Characterization of the interactions of the pneumolysoid, Δ6 PLY, with human neutrophils in vitro.

The pneumolysin toxoid, Δ6 PLY, is a prototype pneumococcal protein vaccine candidate. However, its potentially detrimental residual pro-inflammatory interactions with human neutrophils are unknown. In the current study the effects of the toxoid (8-1000 ng/ml) have been compared with those of wild-type pneumolysin (WT/PLY, 8 ng/ml) on neutrophil cytosolic Ca(2+) fluxes, generation of leukotriene B(4) (LTB(4)), and release of matrix metalloproteinase-9 (MMP-9), using spectrofluorimetric, and ELISA procedures (LTB(4) and MMP-9) respectively. Exposure of neutrophils to WT/PLY resulted in influx of Ca(2+) and significant (P<0.05) release of MMP-9 and generation of LTB(4). However, treatment of the cells with Δ6 PLY at concentrations of up to 1000 ng/ml had only trivial effects on Ca(2+) influx and no effects on either release of MMP-9 or LTB(4) production. The observed absence of pro-inflammatory interactions of Δ6 PLY with neutrophils is clearly an important property of this pneumococcal protein vaccine candidate.

Interactive inhibitory effects of formoterol and montelukast on activated human neutrophils.

The research question addressed in the current study was: do formoterol (1 and 10 nM) and montelukast (2 µM) possess interactive inhibitory effects on activated human neutrophils, particularly in relation to alterations in cyclic AMP and cytosolic Ca²(+) fluxes? Isolated human blood neutrophils were activated with the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 µM) in combination with cytochalasin B (CB; 3 µM). Fura-2-acetoxymethyl ester-based spectrofluorimetry, lucigenin-enhanced chemiluminescence, colorimetric and flow cytometric procedures were used to measure cytosolic Ca²(+) fluxes, production of superoxide, elastase release and beta-2 integrin (CR3) expression, respectively, while cyclic AMP and leukotriene (LT)B4 were assayed using competitive binding ELISA procedures. Activation of the cells with fMLP/CB resulted in abrupt and sustained increases in cytosolic Ca²(+), as well as release of elastase and production of superoxide and LTB4, and expression of CR3, all of which were attenuated by formoterol and montelukast individually, and especially by the combination of these agents. These anti-inflammatory effects of each agent, as well as the combination, were associated with significant increases in cyclic AMP. The findings of the current study may explain the efficacy of montelukast and formoterol when used in combination with inhaled corticosteroids in the treatment of severe asthma, possibly by controlling neutrophil-driven inflammation of the airways.

Pneumolysin induces release of matrix metalloproteinase-8 and -9 from human neutrophils.

The research question addressed in the current study was: does the pneumococcal pore-forming toxin, pneumolysin, mobilise matrix metalloproteinase (MMP) -8 and -9 from isolated human blood neutrophils at sublytic concentrations of 5, 10 and 20 ng.mL(-1)? MMPs were measured in the supernatants of unstimulated neutrophils and of cells exposed to pneumolysin and the chemoattractant N-formyl-L-methionyl-l-leucyl-l-phenylalanine (f-MLP; 0.1 microM), individually and in combination, using ELISA procedures, and alterations in cytosolic Ca(2+) concentrations were monitored using a fura-2 acetoxymethyl ester (fura-2/AM)-based spectrofluorimetric method. Treatment of neutrophils with pneumolysin alone caused dose-related release of both MMPs, whereas f-MLP caused modest increases; the combination of both activators was, however, most effective. Pneumolysin/f-MLP-activated release of the MMPs from the cells was paralleled by increases in cytosolic Ca(2+). Exposure of human neutrophils to pneumolysin is accompanied by mobilisation of MMPs, which is potentiated by f-MLP. If operative in vivo, pneumolysin-mediated release of MMPs from neutrophils and other cell types may contribute to the pathogenesis of severe pneumococcal disease.

Dichloromethane extract of Dicerocaryum senecioides leaves exhibits remarkable anti-inflammatory activity in human T-lymphocytes.

