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ZusammenfassungSeit 2014 ist die Zahl der Neuerkrankungen an Tuberkulose (TB) in Deutschland um rund 20% angestiegen. Diese Entwicklung fällt mit einer hohen Zuwanderung von Asylsuchenden zusammen, die z. T. aus Ländern stammen, in denen die TB deutlich weiter verbreitet ist als hierzulande. Vor diesem Hintergrund setzt sich der vorliegende Beitrag zum Ziel, Hemmnisse bei der Diagnose und der Therapie asylsuchender TB-Patienten in Deutschland zu identifizieren und zu erklären. Zugleich sollen Verbesserungspotenziale in diesen Bereichen aufgezeigt werden. Als Datengrundlage dienen 14 leitfadengestützte, problemzentrierte Interviews mit Ärzten und Bediensteten des öffentlichen Gesundheitswesens, die als Experten die Versorgerseite repräsentieren. Auf der einen Seite bringen strukturelle Faktoren die von den Experten benannten Hemmnisse hervor. So führt bspw. der eingeschränkte Zugang zur Gesundheitsversorgung zu einer verspäteten Diagnose, da Asylsuchende hierdurch später (oder gar nicht) zum Arzt gehen. Entsprechend wird eine flächendeckende Einführung einer elektronischen Gesundheitskarte vorgeschlagen. Auf der anderen Seite spielen individuelle bzw. kulturelle Faktoren eine wichtige Rolle. Hierzu zählen z. B. Sprachbarrieren, welche die Anamnese (und damit die Diagnose), v. a. aber die Aufklärung des Patienten erschweren und damit den Therapieerfolg gefährden. Zudem resultiert aus der fehlenden Vertrautheit von Asylsuchenden mit dem deutschen Gesundheitssystem die Gefahr einer Therapieunterbrechung. Schulungsprogramme nach dem „train the trainer“-Konzept und die Etablierung eines „Lotsen“, der asylsuchende TB-Patienten durch das deutsche Gesundheitssystem navigiert, könnten diese Probleme lindern.

Cryptocurrencies have developed very dynamically although their future role is yet unclear. In any event, they are too big to ignore. The purpose of this article is to contribute to the understanding of cryptocurrencies in an individual and in a portfolio context. The study is based on daily closing prices of leading cryptocurrencies (Bitcoin, Ethereum, Ripple, Litecoin, and Dash) and fiat currencies (EUR, GBP, CHF, CAD, and JPY), all measured against USD. The analysis is threefold: First, the authors analyze basic statistical properties, such as correlation and autocorrelation of returns. Second, they perform a Kolmogorov–Smirnov test (KS test) and a variance ratio test (VRT) with heteroscedasticity adjustment. Third, they solve more than 4,800 optimization problems to analyze the impact of individual crypto- and fiat currencies on portfolio diversification. Among other findings, the authors find that Bitcoin, Ethereum, Dash, CAD, JPY, and EUR contribute most to reduce the variance of a mixed portfolio. In a portfolio consisting of cryptocurrencies only, Bitcoin and Ripple have the largest diversification effect. The findings provide insights for investors who focus on minimum variance portfolios or, more generally, for investors who seek to reduce return volatility exposure, as well as for monetary authorities, cryptocurrency issuers, and providers of market infrastructure. TOPICS:Real assets/alternative investments/private equity, statistical methods, portfolio construction

Since 2014, the number of new cases of tuberculosis (TB) has risen in Germany by about 20%. This coincides with a large number of people applying for asylum in Germany. Some of them are from countries in which TB is much more prevalent than in Germany. The objectives of this contribution are to identify and explain barriers in the diagnosis and treatment of asylum seekers with TB and potential improvements in those fields. Data are derived from 14 problem-centred interviews that were carried out with doctors and staff from public health offices, representing the views of experts in the field of health care. On the one hand, the results suggest that structural factors are responsible for some of the barriers mentioned by the experts. For example, the restricted access to health care for asylum seekers leads to a delayed diagnosis since they visit the doctor too late (if at all). Accordingly, a nationwide implementation of an electronic health card for asylum seekers was proposed. On the other hand, individual and cultural factors play important roles as well. To those belong language barriers: they not only complicate history taking and diagnosis, but also educating patients about their disease and therapy. Moreover, the lack of knowledge concerning the German health care system increases the risk of treatment interruptions. To alleviate those problems, experts propose to carry out train-the-trainer-programmes and to install "guides" who pilot asylum seekers with TB through the German health care system.Seit 2014 ist die Zahl der Neuerkrankungen an Tuberkulose (TB) in Deutschland um rund 20% angestiegen. Diese Entwicklung fällt mit einer hohen Zuwanderung von Asylsuchenden zusammen, die z. T. aus Ländern stammen, in denen die TB deutlich weiter verbreitet ist als hierzulande. Vor diesem Hintergrund setzt sich der vorliegende Beitrag zum Ziel, Hemmnisse bei der Diagnose und der Therapie asylsuchender TB-Patienten in Deutschland zu identifizieren und zu erklären. Zugleich sollen Verbesserungspotenziale in diesen Bereichen aufgezeigt werden. Als Datengrundlage dienen 14 leitfadengestützte, problemzentrierte Interviews mit Ärzten und Bediensteten des öffentlichen Gesundheitswesens, die als Experten die Versorgerseite repräsentieren. Auf der einen Seite bringen strukturelle Faktoren die von den Experten benannten Hemmnisse hervor. So führt bspw. der eingeschränkte Zugang zur Gesundheitsversorgung zu einer verspäteten Diagnose, da Asylsuchende hierdurch später (oder gar nicht) zum Arzt gehen. Entsprechend wird eine flächendeckende Einführung einer elektronischen Gesundheitskarte vorgeschlagen. Auf der anderen Seite spielen individuelle bzw. kulturelle Faktoren eine wichtige Rolle. Hierzu zählen z. B. Sprachbarrieren, welche die Anamnese (und damit die Diagnose), v. a. aber die Aufklärung des Patienten erschweren und damit den Therapieerfolg gefährden. Zudem resultiert aus der fehlenden Vertrautheit von Asylsuchenden mit dem deutschen Gesundheitssystem die Gefahr einer Therapieunterbrechung. Schulungsprogramme nach dem „train the trainer“-Konzept und die Etablierung eines „Lotsen“, der asylsuchende TB-Patienten durch das deutsche Gesundheitssystem navigiert, könnten diese Probleme lindern.

