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1.
Incorporation of 1-oleoyl-2-acetylglycerol (OAG) into multilamellar liposomes composed of egg phosphatidylethanolamine (PE) and arachidonic acid (AA) resulted in a significant enhancement of superoxide release by guinea pig neutrophils when compared to free OAG. OAG incorporated into liposomes containing phosphatidylcholine and arachidonic acid were generally less effective than free OAG. The potency of the liposomes correlates well with the ability of the liposomes to undergo lipid mixing at acidic pH. The enhanced effect of liposome-associated OAG could be related to exposure to an acidic environment in the endosomes/lysosomes once liposomes are endocytosed by neutrophils.  相似文献   

2.
The aim of this study was to investigate the effect of liposomes on docetaxel-induced lipid oxidization and membrane damage in human hepatoma cells. Cytotoxicity of free docetaxel and docetaxel-containing liposomes was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay in human hepatoma cell lines HepG2 and SMMC-7721. To the cell lines, blank liposomes prepared with soybean phosphatidylcholine (SPC), dimyristoylphosphocholine (DMPC), and dioleoylphosphocholine (DOPC) did not show any significant toxicity below a 0.02-mg/mL phospholipid concentration. On the other hand, free docetaxel showed IC50 values of 9.13?×?10?6?±?1.54?×?10?5 and 1.58?×?10?2?±?2.71?×?10?2 mg/mL in HepG2 cells and SMMC-7721 cells, respectively, after of 24 hours of incubation. IC50 values of docetaxel-encapsulating liposomes, measured in terms of total docetaxel concentration, were at least 1.5-fold higher than those of free docetaxel. SPC liposomes reduced cellular damage caused by free docetaxel, as evidenced by the attenuation of docetaxel-induced lactate dehydrogenase (LDH) leakage by over 11% after liposome encapsulation at each dosage. Docetaxel-induced oxidative membrane damage was monitored by the formation of the lipid peroxidation product, malondialdehyde (MDA), and the antioxidative property of SPC liposome was monitored by the suppression of superoxide dismutase (SOD). These data demonstrated that free docetaxel facilitated MDA formation and suppressed SOD, and that these membrane-damaging effects were reduced by SPC liposomes.  相似文献   

3.
In order to determine whether a methylation reaction is involved in the platelet metabolism of arachidonic acid (AA), we investigated the effect of the transmethylase inhibitors 3-deazaadenosine (DZA) and L-homocysteine-thiolactone (Hcy) on the production of immunoreactive thromboxane (TX) B2 by rat platelets. Incubation for at least one hour of the plateletrich plasma with DZA and Hcy led to an inhibition of TX synthesis induced by collagen (5 μg.ml?1). Platelets in plasma were then preincubated for 4 hours with DZA (10?3M) in association with Hcy(5×10?4M), washed, resuspended in buffer, and stimulated with 3 different activators. The formation of TXB2 in response to collagen (25 μg.ml?1) was markedly reduced, whereas no inhibition occurred when AA (5×10?6M) or the calcium ionophore A 23,187 (5×10?6M) were used. In addition labelled AA was incorporated into the platelet phospholipids (PL). Its release induced by collagen (25 μg.ml?1) was inhibited when platelets were preincubated with DZA and Hcy under the same experimental conditions. By contrast, the release of AA induced by A 23187 (10?6M) was unaffected. This results strongly suggest the association of a methylation reaction with platelet activation, at a calcium-independent step of endogenous AA metabolism, before the cyclo-oxygenase level. Its precise biochemical nature remains to be determined.  相似文献   