The leaves of Dicerocaryum senecioides are used in South Africa as a traditional remedy for many ailments, including inflammatory disorders. The purpose of this study was therefore to evaluate the anti-inflammatory potential of a dichloromethane extract of D. senecioides leaves. Methanol extracts of the leaves were sub-fractionated with dichloromethane and the anti-inflammatory potential of this fraction investigated according to its effects on the mitogen-induced proliferative responses and cytokine profiles of isolated human blood mononuclear leucocytes (MNL). The cells were pre-treated with the extract (25-100 microg mL(-1)) followed by addition of the mitogen, phytohaemagglutinin (PHA, 5 microg mL(-1) final), and measurement of lymphocyte activation and proliferation, using flow cytometric detection of up-regulation of expression of CD25 and incorporation of radiolabelled thymidine into newly synthesised DNA, respectively. Cytokine production by unstimulated and PHA-activated cells was measured using multiplex suspension bead array technology. Treatment of cells with the Dicerocaryum extract resulted in dose-related inhibition of PHA-activated lymphocyte proliferation and expression of CD25, as well as decreased production of Th1 (IFN-gamma, TNF-alpha) and Th2 (IL-10) cytokines. These observations not only underscore the anti-inflammatory potential of components of Dicerocaryum leaves, but also provide a basis for future definitive studies.

Comparison of the effects of macrolides, amoxicillin, ceftriaxone, doxycycline, tobramycin and fluoroquinolones, on the production of pneumolysin by Streptococcus pneumoniae in vitro.

OBJECTIVES: To compare the effects of subinhibitory concentrations of amoxicillin, ceftriaxone, azithromycin, clarithromycin, erythromycin, telithromycin, clindamycin, ciprofloxacin, moxifloxacin, tobramycin and doxycycline on pneumolysin production by a macrolide-susceptible strain and two macrolide-resistant strains [erm(B) or mef(A)] of Streptococcus pneumoniae. METHODS: Pneumolysin was assayed using a functional procedure based on the influx of Ca(2+) into human neutrophils. RESULTS: Only the macrolides/macrolide-like agents caused significant attenuation of the production of pneumolysin, which was evident with all three strains of the pneumococcus. CONCLUSIONS: Macrolides, at sub-MICs, but not other classes of antibiotic, subvert the production of pneumolysin, even in the presence of (and irrespective of the mechanism of) macrolide resistance in S. pneumoniae.

Clarithromycin alone and in combination with ceftriaxone inhibits the production of pneumolysin by both macrolide-susceptible and macrolide-resistant strains of Streptococcus pneumoniae.

OBJECTIVES: To investigate the effects of clarithromycin (0.01-0.5 mg/L) alone or in combination with ceftriaxone (0.1 and 0.25 mg/L) on pneumolysin production by both macrolide-susceptible and -resistant [2 erm(B) positive and 2 mef(A) positive] strains of Streptococcus pneumoniae. METHODS: The bacteria were cultured for 6 h at 37 degrees C/5% CO(2) in tryptone soy broth, washed, enumerated and resuspended to 0.5-3 x 10(8) cfu/mL in tissue culture medium, RPMI 1640. After 16 h of incubation at 37 degrees C / 5% CO(2), pneumolysin was assayed in the bacteria-free supernatants, as well as in lysates, using a functional assay based on the influx of calcium into human neutrophils. RESULTS: Exposure of not only macrolide-susceptible strains, but also the macrolide-resistant strains, of S. pneumoniae to sub-MICs of clarithromycin resulted in dose-related inhibition of the pneumolysin production, whereas production of the toxin was unaffected by ceftriaxone. CONCLUSIONS: These observations demonstrate that even in the setting of macrolide resistance the production of pneumolysin, a key virulence factor of the pneumococcus, is attenuated by exposure of this microbial pathogen to clarithromycin.

Hyaluronidase augments pneumolysin-mediated injury to human ciliated epithelium.