Die Angat-Stauanlage befindet sich in den Philippinen in einem hochseismischen Gebiet unmittelbar oberhalb der Metropolregion Manila mit ihren derzeit etwa 13 Mio. EinwohnerInnen. Die Anlage ist eine Mehrzweckanlage und dient der Trinkwasserversorgung von Manila, landwirtschaftlicher Nutzung (Bewasserung), Hochwasserschutz und Energieerzeugung. Der Angat-Stausee mit einem bewirtschaftbaren Speichervolumen von 850 Mio. m3 wird durch einen 131 m hohen Hauptdamm und zwei Nebendamme aufgestaut. Die Anlage verfugt zudem uber eine Hochwasserentlastungsanlage, einen Grundablass und eine Wasserkraftanlage mit einer installierten Leistung von 246 MW. Aufgrund der Neubewertung der Erdbebensituation im Jahre 2013 wurde beschlossen, die Angat-Stauanlage umfangreichen Sicherheitsuntersuchungen zu unterziehen. Mit der Privatisierung der Wasserkraftanlage im Jahr 2015 wurde dieser Plan umgesetzt, und noch im gleichen Jahr wurde Poyry mit dieser Aufgabe betraut. Die Philippinen verfugen uber keine eigenen Richtlinien zur Talsperrensicherheit. Es wurde deshalb ein Sicherheitskonzept gewahlt, das sich im Wesentlichen auf internationale, aber auch auf andere nationale Normen und Richtlinien stutzt. Die vorliegende Veroffentlichung stellt den gewahlten Ansatz vor und beschreibt deren Umsetzung an der Angat-Stauanlage.

LytR-cpsA-Psr (LCP) domain containing proteins fulfil important functions in bacterial cell wall synthesis. In Mycobacterium tuberculosis complex (Mtbc) strains, the causative agents of tuberculosis (TB), the genes Rv3484 and Rv3267 encode for LCP proteins which are putatively involved in arabinogalactan transfer to peptidoglycan. To evaluate the significance of Rv3484 for Mtbc virulence, we generated a deletion mutant in the Mtbc strain H37Rv and studied its survival in mice upon aerosol infection. The deletion mutant failed to establish infection demonstrating that Rv3484 is essential for growth in mice. Following an initial phase of marginal replication in the lungs until day 21, the Rv3484 deletion mutant was almost eliminated by day 180 post-infectionem. Interestingly, the mutant also showed higher levels of resistance to meropenem/clavulanate and lysozyme, both targeting peptidoglycan structure. We conclude that Rv3484 is essential for Mtbc virulence in vivo where its loss of function cannot be compensated by Rv3267.

Human tuberculosis (TB) is a leading global health threat and still constitutes a major medical challenge. However, mechanisms governing tissue pathology during post-primary TB remain elusive, partly because genetically or immunologically tractable animal models are lacking. In human TB, the demonstration of a large relative increase in interleukin (IL)-4 and IL-13 expression, which correlates with lung damage, indicates that a subversive T helper (TH)2 component in the response to Mycobacterium tuberculosis (Mtb) may undermine protective immunity and contribute to reactivation and tissue pathology. Up to now, there has been no clear evidence regarding whether IL-4/IL-13-IL-4 receptor-α (Rα)-mediated mechanisms may in fact cause reactivation and pathology. Unfortunately, the virtual absence of centrally necrotizing granulomas in experimental murine TB is associated with a poor induction of a TH2 immune response. We therefore hypothesize that, in mice, an increased production of IL-13 may lead to a pathology similar to human post-primary TB. In our study, aerosol Mtb infection of IL-13-over-expressing mice in fact resulted in pulmonary centrally necrotizing granulomas with multinucleated giant cells, a hypoxic rim and a perinecrotic collagen capsule, with an adjacent zone of lipid-rich, acid-fast bacilli-containing foamy macrophages, thus strongly resembling the pathology in human post-primary TB. Granuloma necrosis (GN) in Mtb-infected IL-13-over-expressing mice was associated with the induction of arginase-1-expressing macrophages. Indirect blockade of the endogenous arginase inhibitor l-hydroxyarginine in Mtb-infected wild-type mice resulted in a strong arginase expression and precipitated a similar pathology of GN. Together, we here introduce an experimental TB model that displays many features of centrally necrotizing granulomas in human post-primary TB and demonstrate that IL-13/IL-4Rα-dependent mechanisms leading to arginase-1 expression are involved in TB-associated tissue pathology. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Practical Applications Summary In Beyond Bitcoin: A Statistical Comparison of Leading Cryptocurrencies and Fiat Currencies and Their Impact on Portfolio Diversification from the Summer 2019 issue of The Journal of Alternative Investments, authors Stefan Ehlers and Kolja Gauer (both at Volkswagen AG) provide a first-of-its-kind analysis of whether traditional currencies (also known as fiat currencies) and cryptocurrencies act similarly or differently with respect to their fluctuations in value and total return. The authors also explore whether mixing cryptocurrencies and fiat currencies in an investment portfolio can help diversify it and reduce the portfolio’s variance. The authors find no correlation between the fluctuations in value and total return of cryptocurrencies and fiat currencies, so combining them in a mixed portfolio improves diversification. Also, only Bitcoin and XRP play an important role in reducing the variance of a pure cryptocurrency portfolio, while just a few cryptocurrencies and fiat currencies significantly reduce the variance of mixed portfolios. So, those who want to invest in cryptocurrencies and avoid major swings in value and returns should consider including a few specific currencies in their portfolio and should combine cryptocurrencies with fiat currencies in a mixed portfolio. TOPICS:Currency, statistical methods, portfolio construction

The Wnt signaling network, an ancient signaling system governing ontogeny and homeostatic processes, has recently been identified to exert immunoregulatory functions in a variety of inflammatory and infectious disease settings including tuberculosis. In this study, we show that Wnt6 is expressed in granulomatous lesions in the lung of Mycobacterium tuberculosis–infected mice. We identified foamy macrophage-like cells as the primary source of Wnt6 in the infected lung and uncovered a TLR–MyD88–NF-κB–dependent mode of induction in bone marrow–derived macrophages. Analysis of Wnt6-induced signal transduction revealed a pertussis toxin–sensitive, ERK-mediated, but β-catenin–independent induction of c-Myc, a master regulator of cell proliferation. Increased Ki-67 mRNA expression levels and enhanced thymidine incorporation in Wnt6-treated macrophage cultures demonstrate a proliferation-promoting effect on murine macrophages. Further functional studies in M. tuberculosis–infected macrophages using Wnt6 conditioned medium and Wnt6-deficient macrophages uncovered a Wnt6-dependent induction of macrophage Arginase-1 and downregulation of TNF-α. This identifies Wnt6 as a novel factor driving macrophage polarization toward an M2-like phenotype. Taken together, these findings point to an unexpected role for Wnt6 in macrophage differentiation in the M. tuberculosis–infected lung.

