JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability.
J. Microbiol. Methods
PUBLISHED: 09-03-2014
Show Abstract
Hide Abstract
The intracellular (IC) effect of drugs against Mycobacterium tuberculosis (Mtb) is not well established but increasingly important to consider when combining current and future multidrug regimens into the best possible treatment strategies. For this purpose, we developed an IC model based on a genetically modified Mtb H37Rv strain, expressing the Vibrio harvei luciferase (H37Rv-lux) infecting the human macrophage like cell line THP-1. Cells were infected at a low multiplicity of infection (1:1) and subsequently exposed to isoniazid (INH), ethambutol (EMB), amikacin (AMI) or levofloxacin (LEV) for 5days in a 96-well format. Cell viability was evaluated by Calcein AM and was maintained throughout the experiment. The number of viable H37Rv-lux was determined by luminescence and verified by a colony forming unit analysis. The results were compared to the effects of the same drugs in broth cultures. AMI, EMB and LEV were significantly less effective intracellularly (MIC90: >4mg/L, 8mg/L and 2mg/L, respectively) compared to extracellularly (MIC90: 0.5mg/L for AMI and EMB; 0.25mg/L for LEV). The reverse was the case for INH (IC: 0.064mg/L vs EC: 0.25mg/L). In conclusion, this luciferase based method, in which monitoring of cell viability is included, has the potential to become a useful tool while evaluating the intracellular effects of anti-mycobacterial drugs.
Related JoVE Video
Antimycobacterial activity of selected medicinal plants traditionally used in Sudan to treat infectious diseases.
J Ethnopharmacol
PUBLISHED: 06-19-2014
Show Abstract
Hide Abstract
The emergence of multidrug-resistant strains of Mycobacterium tuberculosis underscores the need for continuous development of new and efficient methods to determine the susceptibility of isolates of Mycobacterium tuberculosis in the search for novel antimycobacterial agents. Natural products constitute an important source of new drugs, and design and implementation of antimycobacterial susceptibility testing methods are necessary to evaluate the different extracts and compounds. In this study we have explored the antimycobacterial properties of 50 ethanolic extracts from different parts of 46 selected medicinal plants traditionally used in Sudan to treat infectious diseases.
Related JoVE Video
Replication Rates of Mycobacterium tuberculosis in Human Macrophages Do Not Correlate with Mycobacterial Antibiotic Susceptibility.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
The standard treatment of tuberculosis (TB) takes six to nine months to complete and this lengthy therapy contributes to the emergence of drug-resistant TB. TB is caused by Mycobacterium tuberculosis (Mtb) and the ability of this bacterium to switch to a dormant phenotype has been suggested to be responsible for the slow clearance during treatment. A recent study showed that the replication rate of a non-virulent mycobacterium, Mycobacterium smegmatis, did not correlate with antibiotic susceptibility. However, the question whether this observation also holds true for Mtb remains unanswered. Here, in order to mimic physiological conditions of TB infection, we established a protocol based on long-term infection of primary human macrophages, featuring Mtb replicating at different rates inside the cells. During conditions that restricted Mtb replication, the bacterial phenotype was associated with reduced acid-fastness. However, these phenotypically altered bacteria were as sensitive to isoniazid, pyrazinamide and ethambutol as intracellularly replicating Mtb. In support of the recent findings with M. smegmatis, we conclude that replication rates of Mtb do not correlate with antibiotic tolerance.
Related JoVE Video
Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
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.
Related JoVE Video
Modeling Mycobacterium tuberculosis early granuloma formation in experimental human lung tissue.
Dis Model Mech
PUBLISHED: 11-07-2013
Show Abstract
Hide Abstract
The widely used animal models for tuberculosis (TB) display fundamental differences from human TB. Therefore, a validated model that recapitulates human lung TB is attractive for TB research. Here, we describe a unique method for establishment of TB infection in an experimental human lung tissue model. The model is based on cell lines derived from human lungs and primary macrophages from peripheral blood, and display characteristics of human lung tissue including evenly integrated macrophages throughout the epithelium, production of extracellular matrix, stratified epithelia and mucus secretion. Establishment of experimental infection in the model tissue with Mycobacterium tuberculosis, the bacterium that causes TB, resulted in clustering of macrophages at the site of infection, reminiscent of early TB granuloma formation. We quantitated the extent of granuloma formation induced by different strains of mycobacteria and validated our model against findings in other TB models. We found that early granuloma formation is dependent on ESAT-6, which is secreted via the Type VII secretion machinery of virulent mycobacteria. Our model, which can facilitate the discovery of the interactions between mycobacteria and host cells in a physiological environment, is the first lung tissue model described for TB.
Related JoVE Video
Human Gene Variants Linked to Enhanced NLRP3 Activity Limit Intramacrophage Growth of Mycobacterium tuberculosis.
J. Infect. Dis.
PUBLISHED: 10-24-2013
Show Abstract
Hide Abstract
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.
Related JoVE Video
A mathematical model Prod. Type: FTPof the initial interaction between Mycobacterium tuberculosis and macrophages.