OBJECTIVES: The main objective of this study was to investigate the effects of pneumococcal hyaluronidase (0.1-10microg/ml), alone and in combination with pneumolysin (50 and 100ng/ml), on human ciliated epithelium. METHODS: Ciliary beat frequency (CBF) and structural integrity of human ciliated respiratory epithelium in vitro were studied using a phototransistor technique and a visual scoring index, respectively. RESULTS: Hyaluronidase per se did not affect either CBF or the structural integrity of the epithelium. However, preincubation of the epithelial strips with hyaluronidase (10microg/ml) for 30min at 37 degrees C significantly potentiated pneumolysin-mediated ciliary slowing and epithelial damage. Hyaluronan, a substrate of hyaluronidase, had no effects on the ciliated respiratory epithelium in concentrations up to 100microg/ml and did not antagonize the injurious effects of pneumolysin on the epithelium. However, preincubation of the epithelial strips with hyaluronan (100microg/ml) was associated with attenuation of the ciliary slowing and epithelial damage induced by incubation of the strips with hyaluronidase (10microg/ml) for 30min at 37 degrees C followed by addition of pneumolysin (50ng/ml). CONCLUSIONS: Although having no direct effects alone, hyaluronidase may contribute to pneumolysin-mediated damage and dysfunction to respiratory epithelium, thereby favoring colonization and subsequently extra-pulmonary dissemination of the pneumococcus.

Effects of clofazimine on potassium uptake by a Trk-deletion mutant of Mycobacterium tuberculosis.

OBJECTIVES: This study was designed to investigate the effects of the membrane-active, anti-mycobacterial agent, clofazimine, on potassium (K+)-uptake by a mutant of Mycobacterium tuberculosis (MTB), in which the Trk system, the major K+ transporter of this microbial pathogen, had been selectively inactivated. METHODS: The ceoB and ceoC genes of MTB, which encode the TrkA proteins, CeoB and CeoC, were deleted by homologous recombination, and the double-knockout mutant and wild-type strains compared with respect to K+ uptake and growth in the presence and absence of clofazimine (0.015-2.5 mg/L) using radioassay procedures. RESULTS: Surprisingly, the magnitudes of K+ uptake and rate of growth of the ceoBC-knockout mutant were significantly (P < 0.05) greater than those of the wild-type strain, due, presumably, to induction of a back-up transporter. Exposure of both the wild-type strain and ceoBC-knockout mutant of MTB to clofazimine was accompanied by dose-related decreases in K+ uptake, as well as growth, which were of comparable magnitude for both strains. CONCLUSIONS: These observations demonstrate that the major K+ transporter of MTB, Trk, as well as an uncharacterized inducible back-up system, is equally sensitive to the inhibitory actions of clofazimine.

Pneumolysin-mediated activation of NFkappaB in human neutrophils is antagonized by docosahexaenoic acid.

This study was designed to investigate the relationship between influx of extracellular Ca(2+), activation of NFkappaB and synthesis of interleukin-8 (IL-8) following exposure of human neutrophils to subcytolytic concentrations (8.37 and 41.75 ng/ml) of the pneumococcal toxin, pneumolysin, as well as the potential of the omega-3 polyunsaturated fatty acid, docosahexaenoic acid, to antagonize these events. Activation and translocation of NFkappaB were measured using a radiometric electrophoretic mobility shift assay, while influx of extracellular Ca(2+) and synthesis of IL-8 were determined using a radioassay and an ELISA procedure, respectively. Exposure of neutrophils to pneumolysin was accompanied by influx of Ca(2+), activation of NFkappaB, and synthesis of IL-8, all of which were eliminated by inclusion of the Ca(2+)-chelating agent, EGTA (10 m m), in the cell-suspending medium, as well as by pretreatment of the cells with docosahexaenoic acid (5 and 10 microg/ml). The antagonistic effects of docosahexaenoic acid on these pro-inflammatory interactions of pneumolysin with neutrophils were not attributable to inactivation of the toxin, and required the continuous presence of the fatty acid. These observations demonstrate that activation of NFkappaB and synthesis of IL-8, following exposure of neutrophils to pneumolysin are dependent on toxin-mediated influx of Ca(2+) and that these potentially harmful activities of the toxin are antagonized by docosahexaenoic acid.

Antimicrobial activity of clofazimine is not dependent on mycobacterial C-type phospholipases.

We have used a phospholipase C (PLC)-deletion mutant (plcABC) of the H37Rv strain of Mycobacterium tuberculosis (MTB), as well as a plcA-insertion mutant of Mycobacterium smegmatis, to investigate the possible involvement of PLCs in clofazimine-mediated inhibition of mycobacterial K(+) transport and growth. Inactivation of the PLCs of MTB and insertion of the plcA gene into M. smegmatis resulted in a substantial reduction and increase in hydrolysis of phosphatidylcholine (PC), respectively. However, both the mutant and wild-type strains of MTB and M. smegmatis were equally sensitive to the inhibitory effects of clofazimine on K(+) uptake and growth. These observations demonstrate that the PLCs of MTB are not involved in the antimicrobial activity of clofazimine.