4.
The transport of sterols incorporated into the lecithin bilayer of small unilamellar liposomes through a model membrane was studied. A two-chamber diffusion cell containing liposomes with incorporated [4-14C]cholesterol or β-[4-14C]sitosterol in the donor chamber and liposomes with unlabeled cholesterol in the receiver chamber was used. The permeability coefficients of the sterols through silastic rubber membranes which served as a model membrane were measured. The permeability for cholesterol incorporated into liposomes in a phosphatidyl choline/cholesterol molar ratio of 1 : 1, produced by sonication for 1 h, and subsequent centrifugation at 100000 × g for 1 h, was 1.6 · 10?8 cm sec?1. Dilution of the liposome suspension did not change the permeability coefficient significantly. The permeability coefficient of sitosterol incorporated into liposomes was about 4-times smaller than that of cholesterol. These results suggest that the sterols were delivered to the silastic membrane by the intact liposomes and that free solute was not involved in the transport to the membrane to a significant degree. The large differences in the permeability coefficients between cholesterol and sitosterol indicate that an aqueous interfacial barrier was crossed by the sterol during the delivery to the membrane.  相似文献   

5.
Lactosyl- and melibiosyl-phosphatidylethanolamine prepared by reductive animation with sodium cyanoborohydride were incorporated into small unilamellar liposomes. Lactosyland melibiosyl-phosphatidylethanolamine liposomes are aggregated by Ricinus communis agglutinin whereas Banderiaea simplicifolia isolectin I aggregates only melibiosyl-phosphatidylethanolamine liposomes. The association constant (Ka) values of interactions of R. communis agglutinin and glycolipids were 5 × 105 and 1.2 × 105m?1 for lactosyl- and melibiosyl-phosphatidylethanolamine, respectively, whereas the Ka for the interaction of B. simplicifolia isolectin I for melibiosyl-phosphatidylethanolamine was found to be 6 × 105m?1. The rates of aggregation of these liposomes are strikingly influenced by the amount of glycolipid incorporated into them. In vivo studies indicate that lactosyl-phosphatidyl-ethanolamine-containing liposomes are rapidly taken up by hepatic cells due to binding of their β-d-galactopyranosyl residues by the hepatic galactose-binding lectin.  相似文献   

6.
Phorbol 12-myristate 13-acetate (PMA) treatment elicited an increased 32P incorporation into phospholipids namely phosphatadyl-inositol (PI); phosphatidyl-inositol-4-phosphate (PIP); phosphatidyl-inositol-4,5-bis-phosphate (PIP2); phosphatidyl-acid (PA); phosphatidyl-choline (PC) and phosphatidyl-ethanolamine (PE) particularly at the 20–30th min after treatment. The ratio of members of the phosphoinositol system, especially PIP and PI, related to the total phospholipid content was increased. PMA (2 × 10?7 M ) was the most effective of the three concentrations tested. The results call attention to the presence of a working phosphoinositol system in Protozoa.  相似文献   

7.
Impaired cardiovascular function during acute myocardial infarction (MI) is partly associated with recruitment of activated polymorphonuclear neutrophils. The protective role of arjunolic acid (AA; 2,3,23-trihydroxy olean-12-en-28-oic acid) is studied in the modulation of neutrophil functions in vitro by measuring the reactive oxygen species (ROS) generation. Neutrophils were isolated from normal and acute MI mice to find out the efficacy of AA in reducing oxidative stress. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) resulted in an oxidative burst of superoxide anion (O2) and enhanced release of lysosomal enzymes. The treatment of neutrophils with PMA induced phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase. Furthermore, we observed activated ERK induced phosphorylation of Ser345 in MI neutrophils. Treatment with AA significantly inhibited the phosphorylation of P47phox and ERK in the stimulated controls and MI neutrophils. Oxidative phosphorylation activities in MI cells were lower than in control, while the glycolysis rates were elevated in MI cells compared to the control. In addition, we observed AA decreased intracellular oxidative stress and reduced the levels of O2 in neutrophils. This study therefore identifies targets for AA in activated neutrophils mediated by the MAPK pathway on p47phox involved in ROS generation.  相似文献   

