Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Atypon full text link Atypon Free PMC article
Full text links

Actions

.2016 Aug;14(6):345-54.
doi: 10.1089/adt.2016.717. Epub 2016 Jun 21.

A Macrophage Infection Model to Predict Drug Efficacy Against Mycobacterium Tuberculosis

Affiliations

A Macrophage Infection Model to Predict Drug Efficacy Against Mycobacterium Tuberculosis

Kaitlyn Schaaf et al. Assay Drug Dev Technol.2016 Aug.

Abstract

In the last 40 years, only a single new antituberculosis drug was FDA approved. New tools that improve the drug development process will be essential to accelerate the development of next-generation antituberculosis drugs. The drug development process seems to be hampered by the inefficient transition of initially promising hits to candidate compounds that are effective in vivo. In this study, we introduce an inexpensive, rapid, and BSL-2 compatible infection model using macrophage-passaged Mycobacterium tuberculosis (Mtb) that forms densely packed Mtb/macrophage aggregate structures suitable for drug efficacy testing. Susceptibility to antituberculosis drugs determined with this Mtb/macrophage aggregate model differed from commonly used in vitro broth-grown single-cell Mtb cultures. Importantly, altered drug susceptibility correlated well with the reported ability of the respective drugs to generate high tissue and cerebrospinal fluid concentrations relative to their serum concentrations, which seems to be the best predictors of in vivo efficacy. Production of these Mtb/macrophage aggregates could be easily scaled up to support throughput efforts. Overall, its simplicity and scalability should make this Mtb/macrophage aggregate model a valuable addition to the currently available Mtb drug discovery tools.

PubMed Disclaimer

Figures

<b>Fig. 1.</b>
Fig. 1.
AnMtb infection model to generate largeMtb/macrophage aggregate structures. THP-1 monocytes were infected withMtb-GFP at an MOI of 25 andMtb/macrophage aggregate formation was documented. Representative bright-field and GFP-merged images ofMtb-infected THP-1 aggregate structures are shown(A) on day 4 and(B) day 10 postinfection.(C) Generation ofMtb/macrophage aggregate structures in 96-well plate format. Bright-field and GFP-merged images were acquired 7 days postinfection by automated imaging. Enlarged view of images from(D) well A6 with 2.5 × objective and(E) well B5 with 20 × objective are shown. MOI, multiplicity of infection.
<b>Fig. 2.</b>
Fig. 2.
Biomolecular characterization ofMtb/macrophage aggregate structures.(A)Mtb/macrophage aggregate structures at 2 weeks postinfection were stained with 7AAD and a representative image is shown as merged GFP (Mtb) and RFP (7AAD) channels with bright-field image.(B)Mtb/macrophage aggregate structures at 2 weeks postinfection were stained with α-ICAM-1-PE mAbs and representative images are shown as merged GFP and RFP channels with bright-field images, or in separate channels alone.(C) Representative mergedred andgreen channel fluorescence images of a group of viable macrophages derived fromMtb/macrophage aggregates stained with α-ICAM-1-PE antibody (red) that contain intracellularMtb (green) at 2 weeks postinfection.(D) Intracellular survival ofMtb extracted from aggregate structures over a time course of 11 days was enumerated by CFU plating.(E)Mtb-GFP fromin vitro broth-grown single-cell cultures (top panel) or extracted fromMtb/macrophage aggregates structures (bottom panel) 11 days postinfection were stained with Nile Red and representative images are shown as a merge of GFP and RFP channels, or in each individual channel. 7AAD, 7-amino-actinomycin D.
<b>Fig. 3.</b>
Fig. 3.
Mtb drug sensitivity profiles for rifampicin and other antibiotics in theMtb/macrophage aggregate model. Drug susceptibility ofMtb derived fromMtb/macrophage aggregate cultures (Mtb-aggregate) to(A) rifampicin,(B) moxifloxacin,(C) ciprofloxacin,(D) streptomycin,(E) gentamicin,(F) cycloserine, and(G) clarithromycin compared toin vitro broth-grown single-cellMtb cultures (Mtb-single). All data are represented as killing curves indicating the % survival as normalized to maximal bacterial growth in the absence of drugs and expressed as the mean ± standard deviation of three independent experiments.
<b>Fig. 4.</b>
Fig. 4.
Drug susceptibility of latentMtb compared to actively replicating broth-grown cultures. Drug susceptibility ofMtb from nutrient starvation derived nonreplicatingMtb (Mtb-latent) to(A) rifampicin,(B) moxifloxacin,(C) ciprofloxacin, and(D) streptomycin compared to active growing broth-grown single-cellMtb cultures (Mtb-single). All data are represented as killing curves indicating the % survival as normalized to maximal bacterial growth in the absence of drugs and expressed as the mean ± standard deviation of three independent experiments.
See this image and copyright information in PMC

References

    1. Bass JB, Jr, Farer LS, Hopewell PC, et al. : Treatment of tuberculosis and tuberculosis infection in adults and children. American Thoracic Society and The Centers for Disease Control and Prevention. Am J Respir Crit Care Med 1994;149:1359–1374 - PubMed
    1. Dartois V: The path of anti-tuberculosis drugs: From blood to lesions to mycobacterial cells. Nat Rev Microbiol 2014;12:159–167 - PMC - PubMed
    1. Ramakrishnan L: Revisiting the role of the granuloma in tuberculosis. Nat Rev Immunol 2012;12:352–366 - PubMed
    1. Brennan PJ, Nikaido H: The envelope of mycobacteria. Annu Rev Biochem 1995;64:29–63 - PubMed
    1. Gupta A, Kaul A, Tsolaki AG, Kishore U, Bhakta S: Mycobacterium tuberculosis: Immune evasion, latency and reactivation. Immunobiology 2012;217:363–374 - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full text links
Atypon full text link Atypon Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2026 Movatter.jp