Role of reactive oxygen species in reperfusion injury of the rabbit lung.

We have developed a model of reperfusion injury in Krebs buffer-perfused rabbit lungs, characterized by pulmonary vasoconstriction, microvascular injury, and marked lung edema formation. During reperfusion there was a threefold increase in lung superoxide anion (O2-) production, as measured by in vivo reduction of nitroblue tetrazolium, and a twofold increase in the release of O2- into lung perfusate, as measured by reduction of succinylated ferricytochrome c. Injury could be prevented by the xanthine oxidase inhibitor allopurinol, the O2- scavenger SOD, the hydrogen peroxide scavenger catalase, the iron chelator deferoxamine, or the thiols dimethylthiourea or N-acetylcysteine. The protective effect of SOD could be abolished by the anion channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, indicating that SOD consumes O2- in the extracellular medium, thereby creating a concentration gradient favorable for rapid diffusion of O2- out of cells. Our results extend information about the mechanisms of reperfusion lung injury that have been assembled by studies in other organs, and offer potential strategies for improved organ preservation, for treatment of reperfusion injury after pulmonary thromboembolectomy, and for explanation and therapy of many complications of pulmonary embolism.     

Opsonization of Legionella pneumophila in human serum: key roles for specific antibodies and the classical complement pathway

Legionella pneumophila has previously been shown to require serum factors for efficient uptake by phagocytic cells. In this investigation, the roles of specific antibody and complement in phagocytosis of L. pneumophila by human polymorphonuclear leucocytes (PMN) and tissue macrophages were determined. Opsonization was assessed by quantitating the uptake of [3H]-labelled Legionellae. Compared to other Gram-negative and to Gram-positive bacterial species, L. pneumophila was highly resistant to the opsonic activity of normal pooled human serum (PHS). Of 12 donor sera tested, only four promoted significant L. pneumophila uptake when used at full strength. Experiments with immune antibody, and with human sera deficient in immunoglobulins, or the complement components C2, C3, or C5, revealed that L. pneumophila opsonization was dependent on antibody-mediated activation of the classical complement pathway; activation of the alternative pathway could not be detected. At high concentrations, immune antibody alone could adequately opsonize L. pneumophila. Human alveolar and peritoneal macrophages required very similar amounts and types of opsonins for L. pneumophila phagocytosis as did human PMN. Heating L. pneumophila to temperatures greater than or equal to 80 degrees abolished its resistance to opsonization by diluted PHS; however, activation of complement via the alternative pathway or via other antibody-independent routes remained undetectable. These studies show that, in addition to immune antibody, the classical pathway of complement plays an important role in the opsonization of L. pneumophila. The limited ability of these bacteria to interact with human complement provides a likely explanation for their resistance to opsonization and may be partly based on heat-sensitive structures on the surface of L. pneumophila.     

Human fibronectin binding to staphylococcal surface protein and its relative inefficiency in promoting phagocytosis by human polymorphonuclear leukocytes, monocytes, and alveolar macrophages.

The interaction between human fibronectin and 17 strains of staphylococci was studied in an attempt to elucidate the staphylococcal cell wall component(s) involved in fibronectin binding and to determine the influence of fibronectin upon phagocytosis by three types of phagocytic cells. Purified, radiolabeled fibronectin bound to a similar degree to six laboratory strains and three fresh clinical isolates of Staphylococcus aureus; similar binding of fibronectin was found with S. aureus strains deficient in cell wall teichoic acid or clumping factor and coagulase, as well as with three strains of S. epidermidis. There was minimal binding of fibronectin to encapsulated S. aureus and to Escherichia coli. Fibronectin bound to intact cells and to a crude cell wall preparation of S. aureus H, but not to purified cell walls or peptidoglycan. Trypsinization of staphylococci prevented subsequent fibronectin binding, but binding did not correlate well with the protein A content in S. aureus cell walls. At physiological concentrations, fibronectin binding to staphylococci did not promote phagocytosis of bacteria by human polymorphonuclear leukocytes, monocytes, or alveolar macrophages. Also, depletion of fibronectin from normal human serum did not result in a measurable loss of opsonic activity for staphylococci. It is concluded that fibronectin binding to staphylococci involves a surface protein shared among strains of S. aureus and S. epidermidis, and that in comparison to C3b and IgG, fibronectin plays a relatively minor role as an opsonin for staphylococci.     

