Characterization of a protein from normal human polymorphonuclear leukocytes with bactericidal activity against Pseudomonas aeruginosa.

ABSTRACT Chronic Pseudomonas aeruginosa lung infection is characterized by the presence of endobronchial antibiotic-tolerant biofilm, which is subject to strong oxygen (O2) depletion due to the activity of surrounding polymorphonuclear leukocytes. The exact mechanisms affecting the antibiotic susceptibility of biofilms remain unclear, but accumulating evidence suggests that the efficacy of several bactericidal antibiotics is enhanced by stimulation of aerobic respiration of pathogens, while lack of O2 increases their tolerance. In fact, the bactericidal effect of several antibiotics depends on active aerobic metabolism activity and the endogenous formation of reactive O2 radicals (ROS). In this study, we aimed to apply hyperbaric oxygen treatment (HBOT) to sensitize anoxic P. aeruginosa agarose biofilms established to mimic situations with intense O2 consumption by the host response in the cystic fibrosis (CF) lung. Application of HBOT resulted in enhanced bactericidal activity of ciprofloxacin at clinically relevant durations and was accompanied by indications of restored aerobic respiration, involvement of endogenous lethal oxidative stress, and increased bacterial growth. The findings highlight that oxygenation by HBOT improves the bactericidal activity of ciprofloxacin on P. aeruginosa biofilm and suggest that bacterial biofilms are sensitized to antibiotics by supplying hyperbaric O2.

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Construction and Characterization of a Pseudomonas aeruginosa Mucoid Exopolysaccharide-Alginate Conjugate Vaccine

Infection and Immunity ◽

10.1128/iai.71.7.3875-3884.2003 ◽

2003 ◽

Vol 71 (7) ◽

pp. 3875-3884 ◽

Cited By ~ 58

Author(s):

Christian Theilacker ◽

Fadie T. Coleman ◽

Simone Mueschenborn ◽

Nicolas Llosa ◽

Martha Grout ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Conjugate Vaccine ◽

Significant Proportion ◽

Vaccine Trial ◽

Keyhole Limpet Hemocyanin ◽

Opsonic Activity ◽

Human Sera ◽

Normal Human

ABSTRACT Deterioration of lung function in patients with cystic fibrosis (CF) is closely associated with chronic pulmonary infection with mucoid Pseudomonas aeruginosa. The mucoid exopolysaccharide (MEP) from P. aeruginosa has been shown to induce opsonic antibodies in mice that are protective against this chronic infection. MEP-specific opsonic antibodies are also commonly found in the sera of older CF patients lacking detectable P. aeruginosa infection. When used in a human vaccine trial, however, MEP only minimally induced opsonic antibodies. To evaluate whether conjugation of MEP to a carrier protein could improve its immunogenicity, we bound thiolated MEP to keyhole limpet hemocyanin (KLH) by using succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as a linker. In contrast to the native MEP polymer, the MEP-KLH conjugate vaccine induced high titers of MEP-specific immunoglobulin G (IgG) in C3H-HeN mice and in a rabbit. Sera from mice immunized with MEP-KLH conjugate, but not from animals immunized with comparable doses of native MEP, demonstrated opsonic killing activity. Vaccination with MEP-KLH conjugate induced opsonic antibodies broadly cross-reactive to heterologous mucoid strains of P. aeruginosa. Preexisting nonopsonic antibodies to MEP are found in normal human sera, including young CF patients, and their presence impedes the induction of opsonic antibodies. Induction of nonopsonic antibodies by either intraperitoneal injection of MEP or injection or feeding of the cross-reactive antigen, seaweed alginate, reduced the level of overall IgG elicited by follow-up immunization with the MEP-KLH conjugate. However, the opsonic activity was lower only in the sera of MEP-KLH conjugate-immunized mice with preexisting antibodies induced by MEP but not with antibodies induced by seaweed alginate. Immunization with MEP-KLH elicited a significant proportion of antibodies specific to epitopes involving O-acetate residues, and this subpopulation of antibodies mediated opsonic killing of mucoid P. aeruginosa in vitro. These results indicate that conjugation of MEP to KLH significantly enhances its immunogenicity and the elicitation of opsonic antibodies in mice and rabbits, that the conjugate induces opsonic antibodies in the presence of preexisting nonopsonic antibodies, and that opsonic antibodies to MEP are directed at epitopes that include acetate residues on the uronic acid polymer.

