Uptake and Intracellular Activity of Moxifloxacin in Human Neutrophils and Tissue-Cultured Epithelial Cells

ABSTRACT The penetration by moxifloxacin of human neutrophils (polymorphonuclear leukocytes [PMN]) and tissue-cultured epithelial cells (McCoy cells) was evaluated by a fluorometric assay. At extracellular concentrations of 5 mg/liter, the cellular-to-extracellular concentration ratios (C/E) of moxifloxacin in PMN and McCoy cells were 10.9 ± 1.0 and 8.7 ± 1.0, respectively (20 min; 37°C). The uptake of moxifloxacin by PMN was rapid, reversible, nonsaturable (at extracellular concentrations ranging from 1 to 50 μg/ml), and not affected by cell viability. The uptake of moxifloxacin was affected by external pH and the environmental temperature. The incubation of PMN in the presence of sodium fluoride, sodium cyanide, and carbonyl cyanidem-chlorophenylhydrazone significantly decreased the C/E of this agent. Neither PMN stimulation nor phagocytosis of opsonizedStaphylococcus aureus significantly affected the uptake of moxifloxacin by human PMN. This agent, at concentrations of 0.5, 1, and 5 mg/liter, induced a significant reduction in the survival of intracellular S. aureus in human PMN. In summary, moxifloxacin reaches much higher intracellular concentrations within phagocytic and nonphagocytic cells than extracellular ones, remaining active inside the neutrophils.

Download Full-text

Uptake and intracellular activity of trovafloxacin in human phagocytes and tissue-cultured epithelial cells.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.2.274 ◽

1997 ◽

Vol 41 (2) ◽

pp. 274-277 ◽

Cited By ~ 35

Author(s):

A Pascual ◽

I García ◽

S Ballesta ◽

E J Perea

Keyword(s):

Staphylococcus Aureus ◽

Epithelial Cells ◽

Polymorphonuclear Leukocytes ◽

Peritoneal Macrophages ◽

Extracellular Concentration ◽

Opsonized Zymosan ◽

Intracellular Activity ◽

Cultured Epithelial Cells ◽

Energy Dependent ◽

Human Polymorphonuclear Leukocytes

The penetration of trovafloxacin into human polymorphonuclear leukocytes (PMNs), human peritoneal macrophages, and tissue-cultured epithelial cells (McCoy cells) was evaluated. The cellular concentration to extracellular concentration (C/E) ratios of trovafloxacin were greater than 9 for extracellular concentrations ranging from 0.5 to 25 micrograms/ml. The uptake of trovafloxacin by PMNs was rapid, reversible, nonsaturable, not energy dependent, and significantly increased at 4 degrees C. Ingestion of opsonized zymosan, but not opsonized Staphylococcus aureus, significantly increased the amount of PMN-associated trovafloxacin. This agent at concentrations of 0.5 and 1 microgram/ml induced a greater reduction in the survival of intracellular S. aureus in PMNs than ciprofloxacin and ofloxacin. It was concluded that trovafloxacin reaches concentrations within phagocytic and nonphagocytic cells several times higher than the extracellular ones, while it remains active in PMNs.

Download Full-text

Intracellular Penetration and Activity of Gemifloxacin in Human Polymorphonuclear Leukocytes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.11.3193-3195.2000 ◽

2000 ◽

Vol 44 (11) ◽

pp. 3193-3195 ◽

Cited By ~ 18

Author(s):

Isabel García ◽

Alvaro Pascual ◽

Sofía Ballesta ◽

Providencia Joyanes ◽

Evelio J. Perea

Keyword(s):

Staphylococcus Aureus ◽

Cell Viability ◽

Polymorphonuclear Leukocytes ◽

Environmental Temperature ◽

Intracellular Activity ◽

Intracellular Concentrations ◽

Human Polymorphonuclear Leukocytes

ABSTRACT The intracellular penetration and activity of gemifloxacin in human polymorphonuclear leukocytes (PMN) were evaluated. Gemifloxacin reached intracellular concentrations eight times higher than extracellular concentrations. The uptake was rapid, reversible, and nonsaturable and was affected by environmental temperature, cell viability, and membrane stimuli. At therapeutic extracellular concentrations, gemifloxacin showed intracellular activity against Staphylococcus aureus.

