Tricks to translating TB transcriptomics.

Transcriptomics and other high-throughput methods are increasingly applied to questions relating to tuberculosis (TB) pathogenesis. Whole blood transcriptomics has repeatedly been applied to define correlates of TB risk and has produced new insight into the late stage of disease pathogenesis. In a novel approach, authors of a recently published study in Science Translational Medicine applied complex data analysis of existing TB transcriptomic datasets, and in vitro models, in an attempt to identify correlates of protection in TB, which are crucially required for the development of novel TB diagnostics and therapeutics to halt this global epidemic. Utilizing latent TB infection (LTBI) as a surrogate of protection, they identified IL-32 as a mediator of interferon gamma (IFNγ)-vitamin D dependent antimicrobial immunity and a marker of LTBI. Here, we provide a review of all TB whole-blood transcriptomic studies to date in the context of identifying correlates of protection, discuss potential pitfalls of combining complex analyses originating from such studies, the importance of detailed metadata to interpret differential patient classification algorithms, the effect of differing circulating cell populations between patient groups on the interpretation of resulting biomarkers and we decipher weighted gene co-expression network analysis (WGCNA), a recently developed systems biology tool which holds promise of identifying novel pathway interactions in disease pathogenesis. In conclusion, we propose the development of an integrated OMICS platform and open access to detailed metadata, in order for the TB research community to leverage the vast array of OMICS data being generated with the aim of unraveling the holy grail of TB research: correlates of protection

ANIMA: Association network integration for multiscale analysis [version 1; referees: 2 approved with reservations]

Contextual functional interpretation of -omics data derived from clinical samples is a classical and difficult problem in computational systems biology. The measurement of thousands of data points on single samples has become routine but relating ‘big data’ datasets to the complexities of human pathobiology is an area of ongoing research. Complicating this is the fact that many publically available datasets use bulk transcriptomics data from complex tissues like blood. The most prevalent analytic approaches derive molecular ‘signatures’ of disease states or apply modular analysis frameworks to the data. Here we describe ANIMA (association network integration for multiscale analysis), a network-based data integration method using clinical phenotype and microarray data as inputs. ANIMA is implemented in R and Neo4j and runs in Docker containers. In short, the build algorithm iterates over one or more transcriptomics datasets to generate a large, multipartite association network by executing multiple independent analytic steps (differential expression, deconvolution, modular analysis based on co-expression, pathway analysis) and integrating the results. Once the network is built, it can be queried directly using Cypher, or via custom functions that communicate with the graph database via language-specific APIs. We developed a web application using Shiny, which provides fully interactive, multiscale views of the data. Using our approach, we show that we can reconstruct multiple features of disease states at various scales of organization, from transcript abundance patterns of individual genes through co-expression patterns of groups of genes to patterns of cellular behaviour in whole blood samples, both in single experiments as well as in a meta-analysis of multiple datasets

ANIMA: Association Network Integration for Multiscale Analysis

Contextual functional interpretation of -omics data derived from clinical samples is a classical and difficult problem in computational systems biology. The measurement of thousands of datapoints on single samples has become routine but relating ‘big data’ datasets to the complexities of human pathobiology is an area of ongoing research. Complicating this is the fact that many publically available datasets use bulk transcriptomics data from complex tissues like blood. The most prevalent analytic approaches derive molecular ‘signatures’ of disease states or apply modular analysis frameworks to the data. Here we show, using a network-based data integration method using clinical phenotype and microarray data as inputs, that we can reconstruct multiple features (or endophenotypes) of disease states at various scales of organization, from transcript abundance patterns of individual genes through co-expression patterns of groups of genes to patterns of cellular behavior in whole blood samples, both in single experiments as well as in a meta-analysis of multiple datasets

Neutrophil-Associated Central Nervous System Inflammation in Tuberculous Meningitis Immune Reconstitution Inflammatory Syndrome.