Individually and combined, Toll-like receptors (TLR)-2, -4, -9, nucleotide oligomerization domain (NOD) 2 and NALP3 contribute to the Mycobacterium tuberculosis (Mtb)-induced innate immune response only to a limited extent, particularly in terms of inducing antibacterial protection and granuloma formation in vivo. A singular essential sensory component of this initial response has not been discovered yet. Trehalose-6,6'-dimycolate (TDM), a well known mycobacterial cell wall glycolipid, is believed to be involved in these early inflammatory processes after Mtb infection. Only recently the macrophage inducible C-type lectin (Mincle) was demonstrated as an essential receptor for TDM. However, not much is known about the sensing capacity of Mincle during infection with live mycobacteria. To determine the significance of Mincle during tuberculosis (TB), we analyzed the outcome of Mtb infection in Mincle-deficient mice. Whereas in the absence of Mincle macrophages did not respond to TDM, Mincle-deficient mice were capable of mounting an efficient granulomatous and protective immune response after low and high dose infections with Mtb. Mutant mice generated a normal T helper (TH) 1 and TH17 immune response followed by the induction of efficient macrophage effector mechanisms and control of mycobacterial growth identical to wildtype mice. From our results we conclude that absence of the innate receptor Mincle can be fully compensated for in vivo in terms of sensing Mtb and mounting a protective inflammatory immune response.

Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.

The contribution of interleukin- (IL-) 4 receptor-alpha- (Rα-) dependent events in the pathogenesis of tuberculosis (TB) is controversial. We have recently shown IL-13 overexpression in mice to cause recrudescentMtbreplication and centrally necrotizing granulomas strongly resembling pathology of human TB. A deletion of IL-4Rαcompletely abrogates TB tissue pathology in these mice. To validate our results in human TB patients, we here determined the association of distinct variants of theIL4,IL13,IL4RA,IL13RA1, andIL13RA2genes with cavity formation in a large Ghanaian cohort of HIV-negative individuals with newly diagnosed pulmonary TB. In fact, the structural variant of theIL4RAI50V, previously shown to result in enhanced signal transduction, was significantly associated with greater cavity size, and a variant ofIL13RA2was associated with disease in females. To evaluate whether the human-like TB pathology in IL-13-overexpressing mice is specifically mediated through the IL-4Rαsubunit, we analyzed IL-13 transgenic mice with a genetic ablation of the IL-4Rα. In these mice, the IL-13-mediated increased susceptibility, human-like pathology of collagen deposition around centrally necrotizing granulomas, and alternative macrophage activation were abolished. Together, our genetic association study in human TB patients further supports the assumption that IL-13/IL-4Rα-dependent mechanisms are involved in mediating tissue pathology of human TB.

Regulation of specific immune responses following exposure to Mycobacterium tuberculosis in humans and the role of regulatory T (Treg) cells in the immune control of latent infection with M. tuberculosis are incompletely understood. Latent infection was assayed by an interferon-c release assay (IGRA) in healthcare workers regularly exposed to tuberculosis (TB) patients and in household TB contacts in Germany. Immunophenotypes of bronchoalveolar lavage (BAL) mononuclear cells and peripheral blood mononuclear cells (PBMCs) were analysed by fluorescence-activated cell sorting. All TB contacts with latent infection (n515) had increased (p,0.0001) frequencies of CD4+ CD25+ CD127- Treg cells (median 2.12%, interquartile range (IQR) 1.63-3.01%) among BAL mononuclear cells compared with contacts (n525) with negative IGRA results (median 0.68%, IQR 0.32-0.96%) No differences were seen when PBMC immunophenotypes of IGRA+ and IGRA- TB contacts were compared (IGRA+ median 9.6%, IQR 5.9-10.1%; IGRA- median 7.7%, IQR 4.6- 11.3%; p50.47). Five out of 25 contacts with negative blood IGRAs showed a positive IGRA from BAL cells, possibly indicating a limited local immune response. In Germany, latent infection with M. tuberculosis, as defined by a positive M. tuberculosis- specific IGRA response on cells from the peripheral blood, is characterised by an increased frequency of Treg cells in the BAL.

In light of the marked global health impact of tuberculosis (TB), strong focus has been on identifying biosignatures. Gene expression profiles in blood cells identified so far are indicative of a persistent activation of the immune system and chronic inflammatory pathology in active TB. Definition of a biosignature with unique specificity for TB demands that identified profiles can differentiate diseases with similar pathology, like sarcoidosis (SARC). Here, we present a detailed comparison between pulmonary TB and SARC, including whole-blood gene expression profiling, microRNA expression, and multiplex serum analytes. Our analysis reveals that previously disclosed gene expression signatures in TB show highly similar patterns in SARC, with a common up-regulation of proinflammatory pathways and IFN signaling and close similarity to TB-related signatures. microRNA expression also presented a highly similar pattern in both diseases, whereas cytokines in the serum of TB patients revealed a slightly elevated proinflammatory pattern compared with SARC and controls. Our results indicate several differences in expression between the two diseases, with increased metabolic activity and significantly higher antimicrobial defense responses in TB. However, matrix metallopeptidase 14 was identified as the most distinctive marker of SARC. Described communalities as well as unique signatures in blood profiles of two distinct inflammatory pulmonary diseases not only have considerable implications for the design of TB biosignatures and future diagnosis, but they also provide insights into biological processes underlying chronic inflammatory disease entities of different etiology.

Homeostasis of intracellular pH is a trait critical for survival of Mycobacterium tuberculosis in macrophages. However, mechanisms by which M. tuberculosis adapts to acidic environments are poorly understood. In this study, we analysed the physiological functions of OmpATb, a surface-accessible protein of M. tuberculosis. OmpATb did not complement the permeability defects of a Mycobacterium smegmatis porin mutant to glucose, serine and glycerol, in contrast to the porin MspA. Uptake rates of these solutes were unchanged in an ompATb operon mutant of M. tuberculosis indicating that OmpATb is not a general porin. Chemical analysis of low-pH culture filtrates showed that the proteins encoded by the ompATb operon are involved in generating a rapid ammonia burst, which neutralized medium pH and preceded exponential growth of M. tuberculosis. Addition of ammonia accelerated growth of the ompATb operon mutant demonstrating that ammonia secretion is indeed a mechanism by which M. tuberculosis neutralizes acidic environments. Infection experiments revealed that the ompATb operon was not required for full virulence in mice suggesting that M. tuberculosis has multiple mechanisms of resisting phagosomal acidification. Taken together, these results show that the ompATb operon is necessary for rapid ammonia secretion and adaptation of M. tuberculosis to acidic environments in vitro but not in mice.

