MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Immunology' source. Sun, 27 Nov 2011 19:22:33 +0100 http://www.medworm.com/index.php?rid=5284731&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_19 Polyvinylidene fluoride (PVDF) membrane-bottomed, 96-well plates and 8-well strips constitute the formats in which the overwhelming majority of ELISPOT assays used in research and diagnostic applications are performed. PVDF is well suited for ELISPOT because it has a high antibody-binding capacity and because its white color provides an excellent backdrop for ELISPOT enumeration. Nitrocellulose (NC) and PVDF membranes and 96-well plates containing those membranes used in ELISPOT assays were initially commercialized for filtration applications and later optimized for a range of different protein analytical applications. An overview of the development and biotechnology applications of PVDF membrane is provided. Characteristics and attributes of the membrane that are relevant to ELISPOT are s... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284731 Wed, 28 Sep 2011 04:00:00 +0100 5284731 http://www.medworm.com/index.php?rid=5284730&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_18 The interferon-gamma (IFN-γ) release assay (IGRA) is an in vitro extension of the century-old in vivo tuberculin skin test, better known as the TST. Shortcomings to the TST are multifactorial and include limitations in sensitivity and specificity. IGRAs improve diagnostic specificity by using antigens not found in the Bacille Calmette-Guérin, a vaccine given in most countries. IGRAs capture the IFN-γ produced by T cells in response to antigen stimulation. The ELISPOT immediately captures IFN-γ produced directly from each cell, resulting in the generation of a cellular “footprint.” The dimensions and intensity of the generated footprint indicate the avidity of the secreting cell. We show a further improvement in IGRAs by addition of interleukin-7 (IL-7).... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284730 Wed, 28 Sep 2011 04:00:00 +0100 5284730 http://www.medworm.com/index.php?rid=5284729&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_17 Enumeration of antigen-specific cells after vaccination is one of the prime immunological parameters determined when developing vaccines. Due to their exquisite sensitivity (limits of detection can be below 1/100,000 cells), ELISPOT assays are therefore an important tool in vaccine development programs. This is particularly the case for vaccines against diseases that require protective cell-mediated immunity, such as tuberculosis. This chapter describes ELISPOT assays detecting bovine IFN-γ. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284729 Wed, 28 Sep 2011 04:00:00 +0100 5284729 http://www.medworm.com/index.php?rid=5284728&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_16 High-throughput in vitro assays, which rapidly and succinctly assess the immune status of large cohorts of individuals, are essential tools for conducting population-based studies, including vaccine research. The enzyme-linked immunospot (ELISPOT) assay has emerged as a sensitive, reliable high-throughput tool to measure functional recall immunity by assessing the frequency of antigen-specific cytokine-secreting lymphocytes present in peripheral blood mononuclear cells (PBMCs). For the past 10 years, ELISPOT method has been the dominant platform and a standard for the cell-mediated immune (CMI) assays. ELISPOT assays are used extensively as a measure of CMI response to vaccines, including smallpox (vaccinia), following primary or secondary vaccination. Here, we present detailed methodology... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284728 Wed, 28 Sep 2011 04:00:00 +0100 5284728 http://www.medworm.com/index.php?rid=5284727&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_15 ELISPOT assay readout is often dichomized as positive or negative responses according to prespecified criteria. However, these criteria can vary widely across institutions. The adoption of a common response criterion is a key step toward cross-laboratory comparability. This chapter describes the two main approaches to response determination, identifying the strengths and limitations of each. Nonparametric statistical tests and consideration of data quality are recommended and instructions provided for their ready implementation by nonstatisticians and statisticians alike. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284727 Wed, 28 Sep 2011 04:00:00 +0100 5284727 http://www.medworm.com/index.php?rid=5284726&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_14 Cytokine ELISPOT assays have emerged as a powerful tool for the detection of rare antigen-specific T cells in freshly isolated cell material, such as blood. While ELISPOT assays allow one to directly visualize and count extremely low frequencies of cytokine-secreting T cells among millions of nonsecreting bystander cells, the interpretation of ELISPOT data can become ambiguous when (a) spot numbers in antigen-containing wells are low, (b) spot counts in negative control wells are elevated, and particularly (c) when both of the above occur simultaneously. Thus, the primary task, even before statistics are employed, must be the optimization of the basic assay parameters and reagents such that the assay yields low background signal in the negative-control wells and the maximal number of antig... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284726 Wed, 28 Sep 2011 04:00:00 +0100 5284726 http://www.medworm.com/index.php?rid=5284725&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_13 ELISPOT results used to be evaluated visually which, however, is inevitably subjective, inaccurate, and cumbersome. Even when applying automated image analysis to this end, the results are highly variable if the counting parameters are set subjectively. Since objective, accurate, and reproducible measurements are fundamental to science, major efforts have been undertaken over the last decade at CTL to understand the scientific principles behind ELISPOT data and to develop “intelligent” image analysis algorithms based on these principles. Thus, a spot recognition and gating algorithm was developed to automatically recognize the signatures of defined cell populations, such as T cells, discerning them from irrelevant cell types and noise. In this way, the science of ELISPOT data a... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284725 Wed, 28 Sep 2011 04:00:00 +0100 5284725 http://www.medworm.com/index.php?rid=5284724&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_12 Accuracy of spot detection and quantification plays a critical role in the analysis of ELISPOT data. Differences in staining intensities of spots and their morphological variations make it difficult developing a reliable software application. We have developed an image recognition method allowing for the automatic detection of round objects (spots) on ELISPOT images independently of the registration conditions. The emphasis is done on objects of elliptical shape which is typical for a wide range of spots that can be analyzed by both monochrome and a dual-color version of our software. The method of subdivision of objects into groups is also described which is based on color attributes of spots. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284724 Wed, 28 Sep 2011 04:00:00 +0100 5284724 http://www.medworm.com/index.php?rid=5284723&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_11 Over the past decade, ELISPOT has become well-established as a mainstream technology for the study of immune responses in vivo mainly due to its unique ability to detect rare antigen-specific lymphocytes ex vivo. The primary readout for ELISPOT assays has traditionally been the measurement of the frequency of analyte-secreting cells within a test population. While it has been generally appreciated that ELISPOT is a high-information-content assay system in which spot morphologies provide additional valuable information on the amount of analyte secreted by individual cells as well as the kinetics of the secretory process, the precise relationships involved have not been fully characterized and the specific relevant information ­conveyed by spot morphologies has remained largely unexplore... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284723 Wed, 28 Sep 2011 04:00:00 +0100 5284723 http://www.medworm.com/index.php?rid=5284722&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_9 Mesenchymal stem cells (MSCs) have many regulatory effects (e.g., T-cell suppression) on various immune cells. As aberrant T-cell activation is a primary cause of many diseases, understanding the underlying mechanisms of how MSCs exert immunosuppression is an important issue with potential therapeutic implications. A group of cytokines was shown to be involved as soluble mediators in the immunosuppressive effects of MSCs. An enzyme-linked immunospot (ELISPOT) assay is an ideal method to find potential mediators involved in the MSC-immunosupporessive pathway; additionally, the ELISPOT assay can measure changes in the full spectrum of cytokines produced during T-cell activation in the presence of MSCs. Here, we show that during a mixed lymphocyte reaction, interleukin-10 (IL-10)-secreting sp... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284722 Wed, 28 Sep 2011 04:00:00 +0100 5284722 http://www.medworm.com/index.php?rid=5284721&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_8 The major application of ELISPOT assays is to study secretion of cytokines and chemokines from immune system cells. We adapted this assay to study TNFα secretion from microglial BV2 cells, which are similar in physiology to microglia in the nervous system. Stimulation of BV2 cells with 1 μg/mL LPS resulted in a robust secretion of TNFα. Unlike uniform round spots formed by TNFα secreted by immune system cells, BV2 cells produced spots with short zigzag “tails” indicating that BV2 cells were actively moving during the incubation. In spite of irregular shapes, spots could be easily counted using an ELISPOT reader. Our study has shown the feasibility of employing an ELISPOT assay as a tool for neuroscience research to study the mechanisms underlying protein se... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284721 Wed, 28 Sep 2011 04:00:00 +0100 5284721 http://www.medworm.com/index.php?rid=5284720&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_7 Enzyme-linked immuno spot (ELISPOT) assay is widely used for vaccine development, cancer and AIDS research, and autoimmune disease studies. The output of ELISPOT assay is a formation of colored spots which appear at the sites of cells releasing cytokines, with each individual spot representing a single cytokine-releasing cell. We worked out a protocol to study oxidative stress in human peripheral blood lymphocytes by determining their potency to secrete IFN-gamma, IL-2, IL-4, IL-5, IL-8, and TNF-alpha in response to acute treatment with hydrogen peroxide. We show that hydrogen peroxide-induced oxidative stress can cause a ∼twofold decrease in the number of lymphocytes secreting the TH1 cytokines IFN-gamma and IL-2, as well as chemokines IL-8 and TNF-alpha. However, the number of cells ... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284720 Wed, 28 Sep 2011 04:00:00 +0100 5284720 http://www.medworm.com/index.php?rid=5284719&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_6 Cytokine ELISPOT has become a powerful routine tool for the analysis of disease- as well as vaccine-induced T-cell responses. The method is limited, however, in that only one cytokine at a time is assessed. Fluorospot is based on the principle of ELISPOT, but facilitates the analysis of single cells secreting several cytokines, e.g., polyfunctional T cells, suggested to be of protective importance in various infectious diseases. By detecting each cytokine with a specific fluorophore and analyzing differentially colored spots by fluorophore-­specific filter systems, cells producing single or multiple cytokines are identified. Fluorospot maintains the simplicity and sensitivity of the ELISPOT while taking the analysis a step forward toward multiplex analysis. (Source: Springer protocols ... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284719 Wed, 28 Sep 2011 04:00:00 +0100 5284719 http://www.medworm.com/index.php?rid=5284718&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_10 Enzyme-linked immunospot (ELISPOT) assay allows for the determination of the frequency of ­cytokine-secreting cells, but does not answer the question of how much cytokine is secreted per cell. In our study, we combined ELISPOT and ELISA assays and developed a protocol to calculate the amount of IFN gamma secreted by each cell. A suspension of human peripheral blood mononuclear cells was split into two pools and cells from one pool were cultured in a regular ELISPOT plate, whereas cells from the other pool were cultured in an uncoated, “blank,” ELISPOT plate. After finishing the incubations, the amount of IFN gamma was measured by ELISA in culture media collected from both plates. The “blank” plate served to measure a total amount of secreted IFN gamma, whereas t... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284718 Wed, 28 Sep 2011 04:00:00 +0100 5284718 http://www.medworm.com/index.php?rid=5284717&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_5 Adeno-associated viral (AAV) vector-mediated gene transfer represents a promising gene replacement strategy for treating various genetic diseases. One obstacle in using viral-derived vectors for in vivo gene delivery is the development of host immune responses to the vector. Recent studies have demonstrated cellular immune responses specific to capsid proteins of various AAV serotypes in animal models and in human trials for different diseases. We developed a canine-specific ELISPOT assay to detect such immunity in dogs received AAV treatment. Here, we describe in detail the use of a constructed panel of overlapping peptides spanning the entire VP1 sequence of AAV capsid protein to detect specific T-cell responses in peripheral blood in dogs following intramuscular injection of AAV. This h... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284717 Wed, 28 Sep 2011 04:00:00 +0100 5284717 http://www.medworm.com/index.php?rid=5284716&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_4 A commercial feline immunodeficiency virus (FIV) vaccine consisting of inactivated dual-subtype viruses was released in the USA in 2002 and released subsequently over the next 6 years in Canada, Australia, New Zealand, and Japan. Based on the genetic, morphologic, and biochemical similarities between FIV and human immunodeficiency virus-1 (HIV-1), FIV infection of domestic cats is being used as a small animal model of HIV/AIDS vaccine. Studies on prototype and commercial FIV vaccines provide new insights to the types of immunity and the vaccine epitopes required for an effective human HIV-1 vaccine. ELISPOT assays to detect cytokines, chemokines, and cytolytic mediators are widely used to measure the magnitude and the types of cellular immunity produced by vaccination. Moreover, such appro... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284716 Wed, 28 Sep 2011 04:00:00 +0100 5284716 http://www.medworm.com/index.php?rid=5284715&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_3 Human and mouse immune system cells are the most frequently used specimens in ELISPOT assays. In an effect to expand the application of ELISPOT assay to other species, we developed matched antibody pairs for ready-to-use kits designed for studying the frequency of equine IFNγ- and IL-4-secreting peripheral blood mononuclear cells (PBMCs). Equine PBMCs were stimulated with either concanavalin A (Con A) or calcium ionomycin mixed with phorbol 12-myristate 13-acetate (CaI + PMA). We found that Con A, in general, had a more profound stimulating effect than CaI + PMA on IL-4 secretion, whereas both stimulatory and inhibitory effects were observed on IFNγ secretion. Our data demonstrate a large dynamic range in IFNγ and IL-4 secretion among different donors, which may reflect a... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284715 Wed, 28 Sep 2011 04:00:00 +0100 5284715 http://www.medworm.com/index.php?rid=5284714&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_2 During more than 25 years of application in immunological sciences, ELISPOT has been established as a routine, robust, versatile, and reliable assay. From basic research to clinical immune monitoring, ELISPOT is being used to address the quantification and (to a lesser extent) functional characterization of immune cells secreting different molecules in the context of health and disease, immune intervention, and therapy in humans and other species [Kalyuzhny (Ed.) (2005) Handbook of Elispot: methods and protocols, Vol. 302, Humana Press Inc., Totowa, NJ]. Over the last decade, ELISPOT assays have been increasingly implemented as an immune-monitoring tool in clinical trials [Schmittel et al. J Immunother 23:289–295, 2000; Whiteside Immunol Invest 29:149–162, 2000; Nagata et al. A... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284714 Wed, 28 Sep 2011 04:00:00 +0100 5284714 http://www.medworm.com/index.php?rid=5284713&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-325-7_1 The T cell system plays an essential role in infections, allergic reactions, tumor and transplant rejection, as well as autoimmune diseases. It does so by the selective engagement of different antigen-specific effector cell lineages that differentially secrete cytokines and other effector molecules. These T cell subsets may or may not have cytolytic activity, can preferentially migrate to different tissues, and display variable capabilities to expand clonally. The quest of T cell immune diagnostics is to understand which specific effector function and T cell lineage is associated with a given clinical outcome, be it positive or adverse. No single assay can measure all of the relevant parameters. In this chapter, we review the unique contributions that ELISPOT assays can make toward underst... