Simultaneous cytofluorometric measurement of phagocytosis, burst production and killing of human phagocytes using Candida albicans and Staphylococcus aureus as target organisms

Hr, Salih; Husfeld, L; Adam, Dieter

doi:10.1046/j.1469-0691.2000.00076.x

Cited by 27 publications

(24 citation statements)

References 28 publications

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“…The mixture of PECs and calcein-AM-labeled C. albicans was then reacted with PE-conjugated monoclonal antibody to GR-1. The percentage of double-positive PECs, labeled with GR-1 (orange fluorescence) and calcein-AM (green fluorescence), was recorded as the percentage of GR-1-positive PECs phagocytizing C. albicans (26).…”

Section: Methodsmentioning

confidence: 99%

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Susceptibility of Germfree Phagocyte Oxidase- and Nitric Oxide Synthase 2-Deficient Mice, Defective in the Production of Reactive Metabolites of Both Oxygen and Nitrogen, to Mucosal and Systemic Candidiasis of Endogenous Origin

Balish¹,

Warner

Nicholas³

et al. 2005

Infect Immun

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defective in the production of both reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), were used to investigate the role of phagocytic cells during mucosal and systemic candidiasis of endogenous origin. The alimentary tracts of germfree mice were colonized with Candida albicans wild type or each of two hyphal signaling-defective mutants (efg1/efg1 and efg1/efg1 cph1/cph1). All Candida-colonized gp91 phox؊/؊ /NOS2 ؊/؊ mice were moribund within 12 to 15 days after oral inoculation. C. albicans wild-type and mutant strains colonized the alimentary tracts equally well and were able to translocate, most likely via Peyer's patches and mesenteric lymph nodes, to the internal organs and trigger the formation of abscesses; however, the wild-type and mutant strains did not survive in the abscessed murine tissues. Surprisingly, there was no significant difference in the ability of peritoneal exudate cells from gp91 phox؊/؊ /NOS2 ؊/؊ , NOS2 ؊/؊ , gp91 phox؊/؊ , or immunocompetent C57BL/6 mice to kill C. albicans in vitro. This suggests that anti-Candida factors other than ROI and RNI can control the growth of C. albicans and that gp91 phox؊/؊ /NOS2 ؊/؊ mice die due to the inability of the host to control its inflammatory response to Candida. Correspondingly, reverse transcription-PCR analysis showed increased expression of the cytokines gamma interferon, tumor necrosis factor alpha, and the chemokines MIP-2 and KC at the site of infection, while interleukin-15 expression remained relatively unchanged between germfree and infected tissues. These studies indicate that defects in ROI and RNI enabled C. albicans to translocate and disseminate to the internal organs, resulting in an uncontrolled immune response, severe pathology, and death; however, ROI and RNI were not required for the killing of phagocytized C. albicans, indicating that other anti-Candida factors either compensate or are sufficient for the killing of phagocytized Candida.

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Section: Methodsmentioning

confidence: 99%

“…Briefly, PECs and C. albicans cells (1:1 ratio) were incubated at 37°C with 5% CO 2 as described previously (26). After a 2-h incubation, 2 M ethidium homodimer 1 (400 l) was added to the PECs-C. albicans mixture (200 l) and incubated at room temperature for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Susceptibility of Germfree Phagocyte Oxidase- and Nitric Oxide Synthase 2-Deficient Mice, Defective in the Production of Reactive Metabolites of Both Oxygen and Nitrogen, to Mucosal and Systemic Candidiasis of Endogenous Origin

Balish¹,

Warner

Nicholas³

et al. 2005

Infect Immun

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“…The first step in this method is to load bacterial cells with a fluorescent dye by incubating them with a fluorogenic esterase substrate (2,26), in this case, Calcien AM (CAM). This uncharged, nonfluorescent substrate diffuses passively into the cytoplasm of the cell, where it is acted upon by ubiquitous native esterases.…”

mentioning

confidence: 99%

Direct Visualization of Spatial and Temporal Patterns of Antimicrobial Action within Model Oral Biofilms