In vitro, Toll-like receptors (TLR)2, 4 and 9 as well as NOD-like receptor 2 critically determine macrophage responses to Mycobacterium tuberculosis (Mtb) infection. However, in low-dose experimental murine tuberculosis, single or multiple deficiencies in TLRs 2, 4, 9 or NOD2 have little, if any, impact on early mycobacterial growth containment, granuloma formation and survival. Here, we analyzed the relevance of NALP3, one component of the danger-signaling inflammasome, for (i) Mtb-induced cytokine secretion in vitro and in vivo, (ii) restriction of Mtb replication in infected organs and (iii) granuloma formation. In the absence of functional NALP3, there was no IL-1beta and IL-18 production in Mtb-infected dendritic cells and macrophages in vitro, whereas secretion of IL-1alpha, IL-12p40 and TNF remained unaffected. After three weeks of infection, NALP3-deficient as well as IL-18-deficient mice were as capable as wildtype mice of restricting Mtb loads at a plateau level within well-differentiated granulomas. In conclusion, despite its involvement in cytokine processing, NALP3 is not essential for induction of protective immunity to Mtb.

Anti-tumour necrosis factor (TNF) monoclonal antibodies or soluble TNF receptors have become an invaluable treatment against chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. Individuals who are treated with TNF antagonists are at an increased risk of reactivating latent infections, especially tuberculosis (TB).Following TNF antagonist therapy, the relative risk for TB is increased up to 25 times, depending on the clinical setting and the TNF antagonist used. Interferon-gamma release assays or, as an alternative in individuals without a history of bacille Calmette-Guerin vaccination, tuberculin skin testing is recommended to screen all adult candidates for TNF antagonist treatment for the presence of latent infection with Mycobacterium tuberculosis.Moreover, paediatric practice suggests concomitant use of both the tuberculin skin test and an interferon-gamma release assay, as there are insufficient data in children to recommend one test over the other. Consequently, targeted preventive chemotherapy is highly recommended for all individuals with persistent M. tuberculosis-specific immune responses undergoing TNF antagonist therapy as it significantly reduces the risk of progression to TB.This TBNET consensus statement summarises current knowledge and expert opinions and provides evidence-based recommendations to reduce the TB risk among candidates for TNF antagonist therapy.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is recognized by pattern recognition receptors on macrophages and dendritic cells, thereby triggering phagocytosis, antigen presentation to T cells and cytokine secretion. The dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN) is a calcium-dependent carbohydrate-binding protein with specificity for mannose-containing glycoconjugates and fucose-containing Lewis antigens. Mannosylated moieties of the mycobacterial cell wall, such as mannose-capped lipoarabinomannan (manLAM) or higher-order phosphatidylinositol-mannosides (PIMs) of Mtb, were previously shown to bind to DC-SIGN on immature dendritic cells and macrophage subpopulations. This interaction reportedly impaired dendritic cell maturation, modulated cytokine secretion by phagocytes and dendritic cells and was postulated to cause suppression of protective immunity to TB. However, experimental Mtb infections in mice transgenic for human DC-SIGN revealed that, instead of favoring immune evasion of mycobacteria, DC-SIGN may promote host protection by limiting tissue pathology. Furthermore, infection studies with mycobacterial strains genetically engineered to lack manLAM or PIMs demonstrated that the manLAM/PIM-DC-SIGN interaction was not critical for cytokine secretion in vitro and protective immunity in vivo. The dominant Mtb-derived ligands for DC-SIGN are presently unknown, and a major role of DC-SIGN in the immune response to Mtb infection may lie in its capacity to maintain a balanced inflammatory state during chronic TB.

Elevated IL-10 has been implicated in reactivation tuberculosis (TB). Since macrophages rather than T cells were reported to be the major source of IL-10 in TB, we analyzed the consequences of a macrophage-specific overexpression of IL-10 in transgenic mice (macIL-10-transgenic) after aerosol infection with Mycobacterium tuberculosis (Mtb). MacIL-10 transgenic mice were more susceptible to chronic Mtb infection than nontransgenic littermates, exhibiting higher bacterial loads in the lung after 12 wk of infection and dying significantly earlier than controls. The differentiation, recruitment, and activation of Th1 cells as well as the induction of IFN-γ-dependent effector genes against Mtb were not affected by macrophage-derived IL-10. However, microarray analysis of pulmonary gene expression revealed patterns characteristic of alternative macrophage activation that were overrepresented in Mtb-infected macIL-10 transgenic mice. Importantly, arginase-1 gene expression and activity were strikingly enhanced in transgenic mice accompanied by a reduced production of reactive nitrogen intermediates. Moreover, IL-10-dependent arginase-1 induction diminished antimycobacterial effector mechanisms in macrophages. Taken together, macrophage-derived IL-10 triggers aspects of alternative macrophage activation and promotes Mtb recrudescence independent of overt effects on anti-TB T cell immunity.

Tuberculosis control relies on the identification and preventive treatment of individuals who are latently infected with Mycobacterium tuberculosis. However, direct identification of latent tuberculosis infection is not possible. The diagnostic tests used to identify individuals latently infected with M. tuberculosis, the in vivo tuberculin skin test and the ex vivo interferon-gamma release assays (IGRAs), are designed to identify an adaptive immune response against, but not necessarily a latent infection with, M. tuberculosis. The proportion of individuals who truly remain infected with M. tuberculosis after tuberculin skin test or IGRA conversion is unknown. It is also uncertain how long adaptive immune responses towards mycobacterial antigens persist in the absence of live mycobacteria. Clinical management and public healthcare policies for preventive chemotherapy against tuberculosis could be improved, if we were to gain a better understanding on M. tuberculosis latency and reactivation. This statement by the TBNET summarises knowledge and limitations of the currently available tests used in adults and children for the diagnosis of latent tuberculosis infection. In summary, the main issue regarding testing is to restrict it to those who are known to be at higher risk of developing tuberculosis and who are willing to accept preventive chemotherapy.

Mycobacterium tuberculosis can utilize various nutrients including nitrate as a source of nitrogen. Assimilation of nitrate requires the reduction of nitrate via nitrite to ammonium, which is then incorporated into metabolic pathways. This study was undertaken to define the molecular mechanism of nitrate assimilation in M. tuberculosis. Homologues to a narGHJI-encoded nitrate reductase and a nirBD-encoded nitrite reductase have been found on the chromosome of M. tuberculosis. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a narG mutant of M. tuberculosis failed to grow on nitrate. A nirB mutant of M. tuberculosis failed to grow on both nitrate and nitrite. Mutant strains of Mycobacterium smegmatis mc2155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from M. tuberculosis, and a gene with homology to the response regulator gene glnR of Streptomyces coelicolor was identified. A ΔglnR mutant of M. tuberculosis was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when nirBD was expressed from a plasmid, suggesting a role of GlnR in regulating nirBD expression. A specific binding site for GlnR within the nirB promoter was identified and confirmed by electrophoretic mobility shift assay using purified recombinant GlnR. Semiquantitative reverse transcription PCR, as well as microarray analysis, demonstrated upregulation of nirBD expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of M. tuberculosis mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of nirBD.

