p = 0.0210), TNF-α (p = 0.0251) and IFN-γ (p = 0.0026) than TB(-)HIV(-) pleural fluid ex vivo. Taken together, this suggests a reduced portion of Th17 lymphocytes in TB/HIV pleurisy is independent of locally mediated cytokine polarisation.

Expression Profiles of Cytokine mRNAs in the Pleural Fluid Reveal Differences Among Tuberculosis, Malignancies, and Pneumonia-Exudative Pleural Effusions.

INTRODUCTION: Tuberculosis (TB) and malignant diseases are the most common causes of lymphocytic pleural effusion in adults. Serum and pleural fluid cytokine levels have been analyzed to help in the differential diagnosis, but with limited results. PURPOSE: This study investigates transcription levels of selected cytokine genes in pleural effusion of patients under investigation for TB. METHODS: This was a prospective study that included adult patients under investigation for pleural effusion in Brazil. The expression of 19 cytokine genes was analyzed by RT-qPCR. RESULTS: The majority of cytokine-related genes expressed in pleural fluid of TB patients were similar in non-TB patients, except for RORA and RORC genes, which showed a statistically higher level in TB. All cytokines in the Th17 pattern were induced in TB patients' pleural fluid. Patients with malignant pleural effusion expressed higher levels of IFN-α1, IFN-ß1, TNF-α, IL-4 and IL-6, and suppression of TGFß-1. CONCLUSION: There is still a lot to understand about the cytokine roles in the pro- and anti-inflammatory environment of exudative pleural effusions. The data presented here showed an increased expression of Th17 pattern cytokines genes in TB patients that could be used as markers to differentiate tuberculous pleuritis from other common causes of exudative pleural effusion.

High IL-35 pleural expression in patients with tuberculous pleural effusion.

BACKGROUND: IL-35 is a novel anti-inflammatory and immunosuppressive cytokine primarily produced by Treg cells, and is involved in inflammatory diseases and autoimmune diseases. However, its roles in tuberculous pleural effusion (TPE) remain unknown. We aimed to investigate the potential involvement of IL-35 in TPE. MATERIAL/METHODS: Thirty TPE patients and 20 lung cancer patients with malignant pleural effusion (MPE) were recruited. Samples of pleural effusion (100 mL) were collected after traditional pleurocentesis. Blood was sampled from TPE patients. Mononuclear cells were isolated by Ficoll-Hypaque gradient. Proportions of Th1, Th17, and IL-35-producing cells were analyzed by flow cytometry. IL-35 was assessed by real-time RT-PCR, ELISA, and immunofluorescence. An ELISPOT assay was used to assess the effect of IL-35 on pleural effusion mononuclear cells (PEMCs). RESULTS: Proportions of IL-35-producing cells were higher in TPE compared with MPE (49.4±6.0 vs. 15.8±5.4%, P<0.001) and blood from TPE patients (49.4±6.0% vs. 16.6±3.1, P<0.001). IL-35, IL-17 and IFN-γ were elevated in TPE compared with MPE (all P<0.01). ELISPOT assay showed that IL-35 reduced the proportion of IFN-γ-producing CD4+ T cells in TPE. IL-35 mRNA expression was higher in TPE compared with MPE (P<0.001). Immunofluorescence showed that IL-35-positive cells were present in pleural tissues from TPE patients. CONCLUSIONS: Results suggest that there is an imbalance in IL-35 metabolism in TPE. However, further studies are required to assess the exact relationship with the immune system response to tuberculosis. IL-35 might play a role in TPE and might be targeted as a treatment for TPE.

Profiling the T-cell receptor repertoire of patient with pleural tuberculosis by high-throughput sequencing.

