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Analyzing the Physicodynamics of Immune Cells in a Three-Dimensional Collagen Matrix

The movement of immune cells is an indispensable prerequisite for their function. All essential steps of cellular immunity rely on the ability of cells to migrate and to interact with each other. Although observation of these phenomena in vivo would be the most physiological approach, intra ...

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SAGE Analysis of Cell Types Involved in Tolerance Induction

Investigations into the mechanisms of immunological tolerance are currently hindered by a paucity of convenient markers, both for the identification and isolation of tolerant cell types and for monitoring the establishment of tolerance in in vivo models. Although high-affinity a ...

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Gene Expression Profiling of Dendritic Cells by Microarray

The immune system of vertebrate animals has evolved to respond to different types of perturbations (invading pathogens, stress signals), limiting self-tissue damage. The decision to activate an immune response is made by antigen-presenting cells (APCs) that are quiescent until they ...

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Estimating Thymic Function Through Quantification of T-Cell Receptor Excision Circles

Analysis of immune reconstitution is of major importance in clinical settings such as following bone marrow transplantation or during anti-retroviral treatment of HIV-infected patients. In these patients, thymic function is essential for the reconstitution of a diversified T- ...

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Investigating Central Tolerance With Reaggregate Thymus Organ Cultures

In the thymus, immature CD4+8+ thymocytes expressing randomly rearranged T-cell receptor α- and β-chain genes undergo positive and negative selection events based on their ability to recognize self-peptide/major histocompatibility complex (MHC) molecules expressed by thym ...

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Studying T-Cell Repertoire Selection Using Fetal Thymus Organ Culture

T lymphocytes express receptors (T-cell receptor) that are not only specific for antigenic peptide but also molecules encoded by the major histocompatibility complex (MHC) that present peptide on the surface of cells (MHC-restricted antigen recognition). However, the vast majori ...

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Derivation, Culture, and Characterization of Thymic Epithelial Cell Lines

The major histocompatibility complex (MHC)-restricted presentation of self-peptides, generated from tissue-specific antigens, by thymic epithelial cells (TECs) is essential for development of central tolerance and for generation of the regulatory T-cell repertoire in the ...

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Isolation, Expansion, and Characterization of Human Natural and Adaptive Regulatory T Cells

Regulatory T cells play a central role in controlling homeostasis, and in inducing and maintaining tolerance to both foreign and self-antigens. Several types of T cells with regulatory activity have been described both in mice and humans, and those within the CD4+ subset have been extensively ...

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Generation of Immunocompetent T Cells from Embryonic Stem Cells

Mature hematopoietic cells, like all other terminally differentiated lineages, arise during ontogeny via a series of increasingly restricted intermediates. Hematopoietic progenitors derive from the mesoderm, which gives rise to hemangioblasts that can differentiate in ...

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Genetic Modification of Dendritic Cells Through the Directed Differentiation of Embryonic Stem Cells

Recent years have witnessed a progressive acceptance of the dual role played by dendritic cells (DC) in the initiation of immune responses and their specific attenuation through the induction of immunological tolerance. Nevertheless, as terminally differentiated cells of the mye ...

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High-Throughput Screening of Peptide Deformylase Inhibitors

The emergence of bacterial pathogens resistant to current antibiotics has caused an urgent demand for new treatments. Peptide deformylase (PDF) has become an exciting target for designing novel antibiotics. To facilitate the screening of PDF inhibitors, three robust, coupled assa ...

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A Method to Assay Inhibitors of Lipopolysaccharide Synthesis

This chapter is orgTreatment of Gram-negative bacterial infections is complicated by innate and acquired drug resistance resulting in a limited number of effective antibiotics. Several Gram-negative bacteria, for which current therapies are ineffective, have recently been i ...

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A Method to Assay Penicillin-Binding Proteins

Key enzymes that assemble the bacterial cell wall are also the target of the Β-lactam class of antibiotics. The covalent binding of labeled penicillin to these proteins has been used in numerous studies in drug discovery, antibiotic mechanisms of action and resistance, and cell wall physiolo ...

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Methods for Assessing the Structure and Function of Cationic Antimicrobial Peptides

Widespread resistance to antibiotics in current clinical use is increasing at an alarming rate. Novel approaches in antimicrobial therapy will be required in the near future to maintain control of infectious diseases. An enormous array of small cationic peptides exists in nature as part of ...

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Bacterial Efflux Pump Inhibitors

Infections caused by multidrug-resistant Gram-negative pathogens play a major role in the morbidity and mortality of hospitalized patients. The rise of resistance to current antibiotic therapies has made the discovery of new agents urgent. One of the major antibiotic resistance mec ...

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Flow Cytometry of Bacterial Membrane Potential and Permeability

This chapter describes reliable flow cytometric methods for assessment of two important physiologic characteristics of bacteria, membrane potential and membrane permeability, which can provide indications of the effects of antimicrobial agents on microorganisms.

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Mycobacterium tuberculosis -Ketoacyl Acyl Carrier Protein Synthase III (mtFabH) Assay: Principles and Method

Fatty acid biosynthesis is one of the relatively newer targets in antibacterial drug discovery. The presence of distinct fatty acid synthases (FAS) in mammals and bacteria and the fact that most bacterial FAS enzymes are essential for viability make this a very attractive antimicrobial dr ...

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The Activity of rRNA Resistance Methyltransferases Assessed by MALDI Mass Spectrometry

Resistance to antibiotics that target the bacterial ribosome is often conferred by methylation at specific nucleotides in the rRNA. The nucleotides that become methylated are invariably key sites of antibiotic interaction. The addition of methyl groups to each of these nucleotides is ...

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Screening for Compounds That Affect the Interaction Between Bacterial Two-Component Signal Transduction Response Regulator Protein and Cognate Promote

Bacterial signal transduction systems can be used as drug targets. The signal transduction targets fall into two groups–-sensor kinases and response regulators. Previously reported studies describe hits that were thought to inactivate sensor kinases but on closer examination we ...

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Assays for -Lactamase Activity and Inhibition

The ability, either innate or acquired, to produce β-lactamases, enzymes capable of hydrolyzing the endocyclic peptide bond in β-lactam antibiotics, would appear to be a primary contributor to the ever-increasing incidences of resistance to this class of antibiotics. To date, four dist ...

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