Background. The immunopathogenesis of tuberculosis-associated immune reconstitution inflammatory syndrome (IRIS) remains incompletely understood, and we know of only 1 disease site-specific study of the underlying immunology; we recently showed that Mycobacterium tuberculosis culture positivity and increased neutrophils in the cerebrospinal fluid (CSF) of patients with tuberculous meningitis (TBM) are associated with TBM-IRIS. In this study we investigated inflammatory mediators at the disease site in patients with TBM-IRIS. Methods. We performed lumbar puncture at 3–5 time points in human immunodeficiency virus (HIV)–infected patients with TBM (n = 34), including at TBM diagnosis, at initiation of antiretroviral therapy (ART) (day 14), 14 days after ART initiation, at presentation of TBM-IRIS, and 14 days thereafter. We determined the concentrations of 40 mediators in CSF (33 paired with blood) with Luminex or enzyme-linked immunosorbent assays. Findings were compared between patients who developed TBM-IRIS (n = 16) and those who did not (TBM-non-IRIS; n = 18). Results. At TBM diagnosis and 2 weeks after ART initiation, TBM-IRIS was associated with severe, compartmentalized inflammation in the CSF, with elevated concentrations of cytokines, chemokines, neutrophil-associated mediators, and matrix metalloproteinases, compared with TBM-non-IRIS. Patients with TBM-non-IRIS whose CSF cultures were positive for M. tuberculosis at TBM diagnosis (n = 6) showed inflammatory responses similar to those seen in patients with TBM-IRIS at both time points. However, at 2 weeks after ART initiation, S100A8/A9 was significantly increased in patients with TBM-IRIS, compared with patients with TBM-non-IRIS whose cultures were positive at baseline. Conclusions. A high baseline M. tuberculosis antigen load drives an inflammatory response that manifests clinically as TBM-IRIS in most, but not all, patients with TBM. Neutrophils and their mediators, especially S100A8/A9, are closely associated with the central nervous system inflammation that characterizes TBM-IRIS

Cytotoxic Mediators in Paradoxical HIV-Tuberculosis Immune Reconstitution Inflammatory Syndrome

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) frequently complicates combined antiretroviral therapy and antituberculosis therapy in HIV-1–coinfected tuberculosis patients. The immunopathological mechanisms underlying TB-IRIS are incompletely defined, and improved understanding is required to derive new treatments and to reduce associated morbidity and mortality. We performed longitudinal and cross-sectional analyses of human PBMCs from paradoxical TB-IRIS patients and non-IRIS controls (HIV-TB–coinfected patients commencing antiretroviral therapy who did not develop TB-IRIS). Freshly isolated PBMC stimulated with heat-killed Mycobacterium tuberculosis H37Rv (hkH37Rv) were used for IFN-γ ELISPOT and RNA extraction. Stored RNA was used for microarray and RT-PCR, whereas corresponding stored culture supernatants were used for ELISA. Stored PBMC were used for perforin and granzyme B ELISPOT and flow cytometry. There were significantly increased IFN-γ responses to hkH37Rv in TB-IRIS, compared with non-IRIS PBMC (p = 0.035). Microarray analysis of hkH37Rv-stimulated PBMC indicated that perforin 1 was the most significantly upregulated gene, with granzyme B among the top five (log(2) fold difference 3.587 and 2.828, respectively), in TB-IRIS. Downstream experiments using RT-PCR, ELISA, and ELISPOT confirmed the increased expression and secretion of perforin and granzyme B. Moreover, granzyme B secretion reduced in PBMC from TB-IRIS patients during corticosteroid treatment. Invariant NKT cell (CD3(+)Vα24(+)) proportions were higher in TB-IRIS patients (p = 0.004) and were a source of perforin. Our data implicate the granule exocytosis pathway in TB-IRIS pathophysiology. Further understanding of the immunopathogenesis of this condition will facilitate development of specific diagnostic and improved therapeutic options

HIV-tuberculosis-associated immune reconstitution inflammatory syndrome is characterized by Toll-like receptor and inflammasome signalling

Patients with HIV-associated tuberculosis (TB) initiating antiretroviral therapy (ART) may develop immune reconstitution inflammatory syndrome (TB-IRIS). No biomarkers for TB-IRIS have been identified and the underlying mechanisms are unclear. Here we perform transcriptomic profiling of the blood samples of patients with HIV-associated TB. We identify differentially abundant transcripts as early as week 0.5 post ART initiation that predict downstream activation of proinflammatory cytokines in patients who progress to TB-IRIS. At the characteristic time of TB-IRIS onset (week 2), the signature is characterized by over-representation of innate immune mediators including TLR signalling and TREM-1 activation of the inflammasome. In keeping with the transcriptional data, concentrations of plasma cytokines and caspase-1/5 are elevated in TB-IRIS. Inhibition of MyD88 adaptor and group 1 caspases reduces secretion of cytokines including IL-1 in TB-IRIS patients. These data provide insight on the pathogenesis of TB-IRIS and may assist the development of specific therapies