Pleural tuberculosis (PLTB), a major cause of morbidity and mortality, is the most common extrapulmonary manifestation of active Mycobacterium tuberculosis (Mtb) in developing countries. Gamma delta T-cell receptor (TCR) repertoire of peripheral blood mononuclear cells (PBMCs) and pleural effusion mononuclear cells (PEMCs) and beta TCR repertoire from peripheral blood mononuclear cells (PBMCs) have been reported. However, a detailed different characteristic of beta TCR repertoire of mononuclear cells isolated from peripheral blood and pleural fluid in the immune response to Mtb infection should be further revealed. The TCR ß-chain (TRB) from PBMCs and PEMCs from an untreated pleural tuberculosis patient was sequenced by the Illumina sequencing platform. A total of 96,758 and 124,130 unique complementarity-determining region 3 (CDR3) sequences were identified at the nucleotide level, encoding 69,488 and 99,095 peptide sequences, respectively. TCR profiling showed that TRBV20-1 family and TRBV20-1/TRBJ1-5 gene combination had a dominant expression in PEMCs, but not in PBMCs. Expansive expression of common CDR3 clonotypes was observed in PEMCs. CDR3 spectratyping analysis showed that few TRBV families had a significantly skewed pattern, with one peak or a few prominent peaks in the PBMCs. By contrast, some TRBV families showed oligoclonal or clonal expansion in the PEMCs. Here, we firstly profiled the TRB repertoire differences of PBMCs and PEMCs from one PLTB patient using high-throughput sequencing. And this study may provide new insight for the detailed and efficient study of TCR repertoire of PEMCs in the future.

Induction of CCL8/MCP-2 by mycobacteria through the activation of TLR2/PI3K/Akt signaling pathway.

Pleural tuberculosis (TB), together with lymphatic TB, constitutes more than half of all extrapulmonary cases. Pleural effusions (PEs) in TB are representative of lymphocytic PEs which are dominated by T cells. However, the mechanism underlying T lymphocytes homing and accumulation in PEs is still incompletely understood. Here we performed a comparative analysis of cytokine abundance in PEs from TB patients and non-TB patients by protein array analysis and observed that MCP-2/CCL8 is highly expressed in the TB-PEs as compared to peripheral blood. Meanwhile, we observed that CCR5, the primary receptor used by MCP-2/CCL8, is mostly expressed on pleural CD4(+) T lymphocytes. Furthermore, we found that infection with either Mycobacterium bovis Bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis H37Rv induced production of MCP-2/CCL8 at both transcriptional and protein level in Raw264.7 and THP-1 macrophage cells, mouse peritoneal macrophages as well as human PBMC monocyte-derived macrophages (MDMs). The induction of MCP-2/CCL8 by mycobacteria is dependent on the activation of TLR2/PI3K/Akt and p38 signaling pathway. We conclude that accumulation of MCP-2/CCL8 in TB-PEs may function as a biomarker for TB diagnosis.

Altered microRNA expression levels in mononuclear cells of patients with pulmonary and pleural tuberculosis and their relation with components of the immune response.

Different lines of evidence demonstrate that microRNAs (miRNAs) play an important role in host-pathogen interactions. In this study we investigated the expression patterns of several miRNAs, most of them involved in regulating inflammatory responses, in patients with tuberculosis (TB). In order to understand the events occurring at the site of infection, we employed mononuclear cells obtained from both peripheral blood (PBMC) and pleural fluids (PFMC) of patients. Interestingly, we found that the miRNA signature of each compartment is different, with a strong down-regulation in PFMCs of miR-223, miR-144* and miR-421. In addition, we observed that miR-146a expression is also down-regulated in tuberculosis patients, both in PBMCs and PFMCs while miR-424 levels are elevated only in the peripheral compartments. We also showed that systemic expression of these miRNAs changes upon specific treatment and is associated with IL-6 levels, a cytokine playing a substantial role in TB immunopathology. Present results contribute to a better knowledge of the host responses in TB pathogenesis, pointing out the role of miRNAs in this disease.

Distinct polyfunctional CD4+ T cell responses to BCG, ESAT-6 and CFP-10 in tuberculous pleurisy.