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Enhanced oxidative burst without interleukin 1 production by normal human polymorphonuclear leukocytes primed with muramyl dipeptides

Inflammation ◽

10.1007/bf00916017 ◽

1987 ◽

Vol 11 (2) ◽

pp. 153-161 ◽

Cited By ~ 7

Author(s):

C. Jupin ◽

M. Parant ◽

L. Chedid ◽

C. Damais

Keyword(s):

Oxidative Burst ◽

Polymorphonuclear Leukocytes ◽

Interleukin 1 ◽

Normal Human ◽

Human Polymorphonuclear Leukocytes

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Uptake and intracellular activity of NM394, a new quinolone, in human polymorphonuclear leukocytes.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.3.739 ◽

1996 ◽

Vol 40 (3) ◽

pp. 739-742 ◽

Cited By ~ 15

Author(s):

M Ozaki ◽

K Komori ◽

M Matsuda ◽

R Yamaguchi ◽

T Honmura ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Infection ◽

Polymorphonuclear Leukocytes ◽

Concentration Ratio ◽

Extracellular Concentration ◽

Intracellular Activity ◽

Subsequent Elution ◽

Active Transport System ◽

Human Polymorphonuclear Leukocytes ◽

Kinetics Of

The uptake of NM394, a new quinolone, by and its subsequent elution from human polymorphonuclear leukocytes were studied and compared with those of ofloxacin and ciprofloxacin. The kinetics of the uptake of NM394 was similar to that of ciprofloxacin. The maximum intracellular-to-extracellular concentration ratio was 12.3, compared with 8.6 for ciprofloxacin and 4.9 for ofloxacin at the extracellular concentration of 20 micrograms/ml. The elution of NM394 from human polymorphonuclear leukocytes occurs relatively slowly; 5 min after the removal of extracellular NM394, nearly 100% still remained in polymorphonuclear leukocytes, compared with ofloxacin, which was so rapidly eluted that only 12% remained. The uptake of NM394 was significantly decreased at 4 degrees C and by the presence of NaCN but was not affected by the presence of L-glycine, L-leucine, L-serine, adenosine, or NaF. NM394 showed intracellular activity at a concentration of 0.1 microgram/ml that significantly reduced the number of phagocytosed Pseudomonas aeruginosa cells with 2 h of incubation. These results suggest that uptake of NM394 by human polymorphonuclear leukocytes occurs via an active transport system differing from that of ofloxacin, whose uptake is affected by the presence of L-glycine and L-leucine, and that once accumulated, NM394 remains intracellularly active and participates in protection against bacterial infection.

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Release of a Potent Polymorphonuclear Leukocyte Chemoattractant Is Regulated by White-Opaque Switching in Candida albicans

Infection and Immunity ◽

10.1128/iai.72.2.667-677.2004 ◽

2004 ◽

Vol 72 (2) ◽

pp. 667-677 ◽

Cited By ~ 88

Author(s):

Jeremy Geiger ◽

Deborah Wessels ◽

Shawn R. Lockhart ◽

David R. Soll

Keyword(s):

Saccharomyces Cerevisiae ◽

Candida Albicans ◽

Mating Type ◽

Related Species ◽

Polymorphonuclear Leukocytes ◽

White Phase ◽

Bona Fide ◽

Human Polymorphonuclear Leukocytes ◽

Cell Chemotaxis

ABSTRACT Previous studies employing transmembrane assays suggested that Candida albicans and related species, as well as Saccharomyces cerevisiae, release chemoattractants for human polymorphonuclear leukocytes (PMNs). Because transmembrane assays do not definitively distinguish between chemokinesis and chemotaxis, single-cell chemotaxis assays were used to confirm these findings and test whether mating-type or white-opaque switching affects the release of attractant. Our results demonstrate that C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. glabrata release bona fide chemoattractants for PMNs. S. cerevisiae, however, releases a chemokinetic factor but not a chemoattractant. Characterization of the C. albicans chemoattractant revealed that it is a peptide of approximately 1 kDa. Whereas the mating type of C. albicans did not affect the release of chemoattractant, switching did. White-phase cells released chemoattractant, but opaque-phase cells did not. Since the opaque phase of C. albicans represents the mating-competent phenotype, it may be that opaque-phase cells selectively suppress the release of chemoattractant to facilitate mating.