8.
The effect of the polyene antibiotic, filipin, upon release of the ions Ca2+, Sr2+, SO42? and phosphate out of phospholipid and phospholipid-cholesterol liposomal vesicles was studied. The addition of filipin at concentrations stoichiometrically comparable to the cholesterol concentration in the liposomes, resulted in 2–10 × stimulation of the rate of release of all of these ions. The filipin mediated stimulation of release of ions from liposomes was dependent upon the presence of cholesterol. The relative effectiveness of filipin increased when the mole percent of cholesterol incorporated into the liposomes increased from 10 to 50% and when the molar filipin:cholesterol ratio increased from 0.2 to 1.0. It has been previously shown that there is a 1:1 stoichiometry of interaction between filipin and cholesterol [Biochem. Biophys. Acta339, 57 (1974)]. The present studies suggest that this 1:1 stoichiometric interaction may also be responsible for the increased release of entrapped ions.A possible mechanism of action of polyene antibiotics is discussed which suggests that the rearrangement of membrane constituents occurring upon interaction of filipin with cholesterol is the basis for the enhancement of ion release. This would imply that the ion specificity observed upon interaction of polyene antibiotics with membranes would not only be determined by the polyene antibiotic itself, but also by the intrinsic properties of the membrane.  相似文献   

9.
Abstract

The efficacy of gel formulations containing free and liposomal foscarnet has been evaluated in a murine model of cutaneous Herpes simplex virus type-1 infection. Both formulations were applied topically 3 times daily for 4 days and initiated 24 h post-infection. The penetration of liposomes incorporated into the gel in infected skin tissues was better than that of liposomes dispersed in buffer. Therein, their localization mostly matched that of viral antigen detected by immunoperoxydase staining. Despite these facts, the efficacy of gel formulations of both free and liposomal foscarnet in preventing the development of a zosteriform rash in mice was similar. Electron microscopic examination revealed that liposomes incorporated into the gel formed aggregates together with the micelles of gel. Diffusion studies showed that liposomes were trapped within these aggregates and were hardly able to diffuse across a polycarbonate membrane. In addition, although the liposomes were shown to be highly stable in vitro, the formation of these aggregates destabilized their membrane resulting in a premature release of foscarnet from liposomes. The efficacy of both gel formulations was higher than that of solutions of free or liposomal foscarnet suggesting that the gel formulation is a suitable matrix for the delivery of drugs. Thus, strategies aimed at reducing the interaction of liposomes with the gel could be a convenient approach to improve the efficacy of liposome-encapsulated drug over the free drug.  相似文献   

10.
Context: Pirfenidone (PFD) is an anti-fibrotic and anti-inflammatory agent indicated for the treatment of idiopathic pulmonary fibrosis (IPF). The current oral administration of PFD has several limitations including first pass metabolism and gastrointestinal irritation.

Objective: The aim of this study is to investigate the feasibility of transdermal delivery of PFD using liposomal carrier system.

Materials and methods: PFD-loaded liposomes were prepared using soy phosphatidylcholine (SPC) and sodium cholate (SC). Encapsulation efficiency (EE) of PFD in liposomes was optimized using different preparation techniques including thin film hydration (TFH) method, direct injection method (DIM) and drug encapsulation using freeze–thaw cycles. In vitro drug release study was performed using dialysis membrane method. The skin permeation studies were performed using excised porcine ear skin model in a Franz diffusion cell apparatus.

Results and discussion: The average particle size and zeta-potential of liposomes were 191?±?4.1?nm and ?40.4?±?4.5?mV, respectively. The liposomes prepared by TFH followed by 10 freeze–thaw cycles showed the greatest EE of 22.7?±?0.63%. The optimized liposome formulation was incorporated in hydroxypropyl methyl cellulose (HPMC) hydrogel containing different permeation enhancers including oleic acid (OA), isopropyl myristate (IPM) and propylene glycol (PG). PFD-loaded liposomes incorporated in hydrogel containing OA and IPM showed the greatest flux of 10.9?±?1.04?μg/cm2/h across skin, which was 5-fold greater compared with free PFD. The cumulative amount of PFD permeated was 344?±?28.8?μg/cm2 with a lag time of 2.3?±?1.3?h.