Prevalence, hemodynamics, and cytokine profile of effusive-constrictive pericarditis in patients with tuberculous pericardial effusion

BACKGROUND: Effusive constrictive pericarditis (ECP) is visceral constriction in conjunction with compressive pericardial effusion. The prevalence of proven tuberculous ECP is unknown. Whilst ECP is distinguished from effusive disease on hemodynamic grounds, it is unknown whether effusive-constrictive physiology has a distinct cytokine profile. We conducted a prospective study of prevalence and cytokine profile of effusive-constrictive disease in patients with tuberculous pericardial effusion. METHODS: From July 2006 through July 2009, the prevalence of ECP and serum and pericardial levels of inflammatory cytokines were determined in adults with tuberculous pericardial effusion. The diagnosis of ECP was made by combined pericardiocentesis and cardiac catheterization. RESULTS: Of 91 patients evaluated, 68 had tuberculous pericarditis. The 36/68 patients (52.9%; 95% confidence interval [CI]: 41.2-65.4) with ECP were younger (29 versus 37 years, P=0.02), had a higher pre-pericardiocentesis right atrial pressure (17.0 versus 10.0 mmHg, P 15 mmHg (odds ratio [OR] = 48, 95%CI: 8.7-265; P 200 pg/ml (OR=10, 95%CI: 1.1, 93; P=0.04) were independently associated with ECP. CONCLUSION: Effusive-constrictive disease occurs in half of cases of tuberculous pericardial effusion, and is characterized by greater elevation in the pre-pericardiocentesis right atrial pressure and pericardial and serum IL-10 levels compared to patients with effusive non-constrictive tuberculous pericarditis

HIV-1 infection alters CD4 1 memory T-cell phenotype at the site of disease in extrapulmonary tuberculosis

HIV-1-infected people have an increased risk of developing extrapulmonary tuberculosis (TB), the immunopathogenesis of which is poorly understood. Here, we conducted a detailed immunological analysis of human pericardial TB, to determine the effect of HIV-1 co-infection on the phenotype of Mycobacterium tuberculosis (MTB)-specific memory T cells and the role of polyfunctional T cells at the disease site, using cells from pericardial fluid and blood of 74 patients with (n 5 50) and without (n 5 24) HIV-1 co-infection. The MTB antigen-induced IFN-c response was elevated at the disease site, irrespective of HIV-1 status or antigenic stimulant. However, the IFN-c ELISpot showed no clear evidence of increased numbers of antigen-specific cells at the disease site except for ESAT-6 in HIV-1 uninfected individuals (p 5 0.009). Flow cytometric analysis showed that CD4 1 memory T cells in the pericardial fluid of HIV-1-infected patients were of a less differentiated phenotype, with the presence of polyfunctional CD4 1 T cells expressing TNF, IL-2 and IFN-c. These results indicate that HIV-1 infection results in altered phenotype and function of MTB-specific CD4 1 T cells at the disease site, which may contribute to the increased risk of developing TB at all stages of HIV-1 infection

Разработка интерактивной моделирующей системы технологии низкотемпературной сепарации газа

We present a study of J ψ meson production in collisions of 26.7 GeV electrons with 820 GeV protons, performed with the H1-detector at the HERA collider at DESY. The J ψ mesons are detected via their leptonic decays both to electrons and muons. Requiring exactly two particles in the detector, a cross section of σ(ep → J ψ X) = (8.8±2.0±2.2) nb is determined for 30 GeV ≤ W γp ≤ 180 GeV and Q 2 ≲ 4 GeV 2 . Using the flux of quasi-real photons with Q 2 ≲ 4 GeV 2 , a total production cross section of σ ( γp → J / ψX ) = (56±13±14) nb is derived at an average W γp =90 GeV. The distribution of the squared momentum transfer t from the proton to the J ψ can be fitted using an exponential exp(− b ∥ t ∥) below a ∥ t ∥ of 0.75 GeV 2 yielding a slope parameter of b = (4.7±1.9) GeV −2