A Semiautomated Framework for Integrating Expert Knowledge into Disease Marker Identification

Background. The availability of large complex data sets generated by high throughput technologies has enabled the recent proliferation of disease biomarker studies. However, a recurring problem in deriving biological information from large data sets is how to best incorporate expert knowledge into the biomarker selection process. Objective. To develop a generalizable framework that can incorporate expert knowledge into data-driven processes in a semiautomated way while providing a metric for optimization in a biomarker selection scheme. Methods. The framework was implemented as a pipeline consisting of five components for the identification of signatures from integrated clustering (ISIC). Expert knowledge was integrated into the biomarker identification process using the combination of two distinct approaches; a distance-based clustering approach and an expert knowledge-driven functional selection. Results. The utility of the developed framework ISIC was demonstrated on proteomics data from a study of chronic obstructive pulmonary disease (COPD). Biomarker candidates were identified in a mouse model using ISIC and validated in a study of a human cohort. Conclusions. Expert knowledge can be introduced into a biomarker discovery process in different ways to enhance the robustness of selected marker candidates. Developing strategies for extracting orthogonal and robust features from large data sets increases the chances of success in biomarker identification.     

Phagocytosis by polymorphonuclear leukocytes of Staphylococcus aureus and Pseudomonas aeruginosa adherent to plastic,agar, or glass

Phagocytic cells may encounter bacteria in vivo that are stationary or adherent to a surface. In this study, recently developed in vitro techniques were adapted to evaluate the interaction of polymorphonuclear leukocytes (PMN) with adherent Staphylococcus aureus and Pseudomonas aeruginosa. By measuring the uptake of radiolabeled bacteria, we found that normal human PMN readily phagocytize these organisms when they are attached to plastic or when they are grown on the surface of nutrient agar. Bacteria adherent to glass elicited a chemiluminescent response, and such organisms were phagocytized and killed by PMN. Opsonization of S. aureus and P. aeruginosa was not required for surface phagocytosis, chemiluminescence, or killing. These new methods should allow evaluation of certain biological and clinical aspects of surface phagocytosis in host defense.     

Human alveolar macrophage cytophilic immunoglobulin G-mediated phagocytosis of protein A-positive staphylococci.

Human alveolar macrophages (AM) have recently been reported to ingest and kill a strain of Staphylococcus (502A) in the absence of opsonins. To further investigate the mechanism of non-opsonic recognition, we studied phagocytosis of 23 clinical and laboratory strains of S. aureus and Staphylococcus epidermidis by AM, and by blood polymorphonuclear leukocytes (PMN) and monocytes (MN). In the absence of opsonins, AM phagocytized 18 protein A-positive but not 5 protein A-negative strains of staphylococci, and the efficiency of phagocytosis directly correlated with the amount of protein A present in the bacterial cell wall (r = 0.86, P less than 0.001). Furthermore, AM rosetted around protein A-coated Sepharose beads, but not around beads without protein A. In contrast, PMN did not phagocytize nonopsonized staphylococci, and did not rosette around either type of Sepharose. MN phagocytized protein A-positive staphylococci, but much less efficiently than AM, and showed some rosetting around protein A-coated Sepharose. The nature of the AM receptor for protein A-positive staphylococci was studied. The surface of AM was positively stained with fluorescein-conjugated antibody to human IgG, but not with IgA- or IgM-specific conjugates. No such surface-immunoglobulins were detected on PMN, and MN were only weakly positive for surface IgG. Pretreatment of AM with F(ab')2 fragments specific for human IgG (anti-Fc) inhibited subsequent phagocytosis of protein A-positive staphylococci. There was no evidence that the AM surface IgG was aggregated or immunecomplexed. From these studies we conclude that human AM possess cytophilic IgG antibodies, which can function as receptors for phagocytosis of protein A-positive staphylococci.