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=5284713 Wed, 28 Sep 2011 04:00:00 +0100 5284713 http://www.medworm.com/index.php?rid=4969595&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_9 Numerous immune receptors have the ability to mediate phagocytosis of large particles by triggering dynamic local rearrangement of the cytoskeleton and cell membrane. Different receptors can be differentially recruited to sites of particle binding, which in turn can have important functional consequences with respect to engulfment and downstream signaling. Using Fcγ receptor-mediated phagocytosis of IgG-coated particles as a model, we describe a method for analyzing nascent phagocytic cups and quantifying relative receptor levels at sites of phagocytosis. This technique is based on a ratiometric analysis of subcellular localization of exogenously expressed receptors carrying different fluorescent protein tags. This approach could be applied more generally to the analysis of surface m... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969595 Tue, 21 Jun 2011 23:00:00 +0100 4969595 http://www.medworm.com/index.php?rid=4969594&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_8 Single-particle tracking (SPT) using fluorescent quantum dots (QDs) provides high-resolution spatial–temporal information on receptor dynamics that cannot be obtained through traditional biochemical techniques. In particular, the high brightness and photostability of QDs make them ideal probes for SPT on living cells. We have shown that QD-labeled IgE can be used to characterize the dynamics of the high-affinity IgE Receptor. Here, we describe protocols for (1) coupling QDs to IgE, (2) tracking individual QD-bound receptors, and (3) analyzing one- and two-color tracking data. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969594 Tue, 21 Jun 2011 23:00:00 +0100 4969594 http://www.medworm.com/index.php?rid=4969593&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_7 Nod1 and Nod2 are pattern recognition receptors of the mammalian innate immune system. They respond to bacterial peptidoglycan fragments and are implicated in host defense against a variety of ­different bacterial pathogens. Recent studies furthermore support additional functions of these proteins in the control of adaptive immune responses and intestinal homeostasis. Activation of Nod1 and Nod2 by their cognate elicitors triggers inflammatory responses driven by the activation of NF-κB and MAPK pathways. In this chapter, we describe a quick and reliable cell-based assay using a luciferase reporter to measure Nod1- and Nod2-mediated NF-κB activation. The described protocol was successfully applied to analyze the influences of overexpressed proteins and siRNA-mediated knock-... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969593 Tue, 21 Jun 2011 23:00:00 +0100 4969593 http://www.medworm.com/index.php?rid=4969592&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_6 Many immunological responses are often regulated by cell surface receptors in cell–cell recognition events. Such immune receptors on the cell surface typically exhibit low-affinity and fast-kinetic ligand interactions (e.g., K d in the μM range, k off  =  10−2 to 20 s−1). Real-time surface plasmon resonance (SPR) detection systems are generally useful for determining these binding parameters. However, several technical points should be considered because the determination of low-affinity binding and fast kinetics is often rather difficult. Here, we introduce a general procedure for SPR experiments and, moreover, show typical examples for ligand binding of immune cell surface receptors, including experimentally useful tips. We also show how t... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969592 Tue, 21 Jun 2011 23:00:00 +0100 4969592 http://www.medworm.com/index.php?rid=4969591&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_5 The innate immune system has evolved to detect microbes and sterile tissue damage with the help of a series of signaling receptors. One key strategy is to detect infectious microbes or host cell damage by recognizing nucleic acids that are modified or appear in compartment normally devoid of nucleic acids. Here, we describe two methods that allow studying the molecular interaction between various nucleic acid recognizing signaling receptors with their ligands. A ligand pull-down assay can be used to show a known interaction between a ligand and its receptor or the method can be utilized as a discovery approach to identify an unknown receptor to a given ligand. An AlphaScreen experiment can be set up to assess the ligand binding affinity to a given receptor. (Source: Springer protocols feed... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969591 Tue, 21 Jun 2011 23:00:00 +0100 4969591 http://www.medworm.com/index.php?rid=4969590&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_4 We describe a set of methods for subcloning, expression, and purification of chimeric protein reagents containing a protein domain (or domains) of interest fused to a C-terminal moiety derived from the Fc region of either IgG or IgM. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969590 Tue, 21 Jun 2011 23:00:00 +0100 4969590 http://www.medworm.com/index.php?rid=4969589&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_10 Fractalkine (CX3CL1) is a membrane-anchored chemokine whose N-terminus contains a unique CX3C motif that is cleaved and released. The membrane-bound form functions as an adhesion molecule and the secreted form as a chemotactic factor. Like other chemokines, CX3CL1 is regulated at the levels of transcription and translation. Recent evidence points to additional functional regulation by cellular trafficking owing to the unique transmembrane structure. CX3CL1 is the only chemokine known to undergo constitutive internalization. To understand mechanisms governing the regulation and processing of such membrane-bound proteins, it is vital to study their subcellular distribution and transport. The methods outlined in this chapter describe (1) transfection of mammalian cells with plasmids encoding ... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969589 Tue, 21 Jun 2011 23:00:00 +0100 4969589 http://www.medworm.com/index.php?rid=4969588&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_12 This chapter describes a method to generate plasma membrane sheets that are large enough to visualize the membrane architecture and perform quantitative analyses of protein distributions. This procedure places the sheets on electron microscopy grids, parallel to the imaging plane of the microscope, where they can be characterized by transmission electron microscopy. The basic principle of the technique is that cells are broken open (“ripped”) through mechanical forces applied by the separation of two opposing surfaces sandwiching the cell, with one of the surfaces coated onto an EM grid. The exposed inner membrane surfaces can then be visualized with electron dense stains and specific proteins can be detected with gold conjugated probes. (Source: Springer protocols feed by Immu... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969588 Tue, 21 Jun 2011 23:00:00 +0100 4969588 http://www.medworm.com/index.php?rid=4969587&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_11 We describe here general approaches for FRAP using green fluorescent protein fusion proteins. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969587 Tue, 21 Jun 2011 23:00:00 +0100 4969587 http://www.medworm.com/index.php?rid=4969586&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_13 Phagocytosis is an immune receptor-mediated process whereby cells engulf large particles. The process is dynamic and requires several localized factors acting in concert with and sequentially after the engagement of immune receptors to envelope the particle. Once the particle is internalized, the nascent ­phagosome undergoes a series of events leading to its maturation to the microbicidal phagolysosome. Investigating these dynamic and temporally controlled series of events in live cells requires noninvasive methods. The ability to rapidly recruit the proteins of interest to the sites of phagocytosis or to nascent phagosomes would help dissect the regulatory mechanisms involved during phagocytosis. Here, we describe a general approach to express in RAW264.7 murine macrophages, a genetic... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969586 Tue, 21 Jun 2011 23:00:00 +0100 4969586 http://www.medworm.com/index.php?rid=4969585&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_14 Fc receptor signaling plays a fundamental role in immune responses. A plethora of Fc ­receptors (e.g., Fc gamma, Fc-alpha, and Fc-epsilon) are expressed on different immune cells, including natural killer cells, macrophages, mast cells, and neutrophils. Receptor clustering and activation by multivalent ligands or opsonized particles induce a signaling cascade that leads to targeted secretion of chemical mediators (i.e., histamine, cytokines, and chemokines) and phagocytosis, among other responses. Spatial targeting and compartmentalization are common mechanisms of regulation in Fc receptor signaling. However, the tools for studying these dynamic interactions have been limited. To overcome these limitations in our model system, microfabricated surfaces containing spatially defined ligan... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969585 Tue, 21 Jun 2011 23:00:00 +0100 4969585 http://www.medworm.com/index.php?rid=4969584&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_15 Monoclonal antibodies (mAbs) specific for cell surface antigens are an invaluable tool to study immune receptor expression and function. Here, we outline a generalized reporter cell-based approach to the generation and high-throughput screening of mAbs specific for cell surface antigens. Termed CELLISA, this technology hinges upon the capture of hybridoma supernatants in mAb arrays that facilitate ligation of an antigen of interest displayed on BWZ reporter cells in the form of a CD3ζ-fusion chimeric antigen receptor (zCAR); in turn, specific mAb-mediated cross-linking of zCAR on BWZ cells results in the production of β-galactosidase enzyme (β-gal), which can be assayed colorimetrically. Importantly, the BWZ reporter cells bearing the zCAR of interest may be used for immuniz... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969584 Tue, 21 Jun 2011 23:00:00 +0100 4969584 http://www.medworm.com/index.php?rid=4969583&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_17 Understanding the transcriptome, defined as the complete transcriptional component of the genome, is far more complex than originally considered. Even with the near fully resolved human and mouse genomes, for which extensive databases of transcribed sequence data (e.g., expressed sequence tags) are available, it is presently not possible to experimentally recover or computationally predict the full range of transcription products that derive from multiexon genes. Many genes are tightly regulated, which could include alternative processing of RNA, and lead to significant underrepresentation of many transcripts. A multitude of factors in addition to cell lineage- and developmental stage-specific expression as well as shortcomings in computational methods result in a less than complete unders... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969583 Tue, 21 Jun 2011 23:00:00 +0100 4969583 http://www.medworm.com/index.php?rid=4969582&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_16 Chaney and Jacobson first introduced the colloidal silica-bead protocol for the coating of cellular plasma membranes in the early 1980s. Since then, this method has been successfully incorporated into a wide range of in vitro and in vivo applications for the isolation of cell-surface proteins. The principle is simple – cationic colloidal silica microbeads are introduced to a suspension or monolayer of cells in culture. Electrostatic interactions between the beads and the negatively charged plasma membrane, followed by cross-linking to the membrane with an anionic polymer, ensure attachment and maintain the native protein conformation. Cells are subsequently ruptured, and segregation of the resulting plasma membrane sheets from the remaining­ cell constituents is achieved by ultra... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969582 Tue, 21 Jun 2011 23:00:00 +0100 4969582 http://www.medworm.com/index.php?rid=4969581&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_18 The secondary immunoglobulin gene diversification processes, somatic hypermutation (SHM), immunoglobulin gene conversion (GCV), and class switch recombination, are important for efficient humoral immune responses. They require the action of activation-induced cytidine deaminase, an enzyme that deaminates cytosine in the context of single-stranded DNA. The chicken DT40 B-cell line is an important model system for exploring the mechanisms of SHM and GCV, as both processes occur constitutively without the need for stimulation. In addition, standard gene targeting strategies can be used for defined manipulations of the DT40 genome. Thus, these cells represent an excellent model of choice for genetic studies of SHM and GCV. Problems arising from defects in early B-cell development that are of c... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969581 Tue, 21 Jun 2011 23:00:00 +0100 4969581 http://www.medworm.com/index.php?rid=4969580&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_19 We describe here a computational strategy to characterize homologs of immune receptors and also to identify putative novel receptors from newly assembled genome sequences. The description of these protocols is aimed at a typical immunologist, and a substantial knowledge of bioinformatics is not expected. The approach is based on using low-stringency sequence searches to identify divergent homologs. For receptors with multiple domains, the intersection of low-stringency searches can be used to identify divergent receptor sequences with high confidence. For multigene families, these predictions can be refined using sequence conservation among gene family paralogs. This strategy has recently been useful in identifying novel expansions in immune receptors in a number of animal genomes and will... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969580 Tue, 21 Jun 2011 23:00:00 +0100 4969580 http://www.medworm.com/index.php?rid=4969579&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_2 The variable lymphocyte receptors (VLRs) of lamprey and hagfish comprise leucine-rich repeat modules, instead of the immunoglobulin-like domain building blocks of antibodies and T-cell receptors in jawed vertebrates. Both types of vertebrate-rearranging antigen receptors are similarly diverse, with repertoires that can potentially exceed 1014 unique receptors. In order to characterize antigen-binding properties of the VLRs, we developed a high-throughput yeast surface display platform for the isolation of monoclonal VLRs. We have isolated VLRs that specifically bind hen egg lysozyme, β-galactosidase, cholera toxin subunit B, R-phycoerythrin, and the blood group trisaccharides A and B, with binding affinities in the mid-nanomolar to mid-picomolar range. VLRs may, thus, be excellent sin... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969579 Tue, 21 Jun 2011 23:00:00 +0100 4969579 http://www.medworm.com/index.php?rid=4969578&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_1 In order to execute their immune functions, leukocytes interact with a broad range of cell types through cell surface receptors, such as those of the immunoglobulin and C-type lectin families, or indirectly through soluble factors. The characterization of activating and inhibitory counterparts of NK cell receptors on myeloid cells, as well as the identification of their physiological ligands, has provided new insights into the underlying mechanisms of immunity and homeostasis. Here, we describe methodology that can be employed to screen for endogenous ligands of type-II C-type lectin-like receptors using reporter cells and Fc fusion proteins. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969578 Tue, 21 Jun 2011 23:00:00 +0100 4969578 http://www.medworm.com/index.php?rid=4969577&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-139-0_3 Scavenger receptors (SRs) are structurally diverse but functionally related innate immune receptors involved in defence and clearance mechanisms. Their broad specificity relies on evolutionarily conserved pattern recognition domains which interact with a variety of microbial, apoptotic and modified self ligands. Studies of immune functions of SR-expressing cells require the identification of interaction partners for SRs. We have developed an ELISA-based method which allows for large-scale high-throughput screening of complex mixtures. The assay successfully identified bacterial and plasma ligands for macrophage scavenger receptor A and can be adapted to screen for novel exogenous or endogenous ligands for any immune receptor of interest. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4969577 Tue, 21 Jun 2011 23:00:00 +0100 4969577 http://www.medworm.com/index.php?rid=4442535&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_17 Tregs are absolutely required for the maintenance of self tolerance in mouse and man. Major abnormalities in Treg number or function cause rare but fatal syndromes with autoimmune, allergic and inflammatory features. Whether subtle Treg abnormalities contribute to the pathogenesis of sporadic autoimmune, allergic and immunoinflammatory diseases in man remains controversial. Robust methods for identifying and isolating human Tregs in patients and healthy controls are essential if we are to understand their role in these increasingly common diseases. We have outlined below a flow cytometric technique to detect and isolate the entire human Treg population based on expression of CD4, CD25, and CD127. Use of a number of additional antibodies for defining subsets within the Treg compartment is d... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442535 Mon, 07 Feb 2011 14:55:50 +0100 4442535 http://www.medworm.com/index.php?rid=4442534&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_16 The in vivo proliferation and disappearance kinetics of lymphocytes may be estimated in humans from rates of deuterium-labeled glucose (2H2-glucose) incorporation into DNA. This protocol describes its application to regulatory T cells (Treg). Because Treg divide frequently, 2H2-glucose is a suitable precursor, achieving high levels of enrichment over a short period. Being nonradioactive and readily administered, it is appropriate for human studies. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442534 Mon, 07 Feb 2011 14:55:50 +0100 4442534 http://www.medworm.com/index.php?rid=4442533&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_15 Regulatory T cells are involved in the maintenance of tolerance. Alterations in their functional capacity are implicated in the development of autoimmunity. In the case of common autoimmune disorders the defects in suppression may be partial, and may be due to a loss of Treg function, or a resistance to suppression by responder T cells. Thus in order to assess Treg function, an in vitro assay that is sensitive enough to demonstrate modest alterations in suppression, and which can differentiate between impaired suppression due to Treg dysfunction, and responder cell resistance is ideal. In this chapter we describe a CFSE based proliferation assay that utilizes a bead based activation system, which is reproducible, consistent and able to distinguish between defects in Treg function and the r... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442533 Mon, 07 Feb 2011 14:55:50 +0100 4442533 http://www.medworm.com/index.php?rid=4442532&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_14 Regulatory T cells (Treg) have become increasingly relevant in the study of human disease including cancer. Treg cells have been shown to inhibit anti-tumor immune responses, and elevated Treg levels have been associated with certain types of cancer. Similarly, depletion of Tregs by various methods can also enhance anti-tumor immune responses. We have found a prevalence of Treg in cancer patients when compared to normal volunteers. In addition, we have shown that the depletion of Treg using the IL-2 fusion protein denileukin diftitox decreased Treg function and increased antigen-specific T cell response to a cancer vaccine. These results indicate the potential for combining Treg depletion with anti-cancer vaccines to enhance tumor antigen-specific immune responses and the need to explore t... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442532 Mon, 07 Feb 2011 14:55:50 +0100 4442532 http://www.medworm.com/index.php?rid=4442531&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_13 Naturally occurring regulatory T (nT Reg ) cells play a critical role in the establishment of immunological self-tolerance in humans. Currently, the analysis of nT Reg cell function from bulk PBMC has led to discrepancies, largely due to the failure to discriminate T Reg cells from other antigen-experienced CD4+ T cells in states of inflammation. We developed a novel, multiparametric, single-cell strategy approach, which consists of isolating and expanding individual CD4+CD25+ T cells into clones, in turn allowing us to discriminate bona fide T Reg cells from activated, FOXP3+ T Eff cells, which frequently confound bulk CD25High T Reg functional assays. This approach enabled us to com... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442531 Mon, 07 Feb 2011 14:55:50 +0100 4442531 http://www.medworm.com/index.php?rid=4442530&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_9 We describe five in vivo models that examine the suppressive capacity of Tregs upon different target cell types. The advantages and disadvantages of each model including resources, time, and technical expertise required to execute each model are also described. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442530 Mon, 07 Feb 2011 14:55:50 +0100 4442530 http://www.medworm.com/index.php?rid=4442529&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_8 The discovery of Foxp3 as a reliable marker for murine regulatory T cells has led to an explosion in the development of genetic tools for investigating the biology of regulatory T cells. More than 25 Foxp3-based mouse strains have been published with a variety of characteristics. The effects of Foxp3 expression can be analyzed using null, hypomorphic, conditional, altered control, and over-expression strains. Reporter strains are available to efficiently isolate Foxp3+ cells, with various reporter designs in terms of construct (fusion, replacement, and bicistronic positioning), and reporter system (GFP, YFP, RFP, Luciferase, Thy1.1). Multifunction strain fusion, replacement, and bicistronic positionings add functional proteins under the control of the Foxp3 promoter allowing induced apopto... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442529 Mon, 07 Feb 2011 14:55:50 +0100 4442529 http://www.medworm.com/index.php?rid=4442528&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_12 Regulatory T lymphocytes expressing CD4, high levels of CD25, and the transcription factor Foxp3 play a crucial role in the control of immune responses to self and nonself antigens. In contrast to immunosuppressive drugs currently used to treat immunopathology, these cells act in a very specific manner. Consequently, their clinical potential in the treatment of autoimmune disorders, inflammatory diseases, graft-versus-host disease, and allograft rejection is currently extensively studied in experimental animal models as well as in clinical trials. We have previously shown that appropriately in vitro stimulated CD4+CD25high regulatory T cells can be used to prevent rejection of bone marrow, skin, and heart allografts in the Mouse. We here describe the protocols used in our laboratory to iso... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442528 Mon, 07 Feb 2011 14:55:50 +0100 4442528 http://www.medworm.com/index.php?rid=4442527&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_11 The peripheral induction of Foxp3-expressing regulatory T cells outside the thymus is required in order to maintain local homeostasis in distinct microenvironments such as the gut. Extrathymic induction of Treg may also be exploited to prevent unwanted immune responses. Here, we discuss the methodology allowing for the stable de novo generation of Tregs specific for foreign antigens in peripheral lymphoid tissue via subimmunogenic peptide delivery using either peptide contained in fusion antibodies directed against the DEC205 endocytotic receptor on steady-state dendritic cells or the implantation of peptide-delivering osmotic mini-pumps. Furthermore, we also address methods in order to achieve TGFβ-dependent Treg conversion in vitro, thereby mainly focusing on the role of retinoic ac... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442527 Mon, 07 Feb 2011 14:55:49 +0100 4442527 http://www.medworm.com/index.php?rid=4442526&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_10 In recent years, researchers have increasingly focused on the modulation of regulatory T cell (Treg) function to interfere with the outcome of virtually every type of immune response. For a long time, specific in vivo targeting of Tregs was precluded due to the lack of appropriate markers. Only after the discovery of Foxp3 as a Treg-specific transcription factor, was the development of Treg-specific mouse models feasible. We generated DEREG mice (DEpletion of REGulatory T cells), a BAC (bacterial artificial chromosome) transgenic mouse line, which allows direct in vivo analysis and depletion of this exceedingly important cell type. Our DEREG mice carry a DTR-eGFP transgene under the control of an additional Foxp3 promoter, thereby allowing specific depletion of Treg by application of dipht... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442526 Mon, 07 Feb 2011 14:55:49 +0100 4442526 http://www.medworm.com/index.php?rid=4442525&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_7 The decision to launch an immune response is made during the interaction of helper T cells and regulatory T cells with dendritic cells. Recognition of antigen leads to formation of immunological synapses at the interface between the cells and to activation of the T cells. The length of interaction between the T cells and dendritic cells influences the functional outcome. We have shown that in the absence of proinflammatory stimuli, regulatory T cells and naive helper T cells interact differently with dendritic cells. Neuropilin-1, which is expressed by most regulatory T cells but not naive helper T cells, promotes prolonged interactions with immature dendritic cells, resulting in higher sensitivity to limiting amounts of antigen. We tracked T cell–dendritic cell interactions in real-... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442525 Mon, 07 Feb 2011 14:55:49 +0100 4442525 http://www.medworm.com/index.php?rid=4442524&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_6 Regulatory T (Treg) cells play a key role in dominant suppression of immune response and maintenance of immune homeostasis. Foxp3, a member of the forkhead transcription factor family, is indispensable for Treg cell development and function. Mice and human with Foxp3 mutations are severely impaired in Treg cell generation and develop lethal autoimmune diseases. We combined chromatin immuno-precipitation and mouse whole genome tiling array profiling (ChIP-on-Chip) to identify the direct downstream targets of Foxp3 in regulatory T cells. Our result showed that Foxp3 not only directly determines expression of a number of Treg signature molecules, but also regulates a group of transcription factors, which potentially control the expression of other Treg-specific genes. (Source: Springer protoc... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442524 Mon, 07 Feb 2011 14:55:48 +0100 4442524 http://www.medworm.com/index.php?rid=4442523&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_5 Natural Regulatory T (Treg) cells are a subset of CD4+ T cells characterized by expression of the transcription factor Foxp3 and the ability to suppress immune responses. Treg cells develop in the thymus in response to highly specific interactions between the T cell receptor (TCR) and self-antigens. These processes can be recapitulated in antigen-specific systems using transgenic mice that coexpress a TCR with its cognate peptide as a neoself-antigen. Here, we describe a method for using such a system to establish a flow cytometric profile of phenotype markers expressed by developing and mature Treg cells in the thymus. Our approach is to compare antigen-specific thymocytes developing in the presence or absence of Treg cell-selecting ligands to identify phenotypic changes that characterize... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442523 Mon, 07 Feb 2011 14:55:48 +0100 4442523 http://www.medworm.com/index.php?rid=4442522&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_4 The study of regulatory T cells (Treg) requires methods for both in vivo and in vitro analyses, both of which have different limitations, but which complement each other to give a more complete picture of physiological function than either method alone. Our analyses have focused on Treg-mediated suppression of CD8+ T cells, and in particular Tregs induced by viral infection. One of the unique characteristics of virus-induced Tregs is that they can suppress CD8+ T cell function in vitro without the requirement for additional stimulation. This ability correlates with their suppressive capacity and activated status in vivo. Interestingly, while virus-induced Tregs suppress CD8+ T cell function in vitro and in vivo, they do not suppress proliferation unless they are further activated in vitro.... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442522 Mon, 07 Feb 2011 14:55:48 +0100 4442522 http://www.medworm.com/index.php?rid=4442521&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_3 Generation of regulatory T cells (or Treg) derived hybridomas offers a tool to study their antigen specificity. T cells hybridomas are produced by fusing TCR α-β-thymoma BW5147 with highly dividing T cell population. In vitro anergy of Tregs is an obstacle in generation of highly dividing Treg population for their fusion. In this chapter, we describe a simple and efficient method to generate large number of blasting Treg and their successful fusion with thymoma BW5147. The resultant hybridomas lose Treg-specific transcription factor FoxP3, respond to antigenic stimulation by producing IL-2, and thus allow the evaluation of antigen specific, Tregs-derived TCRs. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442521 Mon, 07 Feb 2011 14:55:48 +0100 4442521 http://www.medworm.com/index.php?rid=4442520&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_2 Determining the activity of a regulatory T-cell population in vitro is often the first step in analyzing its function. To obtain reliable and reproducible results, it is critical to follow the protocol that is most applicable to your experimental question. We have outlined below a basic in vitro suppression assay as well as a variety of alternative/additional protocols that can be utilized alone or in combination as desired. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442520 Mon, 07 Feb 2011 14:55:48 +0100 4442520 http://www.medworm.com/index.php?rid=4442519&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-979-6_1 Despite the skepticism that once prevailed among immunologists, it is now widely accepted that the normal immune system harbors a T-cell population, called regulatory T cells (Treg cells), specialized for immune suppression. It was first shown that depletion of a T-cell subpopulation from normal rodents produced autoimmune disease. Search for a molecular marker specific for such autoimmune-preventive Treg cells has revealed that the majority, if not all, of them constitutively express the CD25 molecule as depletion of CD25+CD4+ T cells spontaneously evokes autoimmune disease in otherwise normal rodents. The expression of CD25 by Treg cells has made it possible to delineate their developmental pathways, in particular their thymic development, and establish simple in vitro assay for assessin... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4442519 Mon, 07 Feb 2011 14:55:48 +0100 4442519 http://www.medworm.com/index.php?rid=4310102&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_1 In recent times vaccine adjuvants, or immunopotentiators, received abundant attention in the media as critical ingredients of current and future vaccines. Indeed, vaccine adjuvants are recognized to make the difference between competing vaccines based on identical antigens. Moreover, it is recognized that vaccines designed for certain indications require a matching combination of selected antigen(s) together with a critical immunopotentiator that selectively drives the required immune pathway with minimal adverse reactions. Recently, the mechanistic actions of some immunopotentiators have become clearer as a result of research focused on innate immunity receptors. These insights enable more rational adjuvant and vaccine design, which, ideally, is based on predictable immunophenotypes follo... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4310102 Sat, 01 May 2010 00:00:00 +0100 4310102 http://www.medworm.com/index.php?rid=4310101&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_3 To ensure the safe administration of vaccines to humans, vaccines (just like any new chemical entity) are evaluated in a series of nonclinical safety assessment studies that aim at identifying the potential toxicities associated with their administration. The nonclinical safety assessment of vaccines, however, is only part of a testing battery performed prior to human administration, which includes (1) the evaluation of the vaccine in efficacy and immunogenicity studies in animal models, (2) a quality control testing program, and (3) toxicology (nonclinical safety assessment) testing in relevant animal models. Although each of these evaluations plays a critical role in ensuring vaccine safety, the nonclinical safety assessment is the most relevant to the evaluation in human clinical trials... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4310101 Sat, 01 May 2010 00:00:00 +0100 4310101 http://www.medworm.com/index.php?rid=4305185&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_27 Ovarian cancer is the most lethal neoplasm of the female genital tract. Despite progress with chemotherapy, surgery and supportive care, the death rate remains extremely high. Gene silencing therapy represents a possible opportunity to advance the management of ovarian cancer patients. The concept of gene silencing therapy, which is based on RNA interference (RNAi) phenomenon, requires selection of targeted genes and development of a strategy for genetic drug development. Recently, plenty of research studies in ovarian cancer genetics have been published. Although they can be analyzed regarding candidate gene selection, the therapeutic effect of particular gene silencing can only be evaluated experimentally at this time. Obviously, the correct choice and application of a genetic drug deliv... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4305185 Sat, 01 May 2010 00:00:00 +0100 4305185 http://www.medworm.com/index.php?rid=4305184&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_26 RNA interference (RNAi) involves sequence-specific downregulation of target genes, leading to gene silencing in vitro and in vivo. Synthetic small interfering RNAs (siRNAs), formulated with appropriate delivery agents, can serve as effective tools for RNAi-based therapeutics. The potential of siRNA to provide antiviral activity has been studied extensively in many respiratory viruses, including influenza virus, wherein specific siRNAs target highly-conserved regions of influenza viral genome, leading to potent inhibition of viral RNA replication. Despite various delivery strategies, such as polycations and liposomes that have been employed to formulate siRNAs, effective delivery modalities are still needed. Although current strategies can provide significant biodistribution and delivery in... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=4305184 Sat, 01 May 2010 00:00:00 +0100 4305184 http://www.medworm.com/index.php?rid=3426003&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_25 Pain is the new burden of the twenty-first century, raising enormous socio-economic costs to developed and underdeveloped countries. Chronic pain is a central nervous system (CNS) pathology, affecting a large proportion of the population. Morphine and its derivatives are still the golden clinical standards for treating pain although they induce severe side effects. To this day, we still have poor understanding of nociceptive pain and its underlying complex mechanisms; furthermore, novelty in clinical analgesics is lacking. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3426003 Thu, 01 Apr 2010 12:39:21 +0100 3426003 http://www.medworm.com/index.php?rid=3426002&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_24 RNA interference is a promising method for silencing specific genes and has great potential for therapeutic applications. However, the major hurdle for therapeutic application is the limited stability of double-strand RNA (dsRNA) and the absence of a reliable delivery method to target cells. Skin appears to be a favorable target for small interfering RNA (siRNA) therapy. Dendritic cells (DCs) exist in the skin and mucosae on the front lines of defense; these cells capture antigens and play a crucial role in inducing immunity and tolerance. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3426002 Thu, 01 Apr 2010 12:39:21 +0100 3426002 http://www.medworm.com/index.php?rid=3426001&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_23 Dendritic cells (DCs) comprise a family of professional antigen-presenting cells responsible for the induction of primary immune responses. DCs are also important for the induction of immunological tolerance. Recent research has revealed that DC maturation is associated with activation of the NF-κB pathway. RelB, one of the five families of Rel proteins involved in the NF-κB pathway, plays a critical role in coordinating the terminal stages of DC maturation and has the ability to induce optimal Th1 T cell responses. DCs generated from mouse bone marrow can be silenced using siRNA specific for the target gene. Silencing RelB in DCs will result in the generation of immunoregulatory dendritic cells that inhibit allogenic T cell responses. The KLH-specific T cell response should al... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3426001 Thu, 01 Apr 2010 12:39:21 +0100 3426001 http://www.medworm.com/index.php?rid=3426000&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_22 RNA interference (RNAi) is a process through which double-stranded RNA induces the activation of endogenous cellular pathways of RNA degradation, resulting in selective and potent silencing of genes that have homology to the double strand. Much of the excitement surrounding small interfering RNA (siRNA)-mediated therapeutics arises from the fact that this approach overcomes many of the shortcomings previously experienced with alternative approaches to selective blocking that use antibodies, antisense oligonucleotides or pharmacological inhibitors. Induction of RNAi through administration of siRNA has been successfully applied to the treatment of hepatitis, viral infections, and cancer. Increased success in addressing issues of siRNA delivery and efficiency will permit this approach to evol... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3426000 Thu, 01 Apr 2010 12:39:21 +0100 3426000 http://www.medworm.com/index.php?rid=3425999&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_21 Over the past few decades, our expanding knowledge of the mammalian immune system - how it is developed, activated, and regulated - has fostered hope that it may be harnessed in the future to successfully treat human cancer. The immune system activated by cancer vaccines may have the unique ability to selectively eradicate tumor cells at multiple sites in the body without inflicting damage on normal tissue. However, progress in the development of cancer vaccines that effectively capitalize on this ability has been limited and slow. The immune system is restrained by complex, negative feedback mechanisms that evolved to protect the host against autoimmunity and may also prevent antitumor immunity. In addition, tumor cells exploit a plethora of strategies to evade detection and elimination b... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425999 Thu, 01 Apr 2010 12:39:20 +0100 3425999 http://www.medworm.com/index.php?rid=3425998&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_20 RNA interference (RNAi) is a gene silencing phenomenon that is induced by ribonucleoprotein complexes containing 21-28 nucleotides (nt) of double-stranded RNA (si/miRNA). Although this phenomenon occurs in an inherent manner, it can also be induced in an artificially manipulated manner. Recently, the understanding of RNAi mechanisms has progressed from that in plants to that in mammals. As RNAi is a highly efficient and readily available procedure to knockdown specific targets, it can possibly be used as a new technique providing many researchers and clinicians with opportunities for its experimental use and prospective clinical application. Consequently, there has been a rush of elucidation of the effective sequences of siRNAs used for the knockdown of the targets in many fields, includin... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425998 Thu, 01 Apr 2010 12:39:20 +0100 3425998 http://www.medworm.com/index.php?rid=3425997&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_19 RNA interference (RNAi), a valuable tool to specifically silence gene expression, is becoming an indispensable weapon in the arsenal of functional genomics, target validation and gene-specific therapeutic research. We have shown that Streptolysin-O (SLO) reversible permeabilization is an efficient method to deliver small interfering RNAs (siRNAs) to hard-to-transfect human myeloma cell lines. In addition, we have shown that transfection of siRNAs, using the SLO reversible permeabilization, specifically and efficiently induces gene silencing in myeloma cell lines. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425997 Thu, 01 Apr 2010 12:39:20 +0100 3425997 http://www.medworm.com/index.php?rid=3425996&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_18 Since its discovery about 10 years ago, RNA interference (RNAi) has become an almost standard method for the knockdown of any target gene of interest. It is mediated by small interfering RNAs (siRNAs), which trigger a catalytic mechanism for mRNA degradation. Consequently, the delivery of intact siRNA is of critical importance for the induction of RNAi. Due to the physicochemical and biological properties of siRNAs, resulting in high instability and poor cellular uptake, siRNA modifications and pharmaceutical formulations have been used to enhance RNAi efficacy. This is particularly relevant for the in vivo delivery of siRNAs, which still poses a major hurdle for the experimental or therapeutic application of RNAi. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425996 Thu, 01 Apr 2010 12:39:20 +0100 3425996 http://www.medworm.com/index.php?rid=3425995&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_17 RNA interference (RNAi)-mediated silencing of specific genes represents a powerful tool for analyzing protein function. It also has profound biotechnological applications for cellular engineering and therapeutics. However, it is necessary to have a method that controls RNAi in response to artificially regulated stimulation. We designed a fluorescently labeled carrier protein to deliver short hairpin RNA (shRNA) with activity that could be regulated via photostimulation. We constructed a cell-permeable RNA-binding protein (RBP) by fusing the U1A RBP and a HIV-1 Tat peptide, which was labeled with an Alexa Fluor 546 fluorophore (TatU1A-Alexa). TatU1A-Alexa bound specifically to shRNA, which contains a U1A-binding sequence. The TatU1A-Alexa/shRNA complex was then internalized into cells via a... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425995 Thu, 01 Apr 2010 12:39:20 +0100 3425995 http://www.medworm.com/index.php?rid=3425994&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_16 Discovery of RNA interference (RNAi)-mediated specific gene silencing has raised hope for cancer therapy. Unfortunately, the execution of RNAi by delivering small-interfering RNA (siRNA) or small hairpin RNA (shRNA) remains a prime challenge. A methodical evaluation of cationic polymers in RNAi-based cancer studies may offer a promising solution to this problem. In this chapter, we report the methodologies for comprehensive characterization of a biodegradable polymeric system for sh/siRNA delivery in cancer studies. The chapter will describe synthesis, characterization, and optimization of biodegradable poly (β-amino ester) for sh/siRNA delivery. The protocols are provided for shRNA and siRNA complex preparation, stability and morphology study. Also, detailed methods are provided for ... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425994 Thu, 01 Apr 2010 12:39:20 +0100 3425994 http://www.medworm.com/index.php?rid=3425993&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_15 Tumor invasion and metastasis are the primary causes of cancer patient mortality, underscoring the need for identification of novel genes and signaling pathways that mediate these prognosis-determining phenomena. To identify and characterize novel lung adenocarcinoma genes associated with lung cancer progression, we created a bioinformatics-based approach that focuses on human cell-cycle-regulated genes that have evolved only in higher organisms but not in lower eukaryotic cells. In siRNA experiments in lung cancer cells, FLJ10540 was identified as one of several novel targets involved in cell migration and invasion. Here, we demonstrate that PI3K inhibition affects FLJ10540-mediated cell migration and invasion and further, that FLJ10540 knockdown ablates AKT-Ser473 phosphorylation. Taken ... