Methods
The NADPH oxidase of phagocytic cells of the immune system plays an important role in the destruction of certain types of microbial pathogens during infections. Its inappropriate activation to release reactive oxidants may also contribute to host tissue damage in inflammatory diseases. In this review, the structure of the NADPH oxidase is described and many of the commonly used methods to detect its...
Filamentous phage particles have been central in the construction of libraries displaying vast numbers of random peptides. These random peptides can be antigenically presented as fusions to coat proteins III and VIII of the phage. The isolation of ligate-reactive phage from an immense background of nonspecific phage is achieved by the biopanning process. Enrichment of reactive phage relative to unreactive...
The multipin method for peptide synthesis was developed for antibody epitope mapping. From its initial invention as a method for testing many short peptides on the solid surface used for their synthesis, it has evolved into a method for producing solutions of peptides of a wide range of lengths in many “formats,” such as tagged (biotinylated) peptides for binding studies or affinity chromatography...
Stimulation of neutrophils with a variety of stimuli can result in the activation of phospholipases A 2 , C, or D with the resultant hydrolysis of plasma membrane phospholipids and the formation of important second messenger molecules. In the neutrophil, the activities of these phospholipases have been implicated in the processes of both stimulating and maintaining oxidase activation. In this...
As the dominant constituents of the active sites, complementarity-determining regions (CDRs) and particularly the CDR3 loops strongly influence the size and shape of this interdomain space. Six sets of CDR3 loops were extracted from our collection of crystal structures and examined for their modes of association. The CDR3 loops of the NC6.8 Fab face each other across a small crevice that is expanded...
Site-directed mutagenesis is a very useful tool for mapping and defining epitopes on protein antigens. This review discusses the methods used in, and the results of, studies on four different protein antigen systems. In addition, computer analyses and molecular modeling were used in an attempt to better understand the structural and energetic mechanisms underlying the effects observed following mutagenesis...
Our understanding of the energetics that govern antigen–antibody recognition lags behind the increasingly rapid accumulation of structural information on antigen–antibody complexes. Thanks to the development of highly sensitive microcalorimeters, the thermodynamic parameters of antigen–antibody interactions can now be measured with precision and using only nanomole quantities of protein. The method...
The diversity of B-cell response to a large immunogen gives rise to a series of antibodies that can be used for epitope mapping of an antigen. This is based on the relative reaction pattern for all antibodies in relation to each other and other ligands to the studied protein. With the introduction of an instrument system, BIAcore, label-free real-time biomolecular interaction analysis (BIA) was made...
NK cells are present mostly in blood and spleen but under certain pathological and physiological conditions rapidly accumulate at extrahematic sites. The present study investigates the responsiveness of NK cells to C–C chemokines and the mechanisms of emigration from the bloodstream. MCP-1 induced migration across polycarbonate filters of IL-2-activated NK cells, whereas it was a weak attractant for...
Three different isoforms of the enzyme nitric oxide synthase (NOS) (EC 1.14.13.39) catalyze the formation of nitric oxide (NO) froml-arginine, which is then converted tol-citrulline. NO released by the constitutive isoforms is involved in a variety of physiologic functions, whereas larger amounts of NO released from the inducible isoform (iNOS) are mostly associated with inflammatory processes. Overproduction...
Three different assays were used to study the distribution of binding sites for IL-8 in human skin and several animal tissues. Anin situbinding assay was designed in which the binding of radiolabeled IL-8 to small intact tissue pieces was studied, and a histological autoradiographic technique was used to detect the bound chemokine in the subsequently prepared tissue sections. A modified assay was...
Chemokines are a family of small proteins that are present in a variety of inflammatory conditions and have been shown to activate and recruit a wide variety of cell types. They bind to a family of seven transmembrane G-protein-coupled receptors. Models for the interaction of the chemokines with their receptors suggest a two-step mechanism. Initially, the main body of the chemokine interacts with...
Despite its small size and transitory nature, nitric oxide (NO) is a very versatile molecule. In addition to its function as a potent vasodilator and neurotransmitter, NO is important in inflammation and immunity. Indeed,in vitroexperiments demonstrated that NO production by cytokine-activated rodent cells is a primary mediator of their antimicrobial and antitumoral activity. NO results from the oxidative...
Triggering of lymphocytes by mitogens leads to a complex biochemical cascade culminating in cellular activation. Data are accumulating that suggest that reactive free radicals participate in signal transduction. We have examined the effects of nitric oxide (NO) on some parameters of lymphocyte activation and have also performed mechanistic studies. Our data suggest that NO initiates a transductional...
The locomotion of leukocytes and its control by environmental agents is crucial, not only for recruiting cells into inflammatory sites, but also for their migration through lymphoid tissues, and thus for the contact interactions of antigen-bearing accessory cells with lymphocytes in induction of the immune response. The methods used to measure locomotion must be capable of giving reliable information...
Leukocyte infiltration into an inflammatory site is one of the pathological hallmarks of inflammatory reaction. Locally produced chemotactic factors are presumed to mediate the sequence of events leading to tissue injury associated with the infiltration of leukocytes. Chemotactic cytokines (chemokines) have been identified as being produced by various types of cells upon stimulation with inflammatory...