The success of Mycobacterium tuberculosis depends on its ability to survive within host macrophages. Here, M. tuberculosis avoids the acidic, hydrolytically competent environment of the phagolysosome by arresting phagosome maturation. Having shown previously that a M. tuberculosis mutant deficient in lipoprotein signal peptidase (LspA) is strongly attenuated in vivo in a mouse model of infection, we now studied putative mechanisms involved in attenuation of the lspA : : aph mutant at a cellular level. In this work we investigated the ability of the mutant to interfere with two host defence mechanisms, i.e. Toll-like receptor (TLR)2-dependent immune response and phagosome maturation. While mycobacterial lipoproteins have been reported to trigger a TLR2 signalling pathway critical for innate immune responses, we found that growth control of the lspA : : aph mutant was independent of TLR2. In addition, the lspA : : aph mutant arrested phagosome maturation to an extent similar to that of the wild-type, as measured by lysosomal-associated membrane protein 1 (LAMP1) co-localization and intraphagosomal pH. These observations demonstrate severe attenuation even in the presence of arrested phagosome maturation, and point to a role for the early phagosome in growth restriction of the M. tuberculosis lspA mutant.

Background. The acute phase of mycobacterial lung infection is characterized by a nearly exponential outgrowth of mycobacteria in the alveolar airspace and lung parenchymal tissue, suggesting insufficient early protective immunity against mycobacterial challenge. In the current study, we tested the hypothesis that a CC chemokine ligand 2 (CCL2)-dependent increased mononuclear phagocyte subset accumulation in distal airspaces would improve the lungs' protective immunity to infection with Mycobacterium bovis bacille Calmette-Guerin (hereafter, "M. bovis BCG"). Methods. Wild-type mice and CCL2-overexpressing mice that exhibited increased pools of alveolar and lung mononuclear phagocytes-due to the lung-specific overexpression of human CCL2 in type-II alveolar epithelial cells-were infected intratracheally with M. bovis BCG and the developing lung inflammatory response was analyzed. Results. CCL2-overexpressing mice demonstrated significantly decreased mycobacterial loads in the bronchoalveolar space, lung parenchymal tissue, and spleen compared with wild-type mice, when both groups of mice were infected with M. bovis BCG. Moreover, in M. bovis BCG-infected mice, later-developing, accelerated resolution of lung granuloma formation was noted, particularly in CCL2-overexpressing mice as compared with wild-type mice. In addition, CCL2-overexpressing mice demonstrated an increased trafficking of mycobacteria-loaded dendritic cells towards lung-draining lymph nodes that was found to coincide with increased mycobacterial loads in this compartment. Conclusions. The data of the current study suggest that CCL2-dependent amplification of endogenous host-defense programs in the lung may improve the lungs' protective immunity against mycobacterial infections.

In many pathogens, degradation of arginine via the arginine deiminase pathway supports anaerobic metabolism. Here we show by deletion of Rv1001 (arcA) in Mycobacterium tuberculosis that this gene functions as an arginine deiminase. Arginine metabolism in the presence of oxygen was not affected by the mutation, indicating a separate pathway for arginine degradation under aerobic conditions. Following aerosol infection in mice, the DeltaarcA mutant and wild-type strain of M. tuberculosis multiplied and persisted in infected organs in a similar fashion.

Mycobacterium tuberculosis (Mtb) parasitizes host macrophages and subverts host innate and adaptive immunity. Several cytokines elicited by Mtb are mediators of mycobacterial clearance or are involved in tuberculosis pathology. Surprisingly, interleukin-1beta (IL-1beta), a major proinflammatory cytokine, has not been implicated in host-Mtb interactions. IL-1beta is activated by processing upon assembly of the inflammasome, a specialized inflammatory caspase-activating protein complex. Here, we show that Mtb prevents inflammasome activation and IL-1beta processing. An Mtb gene, zmp1, which encodes a putative Zn(2+) metalloprotease, is required for this process. Infection of macrophages with zmp1-deleted Mtb triggered activation of the inflammasome, resulting in increased IL-1beta secretion, enhanced maturation of Mtb containing phagosomes, improved mycobacterial clearance by macrophages, and lower bacterial burden in the lungs of aerosol-infected mice. Thus, we uncovered a previously masked role for IL-1beta in the control of Mtb and a mycobacterial system that prevents inflammasome and, therefore, IL-1beta activation.

The role of Toll-like receptors (TLR) and MyD88 for immune responses to Mycobacterium tuberculosis (Mtb) infection remains controversial. To address the impact of TLR-mediated pathogen recognition and MyD88-dependent signaling events on anti-mycobacterial host responses, we analyzed the outcome of Mtb infection in TLR2/4/9 triple- and MyD88-deficient mice. After aerosol infection, both TLR2/4/9-deficient and wild-type mice expressed pro-inflammatory cytokines promoting antigen-specific T cells and the production of IFN-gamma to similar extents. Moreover, TLR2/4/9-deficient mice expressed IFN-gamma-dependent inducible nitric oxide synthase and LRG-47 in infected lungs. MyD88-deficient mice expressed pro-inflammatory cytokines and were shown to expand IFN-gamma-producing antigen-specific T cells, albeit in a delayed fashion. Only mice that were deficient for MyD88 rapidly succumbed to unrestrained mycobacterial growth, whereas TLR2/4/9-deficient mice controlled Mtb replication. IFN-gamma-dependent restriction of mycobacterial growth was severely impaired only in Mtb-infected MyD88, but not in TLR2/4/9-deficient bone marrow-derived macrophages. Our results demonstrate that after Mtb infection neither TLR2, -4, and -9, nor MyD88 are required for the induction of adaptive T cell responses. Rather, MyD88, but not TLR2, TLR4 and TLR9, is critical for triggering macrophage effector mechanisms central to anti-mycobacterial defense.

Controversy exists concerning the role of Toll-like receptors and MyD88 in immunity to tuberculosis (TB). This mini-review argues that (i) Toll-like receptors are not essential for an effective immune response against TB, (ii) MyD88 is essential, but not because it transduces signals from TLRs, (iii) adaptive immunity to TB is largely TLR/MyD88-independent. Some of the discrepancies may be resolved by cogent attribution of distinct immune functions to the individual components of the TLR/MyD88 system. In mice, TLRs and MyD88 are fully dispensable in sensing Mtb infection and instructing T cell-mediated adaptive immunity, and while TLRs are also redundant during macrophage effector immunity, MyD88 is essential for efficient killing of mycobacteria. This distinction should help to molecularly pinpoint the MyD88-dependent, yet TLR-independent critical mechanisms of macrophage activation involved in intracellular growth restriction of Mtb. Disrupted IL-1R and/or IFN-gamma signaling pathways likely play a much more prominent role in explaining the exquisite susceptibility of MyD88-deficient mice to TB than the function of MyD88 as a TLR adaptor.