Conclusion: The hydrogel formulation containing PFD-loaded liposomes can be developed as a potential transdermal delivery system.  相似文献   

11.
Arachidonic acid (AA) at 10?4M and 10?3M produced a phasic contraction in isolated canine basilar arteries that peaked rapidly and then slowly declined. This contraction was evidently due to the conversion of AA to prostanoids because it was blocked by cyclooxygenase inhibitors and because 11, 14, 17 eicosatrienoic acid (10?3M), which is not a cyclooxygenase substrate, failed to produce a contraction. When the artery was exposed to 10?3M AA for 20 min and washed, subsequent contractile responses to 10?6M serotonin (5-HT) were only 10% of control. Contractions produced by prostaglandin E2 (10?5M), uridine triphosphate (10?4M) and potassium (5.5×10?4M) were inhibited to a lesser degree than 5-HT, the response to potassium being the least affected (66% of control). This damaging effect of 10?3M AA did not occur if the artery was washed at peak contraction nor with 10?4M AA. Autooxidation products were evidently not responsible for the damage because prior oxygenation (90 min) of 10?4M AA had no such effect. Pretreatment with superoxide dismutase or ascorbate did not prevent the inhibition, suggesting that free radical reactions were not involved. Pretreatment with indomethacin (3×10?4M) or meclofenamate (10?4M) also failed to prevent the inhibitory phenomenon. Saponin, a detergent, produced similar inhibitory effects but 11, 14, 17 eicosatrienoic acid or oleate (10?3M) did not. The arteries partially recovered from the inhibition with time. In conclusion, AA produced contraction in basilar arteries by inducing prostaglandin synthesis but can produce secondarily by an unidentified mechanism an inhibition of the contractile responses evoked by various agonists that is both time and concentration dependent.  相似文献   

12.
Liposomes containing entrapped horsedish peroxidase were incubated with three human cell lines in vitro. Although these cells did not ingest latex particles, and took up less than 1 minut of free peroxidase/5 · 106 from the medium, significant amounts (41–164 munits/5 · 106) of peroxidase became cell-associated by 30 min if the enzyme was presented in negatively charged liposomes (phosphatidylchloline/dicetyl phosphate/cholesterol, 70 : 20 : 10 molar ratio). Uptake of liposome-entrapped peroxidase by lymphocytes or fibroblasts was enhanced 2–5-fold if one molar percent of lysophosphatidylcholine was incorporated as a “fusogen”, and was not appreciately diminished by cytochalasin B, an inhibitor of phagocytosis. Lysophosphatidylcholine containing liposomes did not release trapped peroxidase into the medium during incubation, and studies employing the metallochromic dye, arsenazo III demonstrated lack of access of external Ca2+ to the internal, enzyme-laden, aqueous compartments; liposome-liposome fusion was also excluded by similar means. Ultrastructural cytochemstry demonstrated peroxidase within liposomes in the free cytosol of cultured cells 15–90 min after apparent liposome-cell fusion. Data provide evidence that multilamellar liposomes can be as vectors for the introjection of missing enzymes into non-phagocytic human cells.  相似文献   

13.
The delivery of benzyl penicillin [penicillin G (pen‐G)] encapsulated in cationic liposomes to a pen‐G‐sensitive strain of Staphylococcus aureus immobilized in biofilms has been investigated. The cationic liposomes prepared by extrusion (VETs, diameter ~ 140 nm) were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and dimethylammonium ethane carbamoyl cholesterol (DC‐chol) at a molar ratio of 1.0 :0.49 :0.43. This composition containing 22 mole% of the cationic lipid DC‐chol has been found previously (Kim et al. Colloids Surfaces A 1999, 149, 561–570) to be optimum for adsorption of the liposomes on S. aureus biofilms. The effectiveness of the liposomes to deliver pen‐G to the biofilms immobilized on microtitre plates was assessed from the rate of growth of the cells after exposure to the liposomal drug carrier relative to free pen‐G at the same concentration. The time to reach maximum growth rate from biofilms was investigated as a function of overall drug concentration in a range 2.9 × 10? 3 mM to 1.09 mM and as a function of time of exposure to liposomal drug in a range 1.5 s to 2 h. Liposomal drug delivery was most effective relative to free drug at low overall drug concentrations and short times of exposure. The time to reach maximum growth rate from S. aureus biofilms could be extended by a factor of approximately 4 relative to free drug by the use of liposomally encapsulated pen‐G. The results were supported by direct measurements of the distribution of pen‐G between biofilm and supernatant which showed enhanced values relative to free drug and a transient preferential uptake of drug induced by the liposomes. The study demonstrates that for low drug concentrations and short exposure times liposomal drug delivery greatly enhances the effectiveness of pen‐G for inhibiting the growth of bacterial biofilms of the potentially pathogenic bacterium Staphylococcus aureus.  相似文献   