J. Theor. Biol.
PUBLISHED: 05-16-2013
Show Abstract
Hide Abstract
There is a large body of literature describing molecular level interactions between Mycobacterium tuberculosis (Mtb) and macrophages. Macrophages initiate a range of anti-bacterial mechanisms in response to infection, and Mtb is capable of surviving and circumventing many of these responses. We apply a computational approach to ask: what are the effects on the cellular level of these opposing interactions? The model considers the interplay between bacterial killing and the pathogens interference with macrophage function. The results reveal an oscillating balance between host and pathogen, but the balance is transient and varies in length, indicating that stochasticity in the bacterial population or host response could contribute to the diverse incubation periods observed in exposed individuals. The model captures host and strain variation and gives new insight into host-pathogen compatibility and co-evolution.
Related JoVE Video
Inside or outside the phagosome? The controversy of the intracellular localization of Mycobacterium tuberculosis.
Tuberculosis (Edinb)
PUBLISHED: 07-04-2011
Show Abstract
Hide Abstract
The localization of Mycobacterium tuberculosis (Mtb) inside the macrophage has been a matter of debate in recent years. Upon inhalation, the bacterium is taken up into macrophage phagosomes, which are manipulated by the bacterium. Subsequent translocation of the bacilli into the cytosol has been observed by several groups, while others fail to observe this phenomenon. Here, we review the available literature in favour of and against this idea, and scrutinize the existing data on how human macrophages control Mtb infection, relating this to the robustness of the host cell. We conclude that both phagosomal maturation inhibition and escape from the phagosome are part of the greater infection strategy of Mtb. The balance between the host cell and the infecting bacterium is an important factor in determining the outcome of infection as well as whether phagosomal escape occurs and can be captured.
Related JoVE Video
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
Show Abstract
Hide Abstract
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.
Related JoVE Video
Importance of phagosomal functionality for growth restriction of Mycobacterium tuberculosis in primary human macrophages.
J Innate Immun
PUBLISHED: 02-13-2011
Show Abstract
Hide Abstract
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.
Related JoVE Video
Validation of a medium-throughput method for evaluation of intracellular growth of Mycobacterium tuberculosis.
Clin. Vaccine Immunol.
PUBLISHED: 01-27-2010
Show Abstract
Hide Abstract
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.
Related JoVE Video
Leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts.
Microbes Infect.
PUBLISHED: 06-06-2009
Show Abstract
Hide Abstract
Lipophosphoglycan (LPG), the major surface glycoconjugate on Leishmania donovani promastigotes, is crucial for the establishment of infection inside macrophages. LPG comprises a polymer of repeating Galbeta1,4Manalpha-PO(4) attached to a lysophosphatidylinositol membrane anchor. LPG is transferred from the parasite to the host macrophage membrane during phagocytosis and induces periphagosomal F-actin accumulation correlating with an inhibition of phagosomal maturation. The biophysical properties of LPG suggest that it may be intercalated into membrane rafts of the host-cell membrane. The aim of this study was to investigate if the effects of LPG on phagosomal maturation are mediated via action on membrane rafts. We show that LPG accumulates in rafts during phagocytosis of L. donovani and that disruption of membrane rafts abolished the effects of LPG on periphagosomal F-actin and phagosomal maturation, indicating that LPG requires intact membrane rafts to manipulate host-cell functions. We conclude that LPG associates with membrane rafts in the host cell and exert its actions on host-cell actin and phagosomal maturation through subversion of raft function.
Related JoVE Video
Combined polymorphisms in genes encoding the inflammasome components NALP3 and CARD8 confer susceptibility to Crohns disease in Swedish men.
Am. J. Gastroenterol.
PUBLISHED: 03-24-2009
Show Abstract
Hide Abstract
Crohns disease (CD) is characterized by overproduction of proinflammatory cytokines like interleukin (IL)-1beta. Production of mature IL-1beta is dependent on a caspase-1-activating protein complex called the NALP3 inflammasome, composed of NALP3, ASC, and CARD8. NALP3 shares structural similarities with Nod2, and both of these proteins are required for bacteria-induced IL-1beta secretion. The combination of the polymorphisms CARD8 (C10X)and NALP3 (Q705K) was recently shown to be associated with rheumatoid arthritis.Our aim was to investigate whether these combined polymorphisms play a role in the susceptibility to CD.
Related JoVE Video
TRIM27 negatively regulates NOD2 by ubiquitination and proteasomal degradation.
PLoS ONE
Show Abstract
Hide Abstract
NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohns disease. We found that TRIM27 expression is increased in Crohns disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus.We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.
Related JoVE Video
Resistance to first-line anti-TB drugs is associated with reduced nitric oxide susceptibility in Mycobacterium tuberculosis.
PLoS ONE
Show Abstract
Hide Abstract
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.
Related JoVE Video
The Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to excessive interleukin-1? and IL-18 production.
PLoS ONE
Show Abstract
Hide Abstract
The Q705K polymorphism in NLRP3 has been implicated in several chronic inflammatory diseases. In this study we determine the functional role of this commonly occurring polymorphism using an in-vitro system.
Related JoVE Video
Common genetic variations in the NALP3 inflammasome are associated with delayed apoptosis of human neutrophils.
PLoS ONE
Show Abstract
Hide Abstract
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.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

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.