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Application of Optical Biosensor Techniques to the Characterization of PorA-Antibody Binding Kinetics

The design of novel vaccines and strategies to combat infectious disease requires an understanding of the interactions between pathogen and host. Biological interactions in vivo often rely on specific recognition mechanisms that begin with a binding step. The development of biosen ...

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Construction of porA Mutants

The PorA or class 1 protein is one of the major meningococcal outermembrane proteins (OMPs). It is one of the two porins found in this organism, the other one being the PorB or class 2/3 protein. It folds into a 16-stranded β-barrel structure, which is now well-established for bacterial porins, in which seven ...

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Construction of LPS Mutants

Lipopolysaccharide (LPS) is a major component of the meningococcal outer membrane. It consists of a hexa-acylated glucosamine disaccharide substituted at both ends with diphosphoethanolamine, to which an oligosaccharide chain of up to 10 sugar residues is attached (1,2). It lacks a lo ...

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Recombinant Proteins in Vaccine Development

The outer membrane of Neisseria meningitidis contains a variety of proteins with the potential for inclusion in new meningococcal vaccines (1). Studies on the vaccine potential of these proteins would be facilitated by the production of pure recombinant protein, free from other compon ...

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Testing Meningococcal Vaccines for Mitogenicity and Superantigenicity

Proteins with intrinsic mitogenic properties are widely represented in prokaryotes, such as in different Streptococcus species (1–3), Candida albicans (4), and Eikenella corrodens (5). Specifically, several bacterial porins of Escherichia coli, Shigella dysenteriae, Salm ...

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Identification of Peptides that Mimic N. meningitidis LOS Epitopes Via the Use of Combinatorial Phage-Display Libraries

Although capsular polysaccharide-based vaccines are effective at reducing the incidence of meningococcal disease caused by serogroups A, C, Y, and W135 (1–3), immunization against serogroup B disease using similar strategies has proven unsuccessful (4,5). The primary reason for th ...

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Animal Models for Meningococcal Disease

There are many in vitro systems for the study of meningococcal pathogenesis, but it is only in animal models of infection that the interactions of the bacteria with whole tissues and the humoral and cellular immune systems can be assessed. Animal-infection models are also of great importance for ...

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Early Experiences with Nasal Vaccines against Meningococcal Disease

In common with most infectious agents, meningococci invade mucosal membranes before causing disease (1). An attractive vaccine strategy is therefore to induce immunological responses at the mucosal surfaces, with the possibility of blocking microbial invasion, as well as to induce ...

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Analysis of SH2 Ligands and Identification of Sites of Interaction

The molecular analysis of cell signaling pathways requires an understanding of what each component in the pathway is, the manner in which they interact, and how these interactions serve to propagate the signal. Cytokine signaling begins with the binding of an extracellular cytokine to a spe ...

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Assays for Cytotoxicity

Among cytokine families, the tumor necrosis factor (TNF) superfamily stands out as the one that contains several members with cytotoxic activity (1–3). The best characterized is the prototypic member TNF-α, which is cytotoxic to a range of tumor cells, but not to most types of normal cells (1,4). How ...

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Assays for Antiviral Activity

It is well known that interferons (IFNs) have antiviral activity (1,2). In fact, the definition of interferon springs from this observation. Although it is reported that other cytokines, e.g., tumor necrosis factor (TNF) (3), may exert antiviral effects, biological assays based on antiviral a ...

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In Vitro and In Vivo Assays for High-Molecular-Weight Pegylated Muteins of Granulocyte Colony-Stimulating Factor

The hematopoietic growth factor granulocyte colony-stimulating factor (G-CSF) has been in the clinic since 1986, when the clinical development of bacterially synthesized recombinant filgrastim began (1). A glycosylated form of G-CSF (lenograstim) has been approved for use in Euro ...

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Assays for Chemotaxis

Leukocytes play an important role in inflammation and cancer. They are attracted to the place of injury by chemotactic factors, e.g., plasma complement factors C5a and C3a, and chemotactic cytokines or chemokines, which are produced locally at the site of infection. Chemokines are produced by a ...

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Development of a Mammalian Tet-On Expression Cell Line: Glucosylceramide Synthase Regulates TNF--Induced Apoptosis

Tumor necrosis factor-α (TNF-α) is one of the most pleiotropic of cytokines, acting as a host defense factor in myriad immunological and inflammatory responses and antitumor activity (1–3). The cytotoxic effects of TNF-α are primarily mediated through TNF-R1 and the receptor-associated ...

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Immunohistochemical Detection of Cytokines in Human Tissue Sections

Cytokines, small soluble molecules, are secreted by many cells including cells of the immune system and the central nervous system (Figs. 1 and 2). Immunohistochemical detection of cytokines in tissues is hampered by the intrinsic properties of these molecules, i.e., their expression lev ...

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Detection of Cytokine Signal Transduction Cross-Talk in Leukocyte Activation

Cytokines are a diverse group of proteins and glycoproteins that promote intercellular communication among leukocytes or between leukocyte and other cell types (1). Although most cytokines are soluble proteins, they can infrequently be found expressed at the cell surface. Cytokin ...

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Detection of Cytokine- and Chemokine-Expressing Cells at the Single Cell Level

Methods for detection of cytokines and chemokines at a protein level in individual cells based on immunofluorescence or immunohistochemical (1–16) staining techniques have been developed in our laboratory. The difficulties in these procedures lie in the critical steps of fixation ...

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Assay System for the Effect of Prostaglandin E2 in the Determination of Polarized Cytokine Production

The activation of CD4+ T-cell subsets, Th1 and Th2, is one of the most important focuses of immunology. Th1 and Th2 are differentiated from naive T cells after antigenic stimulation by the influence of cytokines or costimulatory molecules (CD80/CD86) provided from antigen presenting cells ( ...

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Large-Scale Generation of Plasmids that Express Type I Interferon

Large-scale preparations of plasmid DNA encoding cytokine cassettes are continuously gaining in importance in many areas of gene-therapy (1) including DNA vaccinations with plasmid cytokine adjuvants (2–4), tumor research (5), and treatment of infectious diseases (6,7). Small-s ...

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Identification of trans-Acting Factors by Electrophoretic Mobility Shift Assay

Cell stimulation with a growth factor or cytokine results in a myriad of intracellular activities, including up- and down-regulation of multiple signal transduction pathways, culminating in observable cellular functions, such as modulation of cell motility, alteration of cell pr ...

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