Tuberculous pleurisy (TBP) is a frequent extrapulmonary manifestation characterized by the accumulation of inflammatory cells that can sometimes be spontaneously self-cured. To achieve a greater insight into T cells at a local site, we systematically characterized and compared the numbers of antigen-specific T cells responding to BCG- or MTB-specific antigens. Our results showed that significantly higher levels of Th1 cytokines were produced by pleural fluid cells (PFCs) than PBMCs following stimulation with BCG or peptides from Mycobacterium tuberculosis (MTB)-specific antigens, ESAT-6 and CFP-10. The proportions of Th1 cells producing IL-2 alone or IL-2 and TNF-α were higher than those producing IFN-γ alone, following stimulation with ESAT-6 or CFP-10 peptides. The cells responding to BCG, ESAT-6 and CFP-10 displayed a CD45RA(-)CCR7(-)CD62L(-)CD27(-) effector/effector memory phenotype. The percentages and median fluorescence intensity (MFI) of polyfunctional CD4(+) T cells were significantly higher following stimulation with peptides from ESAT-6 or CFP-10 than BCG. Our results demonstrated that significantly higher levels of polyfunctional CD4(+) T cells for the epitopes of ESAT-6 or CFP-10 in PFCs may play an important role in the local control of tuberculosis (TB) infection.

Polyfunctional Mycobacterium tuberculosis-specific effector memory CD4+ T cells at sites of pleural TB.

Pleural tuberculosis (TB) is a common presentation of Mycobacterium tuberculosis (MTB) infection, and despite spontaneous resolution remains a strong risk factor for reactivation pulmonary TB in a majority of individuals. This study was undertaken to further understand the characteristics of immune cells at sites of pleural TB. A significant shift toward memory CD4+ T cells with an effector phenotype and away from naïve CD4+ T cells in pleural fluid as compared to blood mononuclear cells was found. These data suggest that effector T cells are capable of migrating to sites of active TB infection and/or the differentiation to effector phenotype T cells in situ is highly amplified. Using multi-parameter flow cytometry analysis, a significant portion of MTB-specific CD4+ T cells in the pleural space were polyfunctional demonstrating two, three or four simultaneous functions including IFN-gamma, IL-2, TNF-alpha, and or MIP-1 alpha production. A greater proportion of these polyfunctional cells were of effector memory rather than central memory phenotype. The role of these polyfunctional MTB-specific CD4+ T cells at sites of pleural TB requires further study.

Interleukin-10 gene promoter polymorphisms and their protein production in pleural fluid in patients with tuberculosis.

Associations of interleukin-10 (IL-10) gene promoter polymorphisms and pleural tuberculosis risk remain unclear. The objective of this study was to determine IL-10 gene promoter polymorphisms at -1082, -819 and -592 sites and their protein production in pleural fluid (PF) in patients with and without pleural tuberculosis. IL-10 gene promoter polymorphisms at the -1082, -819 and -592 sites were genotyped using a SNaPshot assay. Protein levels of IL-10 in PF were measured using an enzyme-linked immunosorbent assay. There were no significant differences in the genotype and allele frequencies of IL-10 gene promoter polymorphisms at position -1082 between the pleural tuberculosis and the control groups. However, the frequency of -819 T or -592 A alleles was significantly more common in patients with pleural tuberculosis than controls. The protein levels of IL-10 in PF were statistically higher in the pleural tuberculosis group than in the control group. Moreover, the polymorphisms at the -1082, -819 and -592 sites were associated with protein levels of IL-10 in PF in the pleural tuberculosis group, while in the control group, only the polymorphism at position -1082 correlated with the protein levels. These findings support the association between IL-10 promoter polymorphisms at -819 and -592 sites and their protein production with pleural tuberculosis risk.

Molecular epidemiology of pleural and other extrapulmonary tuberculosis: a Maryland state review.

