Use of Mono Mac 6 human monocytic cell line and J774 murine macrophage cell line in parallel antimycobacterial drug studies

Wright, E. L.; Quenelle, Debra C.; Suling, William J.; Barrow, William W.

doi:10.1128/aac.40.9.2206

Cited by 34 publications

(35 citation statements)

References 13 publications

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“…We next investigated the effects of hypoxia on the expression of the pH-sensing receptors in the human cell line MM6, which shows characteristics and functional features of mature blood monocytes 33, 34. Expression levels of the pH-sensing receptors OGR1, GPR4, and TDAG8, in MM6 cells exposed to severe hypoxia (0.2% O 2 ) or modest hypoxia (2% O 2 ) for 18 hours, were examined by RT-qPCR.…”

Section: Resultsmentioning

confidence: 99%

Hypoxia Positively Regulates the Expression of pH-Sensing G-Protein–Coupled Receptor OGR1 (GPR68)

Vallière

Cosín-Roger

Simmen

et al. 2016

Cellular and Molecular Gastroenterology and Hepatology

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Background & AimsA novel family of proton-sensing G-protein–coupled receptors, including ovarian cancer G-protein–coupled receptor 1 (OGR1) (GPR68) has been identified to play a role in pH homeostasis. Hypoxia is known to change tissue pH as a result of anaerobic glucose metabolism through the stabilization of hypoxia-inducible factor-1α. We investigated how hypoxia regulates the expression of OGR1 in the intestinal mucosa and associated cells.MethodsOGR1 expression in murine tumors, human colonic tissue, and myeloid cells was determined by quantitative reverse-transcription polymerase chain reaction. The influence of hypoxia on OGR1 expression was studied in monocytes/macrophages and intestinal mucosa of inflammatory bowel disease (IBD) patients. Changes in OGR1 expression in MonoMac6 (MM6) cells under hypoxia were determined upon stimulation with tumor necrosis factor (TNF), in the presence or absence of nuclear factor-κB (NF-κB) inhibitors. To study the molecular mechanisms involved, chromatin immunoprecipitation analysis of the OGR1 promoter was performed.ResultsOGR1 expression was significantly higher in tumor tissue compared with normal murine colon tissue. Hypoxia positively regulated the expression of OGR1 in MM6 cells, mouse peritoneal macrophages, primary human intestinal macrophages, and colonic tissue from IBD patients. In MM6 cells, hypoxia-enhanced TNF-induced OGR1 expression was reversed by inhibition of NF-κB. In addition to the effect of TNF and hypoxia, OGR1 expression was increased further at low pH. Chromatin immunoprecipitation analysis showed that HIF-1α, but not NF-κB, binds to the promoter of OGR1 under hypoxia.ConclusionsThe enhancement of TNF- and hypoxia-induced OGR1 expression under low pH points to a positive feed-forward regulation of OGR1 activity in acidic conditions, and supports a role for OGR1 in the pathogenesis of IBD.

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Section: Resultsmentioning

confidence: 99%

Hypoxia Positively Regulates the Expression of pH-Sensing G-Protein–Coupled Receptor OGR1 (GPR68)

Vallière

Cosín-Roger

Simmen

et al. 2016

Cellular and Molecular Gastroenterology and Hepatology

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“…The intracellular activity of the drugs was evaluated in J774 cells (ATCC TIB-67™), as reported previously for mycobacteria, 19–22 but with some modifications. J774 cells were maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence

Andreu

Fletcher

Krishnan

et al. 2011

Journal of Antimicrobial Chemotherapy

107

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ObjectivesTuberculosis drug development is hampered by the slow growth of Mycobacterium tuberculosis. Bioluminescence, light produced by an enzymatic reaction, constitutes a rapid and highly sensitive measurement of cell metabolic function that can be used as an indirect marker of cell viability in drug screening assays. The aim of this work was to validate and standardize the use of luminescent M. tuberculosis strains to test the activity of antibacterial drugs in vitro and inside macrophages in a 96-well format.MethodsWe have used strains that express the bacterial lux operon and therefore do not require exogenous substrate to produce light, as well as strains expressing the firefly luciferase that need luciferin substrate. Results were compared with those obtained using the resazurin reduction assay and cfu plating.ResultsUsing bioluminescence we were able to reduce the time required to measure the MIC and bactericidal concentrations of antimicrobials to just 3 and 6 days, respectively. Furthermore, antibacterial activity against intracellular mycobacteria was detected within 2 days post-infection. Results were comparable to those obtained by conventional methods.ConclusionsWe have developed a simple and rapid method for screening antimycobacterial drugs in culture and in macrophages. The use of autoluminescent bacteria also facilitates the determination of growth and inhibition kinetics. The method is cost-effective, can easily be adapted to a larger scale and is amenable to automation. Current efforts are directed towards applying this technology to drug screening in vivo.

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“…Bacteria and the human BEC line 5637 (ATCC HTB-9) and primary BECs were cultured as described previously [11,18]. Human airway epithelial cells (16-HBE) were cultured in DMEM plus 4 mM glutamine and 10% FBS, and the human monocytic cell line (Mono Mac 6) was cultured as described previously [19]. …”

Section: Methodsmentioning

confidence: 99%

A Novel TLR4-Mediated Signaling Pathway Leading to IL-6 Responses in Human Bladder Epithelial Cells

et al. 2007

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The vigorous cytokine response of immune cells to Gram-negative bacteria is primarily mediated by a recognition molecule, Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide (LPS) and initiates a series of intracellular NF-κB–associated signaling events. Recently, bladder epithelial cells (BECs) were reported to express TLR4 and to evoke a vigorous cytokine response upon exposure to LPS. We examined intracellular signaling events in human BECs leading to the production of IL-6, a major urinary cytokine, following activation by Escherichia coli and isolated LPS. We observed that in addition to the classical NF-κB–associated pathway, TLR4 triggers a distinct and more rapid signaling response involving, sequentially, Ca2+, adenylyl cyclase 3–generated cAMP, and a transcriptional factor, cAMP response element–binding protein. This capacity of BECs to mobilize secondary messengers and evoke a more rapid IL-6 response might be critical in their role as first responders to microbial challenge in the urinary tract.

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Use of Mono Mac 6 human monocytic cell line and J774 murine macrophage cell line in parallel antimycobacterial drug studies

Cited by 34 publications

References 13 publications

Hypoxia Positively Regulates the Expression of pH-Sensing G-Protein–Coupled Receptor OGR1 (GPR68)

Hypoxia Positively Regulates the Expression of pH-Sensing G-Protein–Coupled Receptor OGR1 (GPR68)

Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence

A Novel TLR4-Mediated Signaling Pathway Leading to IL-6 Responses in Human Bladder Epithelial Cells

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