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Find video protocols related to scientific articles indexed in Pubmed.
Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages.
PLoS ONE
PUBLISHED: 01-01-2014
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Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1? signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.
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Human Gene Variants Linked to Enhanced NLRP3 Activity Limit Intramacrophage Growth of Mycobacterium tuberculosis.
J. Infect. Dis.
PUBLISHED: 10-24-2013
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Activation of the NLRP3 inflammasome and subsequent generation of interleukin 1? is initiated in macrophages upon recognition of several stimuli. In the present work, we show that gain-of-function gene variants of inflammasome components known to predispose individuals to inflammatory disorders have a host-protective role during infection with Mycobacterium tuberculosis. By isolation of macrophages from patients and healthy blood donors with genetic variants in NLRP3 and CARD8 and subsequent infection of the cells with virulent M. tuberculosis, we show that these gene variants, combined, are associated with increased control of bacterial growth in human macrophages.
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Mycobacterium tuberculosis- induced neutrophil extracellular traps activate human macrophages.
J Innate Immun
PUBLISHED: 02-06-2013
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Neutrophils activated by Mycobacterium tuberculosis (Mtb) form neutrophil extracellular traps (NETs), containing DNA and several biologically active cytosolic and granular proteins. These NETs may assist in the innate immune defense against different pathogens. We investigated whether the NET-forming neutrophils mediate an activating signal to macrophages during the early multicellular inflammatory reaction and granuloma formation. Mtb-induced NETs were found to be reactive oxygen species dependent and phagocytosis dependent. A neutrophil elastase inhibitor also delayed NET formation. However, NET formation occurred independently of Mtb-induced apoptosis. We observed close interactions between macrophages and Mtb-activated neutrophils, where macrophages bound and phagocytosed NETs. Significant secretion of the cytokines interleukin (IL)-6, tumor necrosis factor-?, IL-1? and IL-10 were detected from macrophages cocultured with NETs from Mtb-activated but not phorbol myristate acetate-activated neutrophils. NETs binding heat shock protein 72 (Hsp72) or recombinant Hsp72 were able to trigger cytokine release from macrophages. Only Mtb-induced NETs contained Hsp72, suggesting that these NETs can transfer this danger signal to adjacent macrophages. We propose that Hsp72 sequestered in NETs plays an important role in the interaction between neutrophils and macrophages during the early innate immune phase of an Mtb infection. The immunomodulatory role of NETs and proteins derived from them may influence not only chronic inflammation during tuberculosis but also immune regulation and autoimmunity.
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Human macrophages infected with a high burden of ESAT-6-expressing M. tuberculosis undergo caspase-1- and cathepsin B-independent necrosis.
PLoS ONE
PUBLISHED: 04-29-2011
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Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1?, while a low MOI gave no IL-1? response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1? release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.
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Importance of phagosomal functionality for growth restriction of Mycobacterium tuberculosis in primary human macrophages.
J Innate Immun
PUBLISHED: 02-13-2011
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The best characterized survival mechanism of Mycobacterium tuberculosis inside the macrophage is the inhibition of phagosomal maturation. Phagosomal maturation involves several steps including fusion with lysosomes and acidification. However, it has not been elucidated which components of phagosomal maturation correlate with growth restriction of virulent mycobacteria in human macrophages, and we aimed to study this. We infected human monocyte-derived macrophages with M. tuberculosis and assessed bacterial replication, translocation of CD63 to the phagosome, and phagosomal acidification. We found that unstimulated human macrophages were able to control infection with M. tuberculosis upon inoculation at a low multiplicity of infection (MOI) of 1, but not at a high MOI of 10. The low MOI resulted in a macrophage-controlled balance between host cells and bacteria. Both H37Rv and H37Ra infection, at high and low MOI, led to equally ineffective translocation of CD63 to the phagosome. On the other hand, acidification of mycobacterial phagosomes was more efficient at MOI 1 than 10 with both mycobacterial strains, consistent with a direct or indirect role for phagosomal acidification in restricting M. tuberculosis growth. Furthermore, inhibition of the vacuolar H(+)-ATPase as well as of cathepsin D led to enhanced mycobacterial replication inside the macrophage. This again shows the importance of phagosomal acidification for control of mycobacterial growth, through the activation of lysosomal hydrolases. We conclude that acidification and related functional aspects of the mature phagosome are important factors for restriction of M. tuberculosis replication in human macrophages.