BACKGROUND: Limited information exists about the current epidemiological characteristics of extrapulmonary tuberculosis. However, pleural tuberculosis is usually considered to be a manifestation of primary tuberculosis. Our objective was to use molecular epidemiological techniques to describe the occurrence of pleural and other extrapulmonary tuberculosis in Maryland, a state with moderate tuberculosis incidence. METHODS: We surveyed tuberculosis cases reported with a single site of disease in Maryland from 1996 through 2001. Genotyping of Mycobacterium tuberculosis isolates was performed with an IS6110-based restriction fragment-length polymorphism analysis. DNA clustering of strains with >5 IS6110 bands, with supporting epidemiologic information on patients, served as a proxy for recent transmission. RESULTS: A total of 1811 patients with tuberculosis were reported (incidence, 5.9 cases per 100,000 population). Of 1411 patients (77.9%) with cultures positive for M. tuberculosis, 1246 (88.3%) had a single site of disease, with 934 (75.0%) of these isolates having >5 IS6110 bands. Of the 934 patients included in the analyses, 729 (78.0%) had pulmonary tuberculosis, and 205 (22.0%) had extrapulmonary tuberculosis; of the latter group, 46 patients had pleural disease, and 159 patients had nonrespiratory disease. In multivariate analyses, patients with pleural tuberculosis were not significantly associated with clustered strains, compared with patients with nonrespiratory or pulmonary tuberculosis disease. Having a DNA-clustered strain was negatively associated with nonrespiratory tuberculosis, compared with pulmonary disease (adjusted odds ratio, 0.48; P = .003). CONCLUSIONS: Nonrespiratory extrapulmonary tuberculosis is less likely than pulmonary tuberculosis to be a result of recent infection. Pleural tuberculosis is not an appropriate indicator for recent transmission among our population.

The association between microsatellite polymorphisms in intron II of the human Toll-like receptor 2 gene and tuberculosis among Koreans.

The observation that Toll-like receptor (TLR)2-deficient mice are highly susceptible to mycobacteria suggests that mutations altering TLR2 expression may impair host response to Mycobacterium tuberculosis. We evaluated the association between guanine-thymine (GT) repeat polymorphism in intron II of the TLR2 gene and the presence of tuberculosis (TB) in Koreans. The numbers of GT repeats were determined by PCR and gene scans for 176 TB patients and 196 controls. The recombinant TLR2 promoter/exonI/exonII/intronII/luciferase constructs including three representative repeats: (GT)13, (GT)20, and (GT)24 were transfected into K562 cells, and luciferase activities were estimated and compared. The expression of TLR2 on CD14+ peripheral blood mononuclear cells (PBMC) from healthy volunteers were measured with flow cytometry. Genotypes with shorter GT repeats were more common among TB patients (49.4 vs 37.7%, P=0.02). This observation was confirmed among 82 other TB patients as a validation cohort. Shorter GT repeats were associated with weaker promoter activities and lower TLR2 expression on CD14+ PBMCs. In conclusion, the development of TB disease in Koreans was associated with shorter GT repeats in intron II of the TLR2 gene. This association is correlated with lower expression of TLR2 through weaker promoter activity for genes with shorter GT repeats.

Cytokine gene polymorphisms in Colombian patients with different clinical presentations of tuberculosis.

Tuberculosis (TB) has different clinical presentations. Pulmonary TB affects only the lungs and exhibits variable anti-mycobacterial immune responses. Pleural TB is a localized disease with a strong immune response. Miliary TB is a disseminated form with poor immune response. Cytokines play a pivotal role in anti-mycobacterial response and may determine the type of TB. Thus, gene polymorphisms associated with cytokine production may be associated with clinical presentations of TB. In this study, 54 tuberculin-negative healthy controls, 81 tuberculin-positive healthy controls, 140 patients with pulmonary TB, 30 with pleural TB and 20 with miliary TB were studied. Single nucleotide polymorphisms were typed for tumour necrosis factor-alpha, interferon-gamma (IFN-gamma), transforming growth factor-beta1, interleukin-10 (IL-10) and interleukin-6 by sequence-specific primer polymerase chain reaction (SSP-PCR). Allelic, genotypic and haplotypic associations with clinical forms of TB were evaluated. IL-10 -1082 A/A genotype and IFNgamma+874 T allele were associated with pleural TB. Seventy-five extended genotypes were found; two differed between patients and controls, and two between groups of patients. Results suggest that IL-10 low-producer polymorphism and IFN-gamma high-producer polymorphism are associated with pleural TB.