Download Full-text

Cellular accumulation of the new ketolide RU 64004 by human neutrophils: comparison with that of azithromycin and roxithromycin.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.10.2099 ◽

1997 ◽

Vol 41 (10) ◽

pp. 2099-2107 ◽

Cited By ~ 37

Author(s):

D Vazifeh ◽

H Abdelghaffar ◽

M T Labro

Keyword(s):

Polymorphonuclear Leukocytes ◽

Inhibitory Concentration ◽

Interindividual Variability ◽

Human Neutrophils ◽

Carrier Mediated Transport ◽

Saturation Kinetics ◽

Km Value ◽

Erythromycin A ◽

Carrier Systems ◽

External Ph

We analyzed the uptake of RU 64004 by human neutrophils (polymorphonuclear leukocytes [PMNs]) relative to those of azithromycin and roxithromycin. RU 64004 was strongly and rapidly accumulated by PMNs, with a cellular concentration/extracellular concentration ratio (C/E) of greater than 200 in the first 5 min, and this was followed by a plateau at 120 to 180 min, with a C/E of 461 +/- 14.8 (10 experiments) at 180 min. RU 64004 uptake was moderately sensitive to external pH, and activation energy was also moderate (63 +/- 3.8 kJ/mol). RU 64004 was mainly located in PMN granules (about 70%) and egressed slowly from loaded cells, owing to avid reuptake. The possibility that PMN uptake of RU 64004 and other macrolides occurs through a carrier-mediated system was suggested by three key results. First, there existed a strong interindividual variability in uptake kinetics, suggesting variability in the numbers or activity of a transport protein. Second, macrolide uptake displayed saturation kinetics characteristic of that of a carrier-mediated transport system: RU 64004 had the highest Vmax value (3,846 ng/2.5 x 10(6) PMNs/5 min) and the lowest Km value (about 28 microM), indicating a high affinity for the transporter. Third, as observed previously with other erythromycin A derivatives, Ni2+ (a blocker of the Na+/Ca2+ exchanger which mediates Ca2+ influx in resting neutrophils) impaired RU 64004 uptake by PMNs, with a 50% inhibitory concentration of about 3.5 mM. In addition, we found that an active process is also involved in macrolide efflux, because verapamil significantly potentiated the release of all three macrolides tested. This effect of verapamil does not seem to be related to an inhibition of Ca2+ influx, because neither EGTA [ethylene glycol-bis (beta-aminoethyl ether)-N,N',N'-tetraacetic acid] nor Ni2+ modified macrolide efflux. The nature and characteristics of the entry- and efflux-mediating carrier systems are under investigation.

Download Full-text

Uptake and Intracellular Activity of Linezolid in Human Phagocytes and Nonphagocytic Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.12.4013-4015.2002 ◽

2002 ◽

Vol 46 (12) ◽

pp. 4013-4015 ◽

Cited By ~ 15

Author(s):

Álvaro Pascual ◽

Sofía Ballesta ◽

Isabel García ◽

Evelio J. Perea

Keyword(s):

Cell Viability ◽

Staphylococcus Epidermidis ◽

Polymorphonuclear Leukocytes ◽

Environmental Temperature ◽

Cultured Cells ◽

Intracellular Activity ◽

Intracellular Concentrations ◽

Human Polymorphonuclear Leukocytes

ABSTRACT The intracellular penetration and activity of linezolid in human polymorphonuclear leukocytes and tissue-cultured cells (McCoy) were evaluated. Linezolid reached intracellular concentrations slightly greater than extracellular ones in both types of cell. The uptake was rapid and not saturable and was affected by environmental temperature and cell viability. Linezolid showed slight intracellular activity against Staphylococcus epidermidis at high extracellular concentrations.