Differential induction of cell death in mycobacteria-infected monocytes and macrophages has been invoked as one possible mechanism by which some tumor necrosis factor (TNF)-targeted biologicals reactivate tuberculosis more frequently than others. We infected primary human monocytes and monocyte-derived macrophages with the virulent Mycobacterium tuberculosis strain H37Rv and followed the rate of cell death in the absence or presence of a wide concentration range of four different TNF-targeted biologicals: infliximab and adalimumab (both monoclonal antibodies to human TNF) and etanercept and polyethylene-glycols TNFR1 (fusion constructs of human TNFR2 and TNFR1, respectively). None of the TNF-targeted biologicals used modulated the death rate of monocytes/macrophages induced by infection with M. tuberculosis alone. Our data support the view that mycobacteria-induced cell death is largely independent of TNF and that the primary target for differential modulation by TNF-targeted biologicals during tuberculosis is not a recently recruited monocyte or freshly differentiated macrophage.

Tumor necrosis factor (TNF) is an important cytokine for host defense against pathogens but is also associated with the development of human immunopathologies. TNF blockade effectively ameliorates many chronic inflammatory conditions but compromises host immunity to tuberculosis. The search for novel, more specific human TNF blockers requires the development of a reliable animal model. We used a novel mouse model with complete replacement of the mouse TNF gene by its human ortholog (human TNF [huTNF] knock-in [KI] mice) to determine resistance to Mycobacterium bovis BCG and M. tuberculosis infections and to investigate whether TNF inhibitors in clinical use reduce host immunity. Our results show that macrophages from huTNF KI mice responded to BCG and lipopolysaccharide similarly to wild-type macrophages by NF-κB activation and cytokine production. While TNF-deficient mice rapidly succumbed to mycobacterial infection, huTNF KI mice survived, controlling the bacterial burden and activating bactericidal mechanisms. Administration of TNF-neutralizing biologics disrupted the control of mycobacterial infection in huTNF KI mice, leading to an increased bacterial burden and hyperinflammation. Thus, our findings demonstrate that human TNF can functionally replace murine TNF in vivo , providing mycobacterial resistance that could be compromised by TNF neutralization. This new animal model will be helpful for the testing of specific biologics neutralizing human TNF.

The interleukin (IL)-27 receptor-alpha WSX-1 is one component of the heterodimeric IL-27 receptor that is expressed on various cell types including macrophages. We previously demonstrated that IL-27 induces STAT-3 and is able to inhibit the production of pro-inflammatory cytokines in activated macrophages suggesting a novel feed-back mechanism by which IL-27 can modulate excessive inflammation. Because IL-4 receptor-alpha (IL-4Ralpha)-induced alternatively activated macrophages have also been described to attenuate pathological inflammatory immune responses, we analyzed the contribution of IL-27 in alternative macrophage activation. In the present study, like IL-10 and IL-4, IL-27 was found to suppress IL-12/23p40 production in activated bone marrow-derived macrophages. Whereas IL-10 induced the upregulation of the IL-4Ralpha on macrophages, receptor expression was not triggered by IL-27. In contrast to IL-4, IL-27 did not induce alternative macrophage activation but IL-4 strongly upregulated the expression of WSX-1 on macrophages and alternative macrophage activation enhanced IL-27-mediated signalling. We therefore conclude from our study that IL-10, IL-4 and IL-27 collaborate in modulating macrophage activation by successive upregulation of the IL-4Ralpha and WSX-1 on alternatively activated macrophages.

Microarray - assisted gene - expression screens of human macrophages revealed WNT5A, a homolog of Wingless, a key regulator of Drosophila melanogaster embryonic segmentation and patterning, to be consistently up-regulated following stimulation with different mycobacterial species and conserved bacterial structures. The expression of WNT5A required Toll-like receptor signaling and NF-κB activation, which identifies a novel induction pathway for a Wingless homolog. We show that human peripheral-blood mononuclear cells express the WNT5A receptor Frizzled-5 (FZD5). Both WNT5A and FZD5 also were detected in granulomatous lesions in the lungs of Mycobacterium tuberculosis–infected patients. Functional studies showed that WNT5A and FZD5 regulate the microbially induced interleukin-12 response of antigen-presenting cells and interferon-γ production by mycobacterial antigenstimulated T cells. Our findings implicate the evolutionarily conserved WNT/Frizzled signaling system in bridging innate and adaptive immunity to infections.

CD8+ T cells are involved in protection against Mycobacterium tuberculosis infection and represent a promising target for new vaccine strategies. Because IL-15 is important for the homeostasis of CD8+ T cells, we studied the immune response in IL-15-deficient mice during tuberculosis. In the absence of IL-15, CD8+ T cells failed to efficiently accumulate in draining lymph nodes and at the site of infection. The expression of antigen-specific effector functions, such as the production of interferon-gamma and cytotoxicity, were impaired in CD8+ T cells, but not CD4+ T cells, from IL-15-deficient mice. This defect was associated with an increased mortality of IL-15-deficient mice during the chronic phase of infection. The lectin-like stimulatory receptor natural killer group 2D (NKG2D) was up-regulated on CD8+ T cells only from wild-type mice, but not from IL-15-deficient mice. Mechanistically, blocking NKG2D function with an mAb inhibited M. tuberculosis-directed CD8+ T cell responses in vitro. We conclude that in addition to regulating the expansion of CD8+ T cells, IL-15 is also necessary for inducing effector mechanisms in CD8+ T cells that depend on NKG2D expression. Hence, our results implicate IL-15 and NKG2D as promising targets for modulating CD8+ T cell-mediated protection against tuberculosis.

Immunity to Mycobacterium tuberculosis infection is critically dependent on the timely priming of T effector lymphocytes and their efficient recruitment to the site of mycobacterial implantation in the lung. E-, P-, and L-selectin counterreceptors control lymphocyte homing to lymph nodes and leukocyte trafficking to peripheral sites of acute inflammation, their adhesive function depending on fucosylation by fucosyltransferases (FucT) IV and VII. To address the relative importance of differentially glycosylated selectin counterreceptors for priming of T cell effector functions in a model of mycobacteria-induced granulomatous pulmonary inflammation, we used aerosol-borne M. tuberculosis to infect FucT-IV−/−, FucT-VII−/−, FucT-IV−/−/FucT-VII−/−, or wild-type control mice. In lymph nodes, infected FucT-IV−/−/FucT-VII−/− and, to a lesser extent, FucT-VII−/− mice had severely reduced numbers of T cells and reduced Ag-specific effector responses. By contrast, recruitment of activated T cells into the lungs was similar in all four groups of mice during infection and expression of T cell, and macrophage effector functions were only delayed in lungs of FucT-IV−/−/FucT-VII−/− mice. Importantly, lungs from all groups expressed CXCL13, CCL21, and CCL19 and displayed organized follicular neolymphoid structures after infection with M. tuberculosis, which suggests that the lung served as a selectin ligand-independent priming site for immune responses to mycobacterial infection. All FucT-deficient strains were fully capable of restricting M. tuberculosis growth in infected organs until at least 150 days postinfection. Our observations indicate that leukocyte recruitment functions dictated by FucT-IV and FucT-VII-dependent selectin ligand activities are not critical for inducing or maintaining T cell effector responses at levels necessary to control pulmonary tuberculosis.