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Identification of drug susceptibility pattern and mycobacterial species in sputum smear positive pulmonary tuberculosis patients with and without HIV co-infection in north west Ethiopia.
Ethiop. Med. J.
PUBLISHED: 11-16-2010
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Ethiopia is among the high-burden countries of tuberculosis (TB) in the world Since mycobacterial culture and susceptibility testing are not routinely performed in Ethiopia, recent data on susceptibility patterns and the mycobacterial species cultured from sputum smear positive patients are limited.
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Kinetics of the QuantiFERON-TB Gold In-Tube test during treatment of patients with sputum smear-positive tuberculosis in relation to initial TST result and severity of disease.
Scand. J. Infect. Dis.
PUBLISHED: 05-15-2010
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The QuantiFERON-TB Gold In-Tube test (QFN) measures interferon-gamma production in response to Mycobacterium tuberculosis antigens. Our aim was to assess the kinetics of the QFN and initial tuberculin skin test (TST) result in relation to severity of disease in a tuberculosis (TB) endemic area. Smear-positive TB patients (n = 71) were recruited at Gondar University Hospital, Ethiopia. The TST, QFN, CD4+ cell count and clinical symptoms (TB score) were assessed and followed up during treatment. From baseline to 7 months after treatment, there was a significant decrease in QFN reactivity (93.8% to 62.5% in HIV-negative/TB; 70.3% to 33.3% in HIV-positive/TB patients) down to a level comparable to a control group of blood donors (51.2%). The agreement between TST and QFN was poor in TB patients compared to healthy controls. A negative TST correlated to more advanced TB in contrast to a negative QFN test. We conclude that the QFN reactivity is significantly reduced at the end of treatment against active TB to the background level of healthy blood donors, and that the agreement between TST and QFN is poor including correlation to the severity of disease.
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Dendritic cell activation by sensing Mycobacterium tuberculosis-induced apoptotic neutrophils via DC-SIGN.
Hum. Immunol.
PUBLISHED: 02-08-2010
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Mycobacterium tuberculosis (Mtb) manipulates cells of the innate immune system to provide the bacteria with a sustainable intracellular niche. Mtb spread through aerosol carrying them deep into the lungs, where they are internalized by phagocytic cells, such as neutrophils (PMNs), dendritic cells (DCs), and macrophages. PMNs undergo accelerated apoptosis after interaction with the bacterium, and apoptotic cells are sequestered by neighboring phagocytes. Removal of aged apoptotic cells because of natural tissue turnover is described as an immunologically silent process facilitating resolution of inflammation and inhibition of DC maturation. Silencing of immune cells could be favorable for intracellular bacteria. The aim of this study was to clarify the interaction between Mtb-induced apoptotic PMNs and DCs, and evaluate whether this interaction follows the proposed anti-inflammatory pathway. In contrast to aged apoptotic cells, Mtb-induced apoptotic PMNs induced functional DC maturation. We found that the cell fraction from Mtb-induced apoptotic PMNs contained almost all stimulatory capacity, suggesting that cell-cell interaction is crucial for DC activation. Inhibitory studies showed that this cell contact-dependent activation required binding of the PMN Mac-1 (CD11b/CD18) to the DC via DC-SIGN and endocytic activity involving the alpha(v)beta(5) but did not involve the scavenger receptor CD36. Taken together, this study demonstrates that the DCs can distinguish between normal and infected apoptotic PMNs via cellular crosstalk, where the DCs can sense the presence of danger on the Mtb-infected PMNs and modulate their response accordingly.
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Validation of a medium-throughput method for evaluation of intracellular growth of Mycobacterium tuberculosis.
Clin. Vaccine Immunol.
PUBLISHED: 01-27-2010
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Intracellular pathogens such as Mycobacterium tuberculosis have adapted to a life inside host cells, in which they utilize host nutrients to replicate and spread. Ineffective methods for the evaluation of growth of intracellular pathogens in their true environment pose an obstacle for basic research and drug screening. Here we present the validation of a luminometry-based method for the analysis of intramacrophage growth of M. tuberculosis. The method, which is performed in a medium-throughput format, can easily be adapted for studies of other intracellular pathogens and cell types. The use of host cells in drug-screening assays dedicated to find antimicrobials effective against intracellular pathogens permits the discovery of not only novel antibiotics but also compounds with immunomodulatory and virulence-impairing activities, which may be future alternatives or complements to antibiotics.