Download Full-text

Intracellular Penetration and Activity of DX-619 in Human Polymorphonuclear Leukocytes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00427-06 ◽

2006 ◽

Vol 50 (9) ◽

pp. 3173-3174 ◽

Cited By ~ 1

Author(s):

Isabel García ◽

Sofía Ballesta ◽

Concepción Murillo ◽

Evelio J. Perea ◽

Álvaro Pascual

Keyword(s):

Staphylococcus Aureus ◽

Polymorphonuclear Leukocytes ◽

Environmental Temperature ◽

Metabolic Inhibitors ◽

Intracellular Activity ◽

Intracellular Concentrations ◽

Human Polymorphonuclear Leukocytes

ABSTRACT The intracellular penetration and activity of DX-619 in human polymorphonuclear leukocytes have been evaluated. DX-619 reached intracellular concentrations 10 times higher than the extracellular concentrations reached. Uptake was rapid, reversible, nonsaturable, and affected by environmental temperature, some metabolic inhibitors, and a soluble membrane activator. DX-619 showed intracellular activity against Staphylococcus aureus.

Download Full-text

Staphylococcus aureus Elaborates Leukocidin AB To Mediate Escape from within Human Neutrophils

Infection and Immunity ◽

10.1128/iai.00095-13 ◽

2013 ◽

Vol 81 (5) ◽

pp. 1830-1841 ◽

Cited By ~ 80

Author(s):

Ashley L. DuMont ◽

Pauline Yoong ◽

Bas G. J. Surewaard ◽

Meredith A. Benson ◽

Reindert Nijland ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Polymorphonuclear Leukocytes ◽

The United States ◽

Growth Conditions ◽

Human Neutrophils ◽

Cytotoxic Potential ◽

Content Type ◽

Human Pmns ◽

Mrsa Strains

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) strains of the pulsed-field type USA300 are primarily responsible for the current community-associated epidemic of MRSA infections in the United States. The success of USA300 is partly attributed to the ability of the pathogen to avoid destruction by human neutrophils (polymorphonuclear leukocytes [PMNs]), which are crucial to the host immune response toS. aureusinfection. In this work, we investigated the contribution of bicomponent pore-forming toxins to the ability of USA300 to withstand attack from primary human PMNs. We demonstrate thatin vitrogrowth conditions influence the expression, production, and availability of leukotoxins by USA300, which in turn impact the cytotoxic potential of this clone toward PMNs. Interestingly, we also found that upon exposure to PMNs, USA300 preferentially activates the promoter of thelukABoperon, which encodes the recently identified leukocidin AB (LukAB). LukAB elaborated by extracellularS. aureusforms pores in the plasma membrane of PMNs, leading to PMN lysis, highlighting a contribution of LukAB to USA300 virulence. We now show that LukAB also facilitates the escape of bacteria engulfed within PMNs, in turn enabling the replication and outgrowth ofS. aureus. Together, these results suggest that upon encountering PMNsS. aureusinduces the production of LukAB, which serves as an extra- and intracellular weapon to protect the bacterium from destruction by human PMNs.

Download Full-text

Proto-Oncogenes and Cell Cycle Gene Expression in Normal and Neoplastic Oral Epithelial Cells Stimulated With Soluble Factors From Single and Dual Biofilms of Candida albicans and Staphylococcus aureus

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.627043 ◽

2021 ◽

Vol 11 ◽

Author(s):

María Isabel Amaya Arbeláez ◽

Ana Carolina Alves de Paula e Silva ◽

Geovana Navegante ◽

Valeria Valente ◽

Paula Aboud Barbugli ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Staphylococcus Aureus ◽