Tumor necrosis factor (TNF) is a critical component of both the antibacterially protective and the inflammatory responses against infections, particularly infections with intracellularly viable microorganisms. It is, therefore, not surprising that some treatment regimens that target TNF function have resulted in an increase in complications associated with infections due to such pathogens as Mycobacterium tuberculosis, Listeria monocytogenes, and Histoplasma capsulatum; organized granuloma formation is required to keep such infections under control. However, treatment with anti-TNF monoclonal antibodies (i.e., infliximab) has been associated with a higher incidence of granulomatous infections than has treatment with a TNF receptor (TNFR) p75 immunoglobulin G-fusion construct (i.e., etanercept). Three hypotheses concerning the mode of action of these 2 agents that might explain this difference are discussed here: differential induction of apoptosis or lysis in membrane TNF-expressing macrophages and T cells, differential inhibition of signaling via TNFRp55 and TNFRp75, and different net neutralizing capacities resulting from different pharmacologic properties.

Produced by macrophages and dendritic cells, interleukin (IL)-12 is composed of a p35 and a p40 subunit and promotes protection against intracellular pathogens through the development of interferon-gamma (IFNγ) -producing T cells. The p40 subunit is also shared by the dimeric cytokines IL-12p40 homodimer and IL-23. In man, genetic defects in IL-12p40-mediated mechanisms are responsible for the familial occurrence of nontuberculous mycobacterial infections, the most common of which is infection with Mycobacterium avium. To experimentally differentiate the contribution of IL-12p40-containing cytokines in the outcome of M. avium infection, we studied wild-type, p35- and p35/p40 doubly deficient mice in an intravenous infection model which reflects many parameters of the disseminated infection in humans. Our study shows that in contrast to p35/p40 doubly deficient mice, p35-deficient mice mount a transient antibacterially protective response against M. avium although such animals were unable to produce detectable levels of IFNγ or generate efficient granulomas. In conclusion, our results identify an antibacterial effector mechanism preserved in p35-deficient mice that is absent in mice devoid of p35 and p40. This phenotype probably reflects an IL-12p40-dependent effect on macrophage activation at the level of innate immunity.

The adenylyl cyclase Rv1625c from Mycobacterium tuberculosis codes for a protein with six transmembrane spans and a catalytic domain, i.e. it corresponds to one half of the pseudoheterodimeric mammalian adenylyl cyclases (ACs). Rv1625c is active as a homodimer. We investigated the role of the Rv1625c membrane domain and demonstrate that it efficiently dimerizes the protein resulting in a 7.5-fold drop in K(m) for ATP. Next, we generated a duplicated Rv1625c AC dimer by a head-to-tail concatenation. This produced an AC with a domain order exactly as the mammalian pseudoheterodimers. It displayed positive cooperativity and a 60% increase of v(max) compared with the Rv1625c monomer. Further, we probed the compatibility of mycobacterial and mammalian membrane domains. The second membrane anchor in the Rv1625c concatamer was replaced with membrane domain I or II of rabbit type V AC. The mycobacterial and either mammalian membrane domains are compatible with each other and both recombinant proteins are active. A M. tuberculosis Rv1625c knockout strain was assayed in a mouse infection model. In vitro growth characteristics and in vivo organ infection and mortality were unaltered in the knockout strain indicating that AC Rv1625c alone is not a virulence factor.

The recently described scaffold model of murein architecture depicts the gram-negative bacterial cell wall as a gel-like matrix composed of cross-linked glycan strands oriented perpendicularly to the plasma membrane while peptide bridges adopt a parallel orientation (B. A. Dmitriev, F. V. Toukach, K. J. Schaper, O. Holst, E. T. Rietschel, and S. Ehlers, J. Bacteriol. 185:3458-3468, 2003). Based on the scaffold model, we now present computer simulation studies on the peptidoglycan arrangement of the gram-positive organism Staphylococcus aureus , which show that the orientation of peptide bridges is critical for the highly cross-linked murein architecture of this microorganism. According to the proposed refined model, staphylococcal murein is composed of glycan and oligopeptide chains, both running in a plane that is perpendicular to the plasma membrane, with oligopeptide chains adopting a zigzag conformation and zippering adjacent glycan strands along their lengths. In contrast to previous models of murein in gram-positive bacteria, this model reflects the high degree of cross-linking that is the hallmark of the staphylococcal cell wall and is compatible with distinguishing features of S. aureus cytokinesis such as the triple consecutive alteration of the division plane orientation and the strictly centripetal mode of septum closure.

Lipoproteins are a subgroup of secreted bacterial proteins characterized by a lipidated N-terminus, processing of which is mediated by the consecutive activity of prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (LspA). The study of LspA function has been limited mainly to non-pathogenic microorganisms. To study a potential role for LspA in the pathogenesis of bacterial infections, we have disrupted lspA by allelic replacement in Mycobacterium tuberculosis, one of the world's most devastating pathogens. Despite the presence of an impermeable lipid outer layer, it was found that LspA was dispensable for growth under in vitro culture conditions. In contrast, the mutant was markedly attenuated in virulence models of tuberculosis. Our findings establish lipoprotein metabolism as a major virulence determinant of tuberculosis and define a role for lipoprotein processing in bacterial pathogenesis. In addition, these results hint at a promising new target for therapeutic intervention, as a highly specific inhibitor of bacterial lipoprotein signal peptidases is available.

Porins mediate the diffusion of hydrophilic solutes across the outer membrane of mycobacteria, but the efficiency of this pathway is very low compared to Gram-negative bacteria. To examine the importance of porins in slow-growing mycobacteria, the major porin MspA of Mycobacterium smegmatis was expressed in Mycobacterium tuberculosis and Mycobacterium bovis. Approximately 20 and 35 MspA molecules per μm2 cell wall were observed in M. tuberculosis and M. bovis BCG, respectively, by electron microscopy and quantitative immunoblot experiments. Surface accessibility of MspA in M. tuberculosis was demonstrated by flow cytometry. Glucose uptake was twofold faster, indicating that the outer membrane permeability of M. bovis BCG to small and hydrophilic solutes was increased by MspA. This significantly accelerated the growth of M. bovis BCG, identifying very slow nutrient uptake as one of the determinants of slow growth in mycobacteria. The susceptibility of both M. bovis BCG and M. tuberculosis to zwitterionic β-lactam antibiotics was substantially enhanced by MspA, decreasing the minimal inhibitory concentration up to 16-fold. Furthermore, M. tuberculosis became significantly more susceptible to isoniazid, ethambutol and streptomycin. Fluorescence with the nucleic acid binding dye SYTO 9 was 10-fold increased upon expression of mspA. These results indicated that MspA not only enhanced the efficiency of the porin pathway, but also that of pathways mediating access to large and/or hydrophobic agents. This study provides the first experimental evidence that porins are important for drug susceptibility of M. tuberculosis.