14.

Introduction

The objective of this study was to evaluate the efficacy of intravenous (i.v.) injection of liposomally encapsulated dexamethasone phosphate (DxM-P) in comparison to free DxM-P in rats with established adjuvant arthritis (AA). This study focused on polyethylene glycol (PEG)-free liposomes, to minimize known allergic reactions caused by neutral PEG-modified (PEG-ylated) liposomes.

Methods

Efficacy was assessed clinically and histologically using standard scores. Non-specific and specific immune parameters were monitored. Activation of peritoneal macrophages was analyzed via cytokine profiling. Pharmacokinetics/biodistribution of DxM in plasma, synovial membrane, spleen and liver were assessed via mass spectrometry.

Results

Liposomal DxM-P (3 × 1 mg/kg body weight; administered intravenously (i.v.) on Days 14, 15 and 16 of AA) suppressed established AA, including histological signs, erythrocyte sedimentation rate, white blood cell count, circulating anti-mycobacterial IgG, and production of interleukin-1beta (IL-1β) and IL-6 by peritoneal macrophages. The suppression was strong and long-lasting. The clinical effects of liposomal DxM-P were dose-dependent for dosages between 0.01 and 1.0 mg/kg. Single administration of 1 mg/kg liposomal DxM-P and 3 × 1 mg/kg of free DxM-P showed comparable effects consisting of a partial and transient suppression. Moreover, the effects of medium-dose liposomal DxM-P (3 × 0.1 mg/kg) were equal (in the short term) or superior (in the long term) to those of high-dose free DxM-P (3 × 1 mg/kg), suggesting a potential dose reduction by a factor between 3 and 10 by liposomal encapsulation. For at least 48 hours after the last injection, the liposomal drug achieved significantly higher levels in plasma, synovial membrane, spleen and liver than the free drug.

Conclusions

This new PEG-free formulation of macrophage-targeting liposomal DxM-P considerably reduces the dose and/or frequency required to treat AA, with a potential to enhance or prolong therapeutic efficacy and limit side-effects also in the therapy of rheumatoid arthritis. Depot and/or recirculation effects in plasma, inflamed joint, liver, and spleen may contribute to this superiority of liposomally encapsulated DxM-P.  相似文献   

15.
A simple methodology based on the differential pulse polarography (DPP) was developed for non-destructive monitoring of drug release from liposomes. The methodology was also capable of determining not only the released material that remained free in the liposomal suspension but also the amount of the drug which was eventually adsorbed on the vesicles surface after its release from the liposomes. Based on this methodology the release kinetics of encapsulated chlorothiazide in 5 mg ml?1 DRV liposomes was studied at 37°C at pH 7.4. The results were compared to those obtained by centrifuging the DRV sample and measuring the free drug in the supernatant solution with UV-spectroscopy. Approximately 70% of the initially encapsulated drug were released within 24 h of which ca. 46% were subsequently adsorbed on vesicles' surface.  相似文献   