The effects of pneumolysin and hydrogen peroxide, alone and in combination, on human ciliated epithelium in vitro.

We have investigated the effects of pneumolysin and H2O2, putative virulence factors of Streptococcus pneumoniae, on the ciliary beat frequency and structural integrity of human ciliated epithelium in vitro. Human ciliated epithelium was obtained by brushing the inferior nasal turbinate of healthy human volunteers. Ciliary slowing (CS) was measured using a photo-transistor technique and epithelial damage (ED) was documented using a visual scoring index. Effects of recombinant pneumolysin (100 ng/ml), a mutant pneumolysin preparation with markedly reduced haemolytic activity (100 ng/ml) and reagent H2O2 (100 microM) were measured alone and in combination, in the absence and presence of catalase (1000 units/ml). When used individually, both recombinant pneumolysin and H2O2 caused significant (P < 0.05) CS and ED. The effects of H2O2 but not those of pneumolysin were almost completely attenuated by catalase, while the mutant pneumolysin preparation did not cause significant CS or ED. When used in combination, the effects of pneumolysin and H2O2 on CS and ED were additive as opposed to synergistic. These actions of pneumolysin and H2O2 may contribute to the pathogenesis of respiratory tract infections caused by the pneumococcus.

Platelet-activating factor and lyso-PAF possess direct antimicrobial properties in vitro.

The effects of platelet-activating factor (PAF) and lyso-platelet-activating factor (L-PAF) at concentrations of 0.25-20 microg/ml on potassium transport and growth of gram-positive and gram-negative bacteria have been investigated in vitro and compared with those of lysophosphatidylcholine (LPC). Potassium transport was determined using 86Rb+ as tracer, while growth was measured according to the extent of uptake of radiolabeled amino acids. All of the test phospholipids caused dose-related inhibition of 86Rb+-uptake and growth of gram-positive bacteria, the order of potency being PAF>LPC>L-PAF. Gram-negative bacteria, on the other hand, were less sensitive to the inhibitory effects of the phospholipids on K+ transport and growth. Some, but not all, of the gram-positive and gram-negative bacteria were able to degrade LPC, but not PAF or L-PAF, demonstrating that enzymatic degradation of phospholipids does not explain the differential sensitivity to these agents. The bioactive phospholipids LPC, PAF and L-PAF may represent an oxygen-independent antimicrobial host defense system operative primarily against gram-positive bacteria.

Pneumolysin potentiates production of prostaglandin E(2) and leukotriene B(4) by human neutrophils.

Exposure to pneumolysin (8.37 and 41.75 ng/ml) caused a calcium-dependent increase in the generation of prostaglandin E(2) and leukotriene B(4) by both resting and chemoattractant-activated human neutrophils in vitro. These interactions of pneumolysin with neutrophils may result in dysregulation of inflammatory responses during pneumococcal infection.

Proinflammatory interactions of pneumolysin with human neutrophils.

The effects of pneumolysin on the proinflammatory activity of human neutrophils, as well as on cation fluxes in these cells, have been investigated. Superoxide production, release of elastase, CR3 expression, phospholipase A2 activity, and alterations in membrane potential were measured by use of lucigenin-enhanced chemiluminescence and colorimetric, flow cytometric, radiometric, and spectrofluorimetric procedures, respectively; and cation fluxes were measured by use of 45Ca2+ and 86Rb+ and by fura-2 spectrofluorometry. Pneumolysin at concentrations >1.67 ng/mL caused influx of Ca2+ and increased phospholipase A2 activity and CR3 expression, which were associated with enhanced superoxide production and release of elastase after activation of the cells with the chemotactic tripeptide FMLP. At the same concentrations, pneumolysin caused efflux of K+ and membrane depolarization. The effects of pneumolysin on cation fluxes were not attributable to inhibition of Ca2+-adenosine triphosphatase (ATPase) or Na+, K+-ATPase. Pneumolysin potentiates the proinflammatory activities of neutrophils by a pore-forming mechanism resulting in Ca2+ influx.