Gene expression patterns associated with resistance and susceptibility to tuberculosis (TB) were investigated at the macrophage level in the well-defined mouse model of infection. Oligonucleotide microarrays were used to analyse the regulation of gene expression in murine bone marrow-derived macrophages infected with Mycobacterium tuberculosis. Four mouse strains, known to differ in terms of growth permissiveness for M. tuberculosis in infected tissues, in the development of pulmonary pathology, and in the rate of premature death due to tuberculosis, were compared: C57BL/6 and BALB/c representing resistant, DBA/2 and CBA/J representing susceptible mouse strains. Genes (55) were regulated more than two-fold in macrophages of all strains investigated following M. tuberculosis infection. Importantly, 18 genes were commonly regulated only in macrophages of the two resistant strains upon infection, and 102 genes were commonly regulated exclusively in macrophages of the two susceptible strains. Using this approach, we have therefore identified more than 100 genes potentially associated with resistance and susceptibility, respectively, to TB at the macrophage level. A tentative interpretation of our microarray data suggests that macrophages from susceptible mice predominantly stimulate the recruitment of cells that contribute disproportionately to tissue damage rather than to microbial elimination. In conclusion, microarray gene chips are useful tools for generating new hypotheses about resistance and susceptibility to TB, and the mouse model can now be used to subject candidate genes identified by this approach to further functional analyses.

Studies in mouse infection models clearly demonstrate tumour necrosis factor (TNF) to be a critical component of both the antibacterially protective and the inflammatory immune response to Mycobacterium tuberculosis. It is therefore not surprising that treatment of patients—for example, those with rheumatoid arthritis—with biological agents interfering with TNF activity have shown an increased risk of reactivating tuberculosis. However, conceivably, TNF targeting biological agents can be developed that because of their particular mode of action and their specific pharmacodynamics may be less likely to have this side effect.

Containment of intracellularly viable microorganisms requires an intricate cooperation between macrophages and T cells, the most potent mediators known to date being IFN-gamma and TNF. To identify novel mechanisms involved in combating intracellular infections, experiments were performed in mice with selective defects in the lymphotoxin (LT)/LT beta R pathway. When mice deficient in LT alpha or LT beta were challenged intranasally with Mycobacterium tuberculosis, they showed a significant increase in bacterial loads in lungs and livers compared with wild-type mice, suggesting a role for LT alpha beta heterotrimers in resistance to infection. Indeed, mice deficient in the receptor for LT alpha(1)beta(2) heterotrimers (LT beta R-knockout (KO) mice) also had significantly higher numbers of M. tuberculosis in infected lungs and exhibited widespread pulmonary necrosis already by day 35 after intranasal infection. Furthermore, LT beta R-KO mice were dramatically more susceptible than wild-type mice to i.p. infection with Listeria monocytogenes. Compared with wild-type mice, LT beta R-KO mice had similar transcript levels of TNF and IFN-gamma and recruited similar numbers of CD3(+) T cells inside granulomatous lesions in M. tuberculosis-infected lungs. Flow cytometry revealed that the LT beta R is expressed on pulmonary macrophages obtained after digestion of M. tuberculosis-infected lungs. LT beta R-KO mice showed delayed expression of inducible NO synthase protein in granuloma macrophages, implicating deficient macrophage activation as the most likely cause for enhanced susceptibility of these mice to intracellular infections. Since LIGHT-KO mice proved to be equally resistant to M. tuberculosis infection as wild-type mice, these data demonstrate that signaling of LT alpha(1)beta(2) heterotrimers via the LT beta R is an essential prerequisite for containment of intracellular pathogens.

Intracellular persistence of mycobacteria may result from an intricate balance between bacterial replication and signaling events leading to antimicrobial macrophage activities. Using human monocyte-derived macrophages, we investigated the relevance of mitogen-activated protein kinase activation for the growth control ofMycobacterium aviumisolates differing in their abilities to multiply intracellularly. The highly replicative smooth transparent morphotype ofM. aviumstrain 2151 induced significantly less p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation than the smooth opaque morphotype of the same strain, which was gradually eliminated from macrophage cultures. Inhibition of the p38 pathway by highly specific inhibitors did not significantly affect mycobacterial replication within macrophages, regardless of the in vitro virulence of theM. aviumstrain. However, repression of the ERK1/2 pathway further enhanced intracellular growth of highly replicativeM. aviumstrains, although it did not increase survival of the poorly replicatingM. aviumisolate. Inhibition of the ERK1/2 pathway resulted in decreased tumor necrosis alpha (TNF-α) secretion irrespective of the virulence of theM. aviumisolate used for infection, revealing that TNF-α could have been only partially responsible for the control of intracellularM. aviumgrowth. In conclusion, ERK1/2- and TNF-α-independent pathways are sufficient to limit intramacrophage growth of less-virulentM. aviumstrains, but early ERK1/2 activation in infected macrophages is critically involved in controlling the growth of highly replicativeM. aviumstrains.

Structural modifications of trivalent cluster mannosides are presented to further elucidate the ligand preferences of the type-1 fimbrial lectin of Escherichia coli. Two types of variations are performed, either regarding the aglycone part of cluster mannosides of type 2, leading to 27, or altering the spacer lengths of mannosyl clusters of type 1, leading to clusters 20–22. Biotinylation of the cluster mannoside with the highest affinity to the type-1 fimbrial lectin is also shown (33). Testing of the inhibitory potencies of the synthesised cluster glycosides as inhibitors of mannose-specific (type-1 fimbriae-mediated) binding of E. coli to mannan in an enzyme-linked immunosorbent assay (ELISA) suggests that a structural preorganisation as given in cluster 2a can be favourably combined with greater spacer flexibility as in cluster 22.

Focal accumulations of mononuclear cells, called granulomas, are a hallmark of mycobacterial infections. A common misconception is that granulomas are uniformly protective. In transparent zebrafish larvae infected with Mycobacterium marinum , Volkman et al. demonstrate an interaction of mycobacteria with epithelial cells that helps recruit macrophages to the granuloma as "feeder cells."