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Induction of apoptosis in human neutrophils by Mycobacterium tuberculosis is dependent on mature bacterial lipoproteins.
Microb. Pathog.
PUBLISHED: 02-19-2009
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Modulation of immune cell apoptosis is a key evasion strategy utilized by Mycobacterium tuberculosis (Mtb). To be able to multiply within macrophages, the bacterium delays apoptosis and down-regulates pro-inflammatory activation in these cells, whereas apoptosis is rapidly induced in the potently bactericidal neutrophils. Initial host-pathogen interactions between neutrophils and Mtb, subsequently leading to apoptosis, need to be investigated to understand the early features during Mtb infections. Opsonized Mtb were readily phagocytosed, and the immuno-mediated phagocytosis triggered early activation of anti-apoptotic Akt in the neutrophils but the bacteria still induced apoptosis to the same extent as non-phagocytosed Mtb. Mtb-induced apoptosis was strictly dependent on NADPH oxidase-generated reactive oxygen species, compounds shown to damage lysosomal granules. Despite this, we found no involvement of damaged azurophilic granules in Mtb-induced apoptosis in human neutrophils. Instead, the Mtb-induced apoptosis was p38 MAPK dependent and induced through the mitochondrial pathway. Moreover, Mtb deficient of mature lipoproteins lacked the determinants required for induction of neutrophil apoptosis. These results show that Mtb exert a strong intrinsic capacity to induce apoptosis in neutrophils that is capable of overcoming the anti-apoptotic signaling in the cell.
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The impact of asymptomatic helminth co-infection in patients with newly diagnosed tuberculosis in north-west Ethiopia.
PLoS ONE
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Areas endemic of helminth infection, tuberculosis (TB) and HIV are to a large extent overlapping. The aim of this study was to assess the impact of asymptomatic helminth infection on the immunological response among TB patients with and without HIV, their house hold contacts and community controls.
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Early treatment response evaluated by a clinical scoring system correlates with the prognosis of pulmonary tuberculosis patients in Ethiopia: a prospective follow-up study.
Scand. J. Infect. Dis.
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In resource-limited settings the monitoring of tuberculosis (TB) patients is challenging, and early identification of TB patients with a high mortality risk is important. The aim of this study was to investigate prospectively whether early changes in a clinical scoring system (TB score) can predict treatment outcome in Ethiopian patients with pulmonary tuberculosis.
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Resistance to first-line anti-TB drugs is associated with reduced nitric oxide susceptibility in Mycobacterium tuberculosis.
PLoS ONE
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The relative contribution of nitric oxide (NO) to the killing of Mycobacterium tuberculosis in human tuberculosis (TB) is controversial, although this has been firmly established in rodents. Studies have demonstrated that clinical strains of M. tuberculosis differ in susceptibility to NO, but how this correlates to drug susceptibility and clinical outcome is not known.
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Common genetic variations in the NALP3 inflammasome are associated with delayed apoptosis of human neutrophils.
PLoS ONE
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Neutrophils are key-players in the innate host defense and their programmed cell death and removal are essential for efficient resolution of inflammation. These cells recognize a variety of pathogens, and the NOD-like receptors (NLRs) have been suggested as intracellular sensors of microbial components and cell injury/stress. Some NLR will upon activation form multi-protein complexes termed inflammasomes that result in IL-1? production. NLR mutations are associated with auto-inflammatory syndromes, and our previous data propose NLRP3 (Q705K)/CARD-8 (C10X) polymorphisms to contribute to increased risk and severity of inflammatory disease by acting as genetic susceptibility factors. These gene products are components of the NALP3 inflammasome, and approximately 6.5% of the Swedish population are heterozygote carriers of these combined gene variants. Since patients carrying the Q705K/C10X polymorphisms display leukocytosis, the aim of the present study was to find out whether the inflammatory phenotype was related to dysfunctional apoptosis and impaired clearance of neutrophils by macrophages.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.