[Study of the expression profile of immunogenesis associated genes in tuberculosis by microarray].

OBJECTIVE: To explore the difference of gene expression profile in tuberculosis patients. METHODS: mRNA levels of pleural fluid and peripheral blood mononuclear cells (PBMC) in tuberculous pleurisy and lung cancer patients were compared by cDNA microarray. Paired mRNAs from fluid specimens of tuberculosis and lung cancer cases were labeled with different fluorochromes during cDNA probe synthesis in a reverse-transcription reaction. The signal intensity of each spot was measured by laser scanner and gene expression was quantified as the tubercle-to-normal fluorescence ratio (T:N ratio). The gene was defined as over expression when the T:N ratio was greater than 2.0 and under expression when the ratio was less than 0.5. RESULTS: Among 626 immunogenesis associated genes there were 53 differences, of which 31 (tnf-alpha, ig-lambda, il-17, il-17r, hla-dp, lcp1, tcralpha, tcrbeta, hsp75, cxcr4, fyb, hla-g, hla-a, il18bp, il-2r, lt-beta, il-8, ip-10, mcp-1, il-12, il-12r, il-10, canx, irf2, ifn-gamma, tlr, il-1, il-7, tlr, lsp-1, il-14)were higher and 22 (il-4, il-18, il-15, ifg-1, scya14, ablim, peci, ppid, hsf 2, actg2, maoa, ttid, gatm, tgfb3, insr, thbd, trap1, tcrgamma, tcrdelta, il-13r, il-11, igf1, a2m)were lower in tuberculous pleurisy than those in the control. CONCLUSIONS: The immunogenesis of tuberculosis involves multi-genetic expression changes, such as tnf-alpha, il-17, il-12, tcralpha, tcrbeta, hsp75, cxcr4, il-4, il-18, il-15 etc., the expression profile of which changed dramatically. The results provide new insight for understanding of the pathogenic mechanisms of tuberculosis and exploring new therapeutic strategies.

[The phenotypes of haptoglobin and the level of acute-phasic proteins in the sera of children with local form of intrathoracic tuberculosis]. / Fenotipy gaptoglobina i uroven' ostrofaznykh belkov v syvorotke krovi u detei s lokal'nymi formami vnutrigrudnogo tuberkuleza.

Seventy-seven children aged 4-12 years who had local forms of primary intrathoracic tuberculosis were examined. On admission to and discharge from hospital, haptoglobin (Hp) was phenotyped and the content of Hp was measured, and the activity of alpha1-antitrypsin (alpha1-AT) was determined. In all ill children, the distribution of Hp phenotypes did not differ from the normal level, but all patients with tuberculous pleurisy were found to be carriers of Hp1 gene (among them the phenotype Hp 2-2 was absent and the minor variant of Hp 1-1 was detectable in half the cases). In patients of this group, alpha1-AT acted as a typical acute phase reagent and remained moderately increased by the termination of treatment in the vast majority of the examinees. On the contrary, on admission the content of Hp was to be decreased. Its increase was natural only in patients with tuberculous pleurisy. The level of Hp was associated with its phenotype. Carriers of Hp1-1 had elevated levels of Hp in the overwhelming majority of cases whereas those of Hp 2-2 had its decreased levels. It is concluded that in children with primary tuberculosis, the serum level of Hp may not be used as an indicator of the activity of the process. Possible causes and the values of decreased levels of circulating Hp in children with primary tuberculosis are discussed in the paper.