16.
This study describes the encapsulation of the local anaesthetic lidocaine (LDC) in large unilamellar liposomes (LUV) prepared in a scalable procedure, with hydrogenated soybean phosphatidylcholine, cholesterol and mannitol. Structural properties of the liposomes were assessed by dynamic light scattering, nanoparticle tracking analysis and transmission electron microscopy. A modified, two-compartment Franz-cell system was used to evaluate the release kinetics of LDC from the liposomes. The in vivo anaesthetic effect of liposomal LDC 2% (LUVLDC) was compared to LDC 2% solution without (LDCPLAIN) or with the vasoconstrictor epinephrine (1:100 000) (LDCVASO), in rat infraorbital nerve blockade model. The structural characterization revealed liposomes with spherical shape, average size distribution of 250?nm and low polydispersity even after LDC incorporation. Zeta potential laid around –30?mV and the number of suspended liposomal particles was in the range of 1012 vesicles/mL. Also the addition of cryoprotectant (mannitol) did not provoke structural changes in liposomes properties. In vitro release profile of LDC from LUV fits well with a biexponential model, in which the LDC encapsulated (EE%?=?24%) was responsible for an increase of 67% in the release time in relation to LDCPLAIN (p?<?0.05). Also, the liposomal formulation prolonged the sensorial nervous blockade duration (~70?min), in comparison with LDCPLAIN (45?min), but less than LDCVASO (130?min). In this context, this study showed that the liposomal formulations prepared by scalable procedure were suitable to promote longer and safer buccal anaesthesia, avoiding side effects of the use of vasoconstrictors.  相似文献   

17.
Abstract

The motive behind present work was to discover a solution for overcoming the problems allied with a deprived oral bioavailability of salbutamol sulfate (SS) due to its first pass hepatic metabolism, shorter half-life, and systemic toxicity at high doses. Pulmonary delivery provides an alternative route of administration to avoid hepatic metabolism of SS, moreover facilitated diffusion and prolonged retention can be achieved by incorporation into liposomes. Liposomes were prepared by thin film hydration technique using 32 full factorial design and formulation was optimized based on the vesicle size and percent drug entrapment (PDE) of liposomes. Optimized liposomal formulation exhibited an average size of about 167.2?±?0.170?nm, with 80.68?±?0.74% drug entrapment, and 9.74?±?1.10?mV zeta potential. The liposomal dispersion was then spray dried and further characterized for in-vitro aerosol performance using Andersen Cascade Impactor. Optimized liposomal formulation revealed prolonged in-vitro drug release of more than 90% up to 14?h following Higuchi’s controlled release model. Thus, the proposed new-fangled liposomal formulation would be a propitious alternative to conventional therapy for efficient and methodical treatment of asthma and alike respiratory ailments.  相似文献   

18.
The possible involvement of arachidonic acid (AA) or its metabolites in β-adrenoceptor desensitization has been studied in rat lung parenchyma both from a functional and a biochemical point of view. In vitro perfusion of rat lungs with AA (3×10?5M for 20 min) reduced the relaxant effect of isoproterenol (ISO) on lung parenchymal strips, shown by a shift to the right of ISO dose-response curve, similar to that obtained using desensitizing concentration of specific β-agonist. Moreover, AA treatment reduced the capacity of ISO to stimulate adenylate-cyclase activity, whereas the number of β-receptor binding sites was not significantly modified. Inhibition of cyclo-oxygenase pathway by indomethacin (INDO) (1.5 × 10?5M) prevented both the loss of ISO-relaxing capacity and the decrease of adenylate-cyclase activity induced by AA treatment. In order to support the role of eicosanoids in β-adrenoceptor desensitization, changes of endogenous free AA levels have also been studied in lung homogenates. Perfusion of rat lung with ISO (10?6M for 20 min) decreased by about 50% the levels of free AA and the pretreatment with BW755C (9×10?5M), a lipo- and cyclo-oxygenase inhibitor, prevented this phenomenon. On the basis of these results, we suggest that the activation of AA cascade is actually involved in β-adrenoceptor desensitization in lung tissues with a possible interference at the site beyond the drug-receptor interaction.  相似文献   

19.
Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow?), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow? to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.  相似文献   

20.
Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O2- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O2- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O2- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O2- production and MPO release from stimulated neutrophils.  相似文献   

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