Reconstitution of bactericidal activity in chronic granulomatous disease cells by glucose-oxidase-containing liposomes.

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by phagocytes devoid of a functioning nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The failure of CGD phagocytes to produce reactive oxygen species (ROS) results in a marked increase in the susceptibility of affected patients to life-threatening bacterial and fungal infections. This study investigated whether loading of CGD phagocytes with glucose oxidase (GO)-containing liposomes (GOLs) could restore cellular production of bactericidal ROS (eg, H2O2 and HOCl) in vitro. Results indicate that GO encapsulated in liposomes enabled NADPH oxidase-deficient phagocytes to use H2O2 for the production of highly bactericidal HOCl. The intracellular colocalization of bacteria and liposomes (or liposome-derived ferritin) was demonstrated by confocal laser microscopy and electron microscopy. After uptake of GOLs (approximately 0.2 U/mL at 1 mM total lipid concentration, size approximately 180 nm), CGD granulocytes produced HOCl levels comparable to those of normal phagocytes. Remarkably, after treatment with GOLs, CGD phagocytes killed Staphylococcus aureus as efficiently as normal granulocytes. Moreover, treated cells retained sufficient motility toward chemotactic stimuli as measured by chemotaxis assay. Side effects were evaluated by measuring the H2O2 concentrations and the production of methemoglobin in whole blood. These studies revealed that H2O2 produced by GOLs was degraded immediately by the antioxidative capacity of whole blood. Elevated methemoglobin levels were observed only after application of extremely high amounts of GOLs (2 U/mL). In summary, the application of negatively charged GOLs might provide a novel effective approach in the treatment of patients with CGD at high risk for life-threatening infections.

Detection by ELISA of low transferrin levels in bronchoalveolar secretions of preterm infants.

Transferrin levels in bronchoalveolar secretions (BAS) are very low compared to serum levels in humans. For the exact measurement of transferrin concentrations in BAS a very sensitive assay was developed as a double sandwich enzyme immunoassay using the combination of a polyclonal and a monoclonal antibody against human transferrin. The measurable range of the assay was 1.5 to 100 ng/ml of human transferrin. The lowest measurable value was 0.84 ng/ml and the sensitivity of the assay was 0.88 ng/ml. The coefficient of variation was 14.1% for 25 ng/ml (intra-assay) and 11-20% (inter-assay). The levels measured in 123 samples of BAS of preterm infants ranged between 0.03 and 8.93 (microgram/microgram secretory component (SC)). The determination of transferrin in BAS of preterm infants is helpful in determining oxidative damage, e.g. the availability of free iron, in the neonatal lung. The transferrin concentration in BAS of neonates who recovered from respiratory distress syndrome (RDS) in the first six days of life was 0.48 compared to 0.52 ((microgram/microgram SC), median range) for infants who developed chronic lung disease.

Presence of bleomycin-detectable free iron in the alveolar system of preterm infants.

Chronic lung disease (CLD) is a major cause of long term morbidity in preterm infants. Reactive oxygen species (ROS) play an important role in the pathogenesis of CLD. We show that a high percentage (63 to 83%) of the investigated bronchoalveolar secretions (BAS) of neonates contain bleomycin-detectable free iron concentrations (0. 04-0.124 nmol/micrograms SC, median range). Beside the presence of redox-active iron several iron-binding proteins like transferrin, ferritin and lactoferrin were determined in BAS. Comparison of protein distribution within the first three days of life showed slight differences between the group of preterm infants who developed CLD and the neonates who recovered from RDS. Because of the existence of free iron we suggest a higher risk of hydroxyl radical formation in the alveolar space. In an artificial system with addition of iron and hydrogen peroxide we were able to demonstrate OH-radical production in BAS by electron paramagnetic resonance (EPR). OH-radical formation by H2O2 and iron in buffer solution was slightly enhanced in the presence of BAS, indicating the absence of OH-radical-scavengers in BAS.

Phagocytic activity and oxidative burst of granulocytes in persons with myeloperoxidase deficiency.

In the present study, phagocytosis and the oxidative metabolism of neutrophil granulocytes from five clinically healthy persons with different degrees of myeloperoxidase deficiency were investigated and compared to those of normal persons. The identification of individuals with myeloperoxidase deficiency was performed with the Bayer/Technicon H3 blood cell counter, which differentiates the leukocytes by measuring the peroxidase activity. Neutrophils of three out of five investigated myeloperoxidase deficient persons showed extremely low peroxidase indices (-53 and lower), but only the neutrophils of one person totally lacked myeloperoxidase. This was demonstrated by comparing myeloperoxidase mass concentration measured with an enzyme immunoassay, lack of HOCl production, and was further confirmed by measuring luminol- and lucigenin-enhanced chemiluminescence. Characteristically, myeloperoxidase deficient granulocytes showed a strikingly decreased luminol-enhanced chemiluminescence while the lucigenin-enhanced chemiluminescence was significantly increased compared to normal granulocytes. Although there is a DNA sequence homology of about 70%, the activity of peroxidase in eosinophils was not affected in any myeloperoxidase deficient person investigated. Moreover, a person with a very rare defect of eosinophil peroxidase had completely normal myeloperoxidase activity. The lack of myeloperoxidase activity is compensated for by an increased phagocytic activity, an increased production of superoxide anion (lucigenin-chemiluminescence) and probably by an alternative metabolism of H2O2; since persons lacking myeloperoxidase activity do not normally suffer from severe infections, H2O2 is obviously metabolized to other reactive oxygen substrates than HOCl, e.g. to OH-radicals.