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Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate

Blood ◽

10.1182/blood.v56.3.442.442 ◽

1980 ◽

Vol 56 (3) ◽

pp. 442-447 ◽

Cited By ~ 77

Author(s):

PC Amrein ◽

TP Stossel

Keyword(s):

Polymorphonuclear Leukocytes ◽

Actin Binding ◽

Intact Cells ◽

Homogenization Treatment ◽

Human Polymorphonuclear Leukocyte ◽

Correlating Functions ◽

Alpha 1 Antitrypsin ◽

Human Polymorphonuclear Leukocytes ◽

Neutral Proteases

Abstract Proteases can complicate the characterization of proteins from cells, especially human polymorphonuclear leukocytes (PMN), which contain abundant neutral proteases. We tested the ability of agents to inhibit proteolysis, with special reference to the subunit polypeptides of the contractile proteins actin, myosin, and actin-binding protein (ABP). Phenylmethylsulfonyl fluoride (PMSF), O-phenanthroline, EGTA, EDTA, N- ethylmaleimide, alone or in combinations, failed to prevent extensive proteolysis of the PMN proteins during solubilization of cells with dodecyl sulfate. These inhibitors and also alpha-1-antitrypsin and soybean trypsin inhibitor similarly could not prevent proteolysis during homogenization of cells in cold isosomolar sucrose. Treatment of PMN with greater than or equal to mM diisopropylfluorophosphate (DFP) prior to solubilization or homogenization markedly inhibited proteolysis. PMSF and DFP were equally effective in inhibiting proteolysis in PMN extracts, suggesting that the efficacy of DFP may result from its permeation of intact cells and granules before barriers are disrupted by detergents or homogenization. Treatment of PMN with DFP under conditions inhibiting proteolysis did not affect their rate of phagocytosis. We recommend the use of DFP in future studies correlating functions and protein structure of PMN.

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Characterization of an oligopeptide chemoattractant receptor on human blood monocytes using a new radioligand

Blood ◽

10.1182/blood.v63.3.588.588 ◽

1984 ◽

Vol 63 (3) ◽

pp. 588-592 ◽

Cited By ~ 10

Author(s):

MC Benyunes ◽

R Snyderman

Keyword(s):

Human Blood ◽

Polymorphonuclear Leukocytes ◽

Specific Radioactivity ◽

Blood Monocytes ◽

Chemotactic Peptide ◽

Peripheral Blood Monocytes ◽

Peptide Receptor ◽

Human Polymorphonuclear Leukocytes ◽

Dissociation Constant Kd

Abstract The study of chemoattractant receptors on human monocytes had been limited by the lack of a radioligand suitable for use with the small numbers of cells routinely available from human donors. A new synthetic oligopeptide radioligand f[35S]met-leu-phe, with a higher specific radioactivity than was available with the tritiated compound, was used to characterize a chemoattractant receptor on freshly isolated human blood monocytes. These cells bind f[35S]met-leu-phe with a dissociation constant (KD) of 30.2 +/- 5.6 nM and contain 84,000 +/- 11,300 receptors per cell. f[35S]met-leu-phe does not bind specifically to blood lymphocytes. The specificity of the oligopeptide receptor on monocytes is indistinguishable from the oligopeptide chemoattractant receptor on human polymorphonuclear leukocytes. Using f[35S]met-leu- phe, it will now be feasible to study the chemotactic peptide receptor on small numbers of partially purified peripheral blood monocytes from patients with defects of immune function.

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