Candida Albicans ◽

Epithelial Cells ◽

Cell Viability ◽

Cell Cycle Gene ◽

Soluble Factors ◽

Oral Epithelial Cells ◽

Oral Epithelial

This study was aimed at analyzing proto-oncogenic signaling pathway activation in normal oral keratinocytes (NOK-si) and neoplastic cell lines (SCC 25 and Detroit 562) stimulated with metabolites (soluble factors) from single and dual biofilms of Candida albicans and Staphylococcus aureus. Soluble factors (SF) from early (16-h) and mature (36-h) biofilms of C. albicans and S. aureus were collected and incubated with cell cultures, which were subsequently evaluated using gene expression via RT-qPCR, cell viability via AlamarBlueTM, and flow cytometry cell cycle analysis. In general, exposure to the SF of early and mature biofilms from C. albicans and dual species caused a major reduction in NOK-si cell viability and enhanced the sub G0 phase. This led to a decrease in gene expression. However, in this cell line, SF of S. aureus biofilms upregulated the CDKN1A gene followed by the maintenance of cell viability and a significant increase in the G2/M population. For tumor cells, SCC 25 and Detroit 562, the stimuli of SF biofilms upregulated oncogenes such as hRAS and mTOR, as well as Bcl-2 and CDKN1A. SCC 25 and Detroit 562 cells could survive even after 24 h of stimuli from both SF (early and mature). This occurred without significant changes taking place in the cell cycle progression for SCC 25, and with a significant tendency to increase the G2/M phase for Detroit 562. These results point to the fact that metabolites from prevalent clinical fungal and bacterial biofilms, C. albicans and S. aureus, can disrupt the homeostasis of normal and neoplastic oral epithelial cells. This changes proto-oncogenes’ expression, specifically PI3KCA, hRAS, mTOR, BRAF, and cell cycle genes CDKN1A and Bcl-2, thus causing a disturbance in cell viability, survival, and the cell cycle profile.

Download Full-text

Protein A Modulates Neutrophil and Keratinocyte Signaling and Survival in Response to Staphylococcus aureus

Frontiers in Immunology ◽

10.3389/fimmu.2020.524180 ◽

2021 ◽

Vol 11 ◽

Author(s):

Camila Ledo ◽

Cintia D. Gonzalez ◽

Ailin Garofalo ◽

Florencia Sabbione ◽

Irene A. Keitelman ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Death ◽

Epithelial Cells ◽

Inflammatory Response ◽

Protein A ◽

Human Neutrophils ◽

Potent Inducer ◽

Tnf Α

The type 1 TNF-α receptor (TNFR1) has a central role in initiating both pro-inflammatory and pro-apoptotic signaling cascades in neutrophils. Considering that TNFR1 signals Staphylococcus aureus protein A (SpA), the aim of this study was to explore the interaction of this bacterial surface protein with neutrophils and keratinocytes to underscore the signaling pathways that may determine the fate of these innate immune cells in the infected tissue during staphylococcal skin infections. Using human neutrophils cultured in vitro and isogenic staphylococcal strains expressing or not protein A, we demonstrated that SpA is a potent inducer of IL-8 in neutrophils and that the induction of this chemokine is dependent on the SpA-TNFR1 interaction and p38 activation. In addition to IL-8, protein A induced the expression of TNF-α and MIP-1α highlighting the importance of SpA in the amplification of the inflammatory response. Protein A contributed to reduce neutrophil mortality prolonging their lifespan upon the encounter with S. aureus. Signaling initiated by SpA modulated the type of neutrophil cell death in vitro and during skin and soft tissue infections (SSTI) in vivo triggering the apoptotic pathway instead of necrosis. Moreover, SpA induced pro-inflammatory cytokines in keratinocytes, modulating their survival in vitro and preventing the exacerbated necrosis and ulceration of the epithelium during SSTI in vivo. Taken together, these results highlight the importance of the inflammatory signaling induced by protein A in neutrophils and skin epithelial cells. The ability of protein A to modulate the neutrophil/epithelial cell death program in the skin is of clinical relevance considering that lysis of neutrophils and epithelial cells will promote an intense inflammatory response and contribute to tissue damage, a non-desirable feature of complicated SSTI.

Download Full-text