Takenaka

Trivedi

Corbin

et al. 2008

Appl Environ Microbiol

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A microscopic method for noninvasively visualizing the action of an antimicrobial agent inside a biofilm was developed and applied to describe spatial and temporal patterns of mouthrinse activity on model oral biofilms. Three species biofilms of Streptococcus oralis, Streptococcus gordonii, and Actinomyces naeslundii were grown in glass capillary flow cells. Bacterial cells were stained with the fluorogenic esterase substrate Calcien AM (CAM). Loss of green fluorescence upon exposure to an antimicrobial formulation was subsequently imaged by time-lapse confocal laser scanning microscopy. When an antimicrobial mouthrinse containing chlorhexidine digluconate was administered, a gradual loss of green fluorescence was observed that began at the periphery of cell clusters where they adjoined the flowing bulk fluid and progressed inward over a time period of several minutes. Image analysis was performed to quantify a penetration velocity of 4 m/min. An enzyme-based antimicrobial formulation led to a gradual, continually slowing loss of fluorescence in a pattern that was qualitatively different from the behavior observed with chlorhexidine. Ethanol at 11.6% had little effect on the biofilm. None of these treatments resulted in the removal of biomass from the biofilm. Most methods to measure or visualize antimicrobial action in biofilms are destructive. Spatial information is important because biofilms are known for their structural and physiological heterogeneity. The CAM staining technique has the potential to provide information about the rate of antimicrobial penetration, the presence of tolerant subpopulations, and the extent of biomass removal effected by a treatment.Direct time-lapse microscopic observation has proven to be an extremely powerful tool over the last decade in developing insight into the complex, spatially structured behaviors of microbial biofilms. This approach has revealed, for example, fluid flow patterns in the channels and interstices of heterogeneous biofilm structures (31), dynamic biomass movement and viscoelastic flow (17,30), the contribution of cell migration to the formation of mushroom-like structures (18), transient patterns of gene expression (25, 37), the time course of diffusive penetration of solutes into biofilms (15,24), and rapid motility of cells trapped in hollow biofilm cell clusters (14, 34).There have been few applications of time-lapse microscopy to investigate the action of antimicrobial agents against biofilms (4, 11, 16). Most of the techniques used to assess the action of antimicrobial agents on biofilms involve sacrificial sampling followed by plating or endpoint staining. By these methods it is difficult to gain insight into spatiotemporal patterns because it is not possible to observe a single spot in time.We have developed a microscopic method for noninvasively visualizing the action of an antimicrobial agent inside a biofilm. The first step in this method is to load bacterial cells with a fluorescent dye by incubating them with a fluorogenic esterase substrate (2...

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“…The aim of this study was to evaluate the influence of moxifloxacin on PMN function using a whole-blood flow-cytometric method. The assay, which has been described previously (5,9,10) is designed to investigate effects on the phagocytosis of Candida albicans and Staphylococcus aureus (the percentages of phagocytosing PMN were determined), the percentage of PMN producing respiratory burst, and the percentage of C. albicans that has been killed by PMN. All three parameters were measured at multiple time points: for phagocytosis and burst, at 0, 2, 4, 6, 8, 10, 15, 20, and 25 min; for killing, at 0, 5, 10, 15, 20, 30, and 40 min.…”

mentioning

confidence: 99%

Effects of Moxifloxacin on Neutrophil Phagocytosis, Burst Production, and Killing as Determined by a Whole-Blood Cytofluorometric Method

Fischer

Adam

2001

Antimicrob Agents Chemother

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Simultaneous cytofluorometric measurement of phagocytosis, burst production and killing of human phagocytes using Candida albicans and Staphylococcus aureus as target organisms

Abstract: The described method provides a new whole blood cytofluorometric assay, which combines rapid and simple handling with high reproducibility of results obtained by investigation of PMN activity using Candida albicans and Staphylococcus aureus as target organisms.

Cited by 27 publications

References 28 publications

Susceptibility of Germfree Phagocyte Oxidase- and Nitric Oxide Synthase 2-Deficient Mice, Defective in the Production of Reactive Metabolites of Both Oxygen and Nitrogen, to Mucosal and Systemic Candidiasis of Endogenous Origin

Susceptibility of Germfree Phagocyte Oxidase- and Nitric Oxide Synthase 2-Deficient Mice, Defective in the Production of Reactive Metabolites of Both Oxygen and Nitrogen, to Mucosal and Systemic Candidiasis of Endogenous Origin

Direct Visualization of Spatial and Temporal Patterns of Antimicrobial Action within Model Oral Biofilms

Effects of Moxifloxacin on Neutrophil Phagocytosis, Burst Production, and Killing as Determined by a Whole-Blood Cytofluorometric Method

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