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425993 Thu, 01 Apr 2010 12:39:20 +0100 3425993 http://www.medworm.com/index.php?rid=3425992&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_14 Linking genes with the underlying mechanisms of diseases is one of the biggest challenges of genomics-driven drug discovery research. Designing an inhibitor for any neurodegenerative disease that effectively halts the pathogenicity of the disease is yet to be achieved. The challenge lies in crossing the blood-brain barrier (BBB)/blood-cerebrospinal fluid barrier (BCSFB) to reach the catalytic pockets of the enzyme/protein involved in the molecular mechanism of the disease process. Designing siRNA with exquisite specificity may result in selective suppression of the disease-linked gene. Although siRNA is the most promising method, it loses its potency in downregulating the gene due to its inherent instability, off-target effects, and lack of on-target effective delivery systems. Viral as we... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425992 Thu, 01 Apr 2010 12:39:20 +0100 3425992 http://www.medworm.com/index.php?rid=3425991&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_13 We describe two efficient and inexpensive methods for reverse transfection with siRNA from a solid surface. One method involves localized reverse transfection from spots on a glass slide, which is mainly useful for making “transfection microarrays” (TMAs). The other involves reverse transfection in multiple wells of microtiter plates. Conditions for cell culture, preparation of reagents, and details of reverse transfection have been determined for several lines of cells, but we focus here on experiments with HeLa cells. In particular, we evaluated the efficiency of transfection, the cytotoxic effects of reverse transfection, and the efficiency of gene “knockdown” by transfection. We also performed phenotypic screening for a functional gene, during which cell viabili... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425991 Thu, 01 Apr 2010 12:39:19 +0100 3425991 http://www.medworm.com/index.php?rid=3425990&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_12 We describe the use of hydrodynamic administration as a technique to deliver naked siRNA constructs into experimental animals as a method of transient gene knockdown. This approach demonstrates that RNAi can be used to silence endogenous genes, involved in the cause of human diseases, with a clinically acceptable formulation and route of administration. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425990 Thu, 01 Apr 2010 12:39:19 +0100 3425990 http://www.medworm.com/index.php?rid=3425989&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_11 siRNA therapy offers immense potential for clinical application. Under physiological conditions, however, siRNA was demonstrated to have a short half-life. Additionally, it may also cause ubiquitous gene silencing as it does not possess a tissue-specific homing mechanism. Thus, the rate-limiting step in the emergence of siRNA as a potential therapeutic agent is the current lack of a safe and tissue- or cell-specific in vivo delivery system. Herein, we propose a novel, cell-specific method for the in vivo delivery of siRNA to dendritic cells (DCs) with the purpose of inducing immune modulation. CD40 siRNA was incorporated within the interior of 86 nm liposomes, which were decorated with surface-bound mAb NLDC-145 as a targeting mechanism. The siRNA encapsulation efficiency was determined to... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425989 Thu, 01 Apr 2010 12:39:19 +0100 3425989 http://www.medworm.com/index.php?rid=3425988&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_9 RNA interference, mediated by small interfering RNAs (siRNAs), is a powerful tool for investigation of gene functions and it is increasingly being used as a therapeutic agent. However, not all siRNAs are equally potent - although simple rules for the selection of good siRNAs were proposed early on, siRNAs are still plagued with widely fluctuating efficiency. Recently, new design tools that incorporate both the structural features of the targeted RNAs and the sequence features of the siRNAs have substantially improved the efficacy of siRNAs. In this chapter, we present the algorithms behind these accessibility-aided tools and show how to design efficient siRNAs with their help. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425988 Thu, 01 Apr 2010 12:39:19 +0100 3425988 http://www.medworm.com/index.php?rid=3425987&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_8 The treatment of viral diseases such as HIV and HCV is limited by the genetic diversity of the viruses, especially when they are under the selective pressure of drugs. This problem holds true for gene-based therapies using RNAi in which there is evolution of drug-resistant strains under the discriminating pressure of treatment (1, 2). In a gene therapy setting for treatment of HIV, the incorporation of multiple effector molecules, targeting different viral and cellular sequences, can improve viral inhibition by substantially delaying the emergence of escape mutants (3-8). However, for short hairpin RNA triggers of RNAi, high levels of expression by strong Pol III promoters has led to cell toxicity, and even death in experimental animals (9, 10). Here, we describe a new combinatorial anti-H... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425987 Thu, 01 Apr 2010 12:39:19 +0100 3425987 http://www.medworm.com/index.php?rid=3425986&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_7 Currently, RNA interference technology is one of the most powerful tools in molecular biology and has been widely used in genetic manipulation. In addition to chemically synthesized small interfering RNA (siRNA), vector-based methods have been developed for stable gene silencing by the expression of a single short-hairpin RNA (shRNA). The artificially expressed RNA molecules are processed to form a silencing complex that causes the specific degradation of its target mRNA. However, silencing vectors containing a single shRNA-expressing sequence sometimes induce only poor knockdown. In order to improve the knockdown efficiency using shRNA, the multiple shRNA-expressing sequences were introduced into a single plasmid vector. Compared with the conventional single shRNA-expression vector, the m... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425986 Thu, 01 Apr 2010 12:39:19 +0100 3425986 http://www.medworm.com/index.php?rid=3425985&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_6 RNA interference mediated by short interfering RNA (siRNA) molecules represents a powerful genetic tool with an increasing interest as potential therapeutics. Current bioinformatic approaches to design functional siRNA molecules take into account both empirical and rational approaches to identify selectable characteristics of active and specific siRNA molecules and focusing the downstream events in the RNAi pathway, such as target messenger RNA accessibility. The design of effective siRNA molecules is the key to successful experimentation with RNAi. Here, we show advanced siRNA design parameters and options for highly efficient siRNA candidate search. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3425985 Thu, 01 Apr 2010 12:39:19 +0100 3425985 http://www.medworm.com/index.php?rid=3351367&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_5 RNA interference (RNAi) with small interfering RNA (siRNA) has become a powerful tool in functional and medical genomic research through directed post-transcriptional gene silencing. In order to apply RNAi technique to eukaryotic organisms, where frequent alternative splicing results in diversification of mRNAs and finally of proteins, we need spliced mRNA isoform silencing to study the function of individual proteins. AsiDesigner is a web-based siRNA design software system, which provides siRNA design capability to account for alternative splicing in mRNA level gene silencing. It provides numerous novel functions, including designing common siRNAs for the silencing of more than two mRNAs simultaneously, a scoring scheme to evaluate the performance of designed siRNAs by adopting state-of-t... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3351367 Thu, 11 Mar 2010 13:39:06 +0100 3351367 http://www.medworm.com/index.php?rid=3351366&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_4 Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital; however, it is also difficult. We have developed an assay system with reporter alleles that encode the Photinus and Renilla luciferase genes carrying mutant and wild-type allelic sequences in their 3′-untranslated regions. The assay system allows us to evaluate designed siRNAs and also short hairpin RNAs for allele-specific silencing against target mutant alleles as well as off-target silencing against corresponding... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3351366 Thu, 11 Mar 2010 13:39:06 +0100 3351366 http://www.medworm.com/index.php?rid=3351365&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_3 Within the past few years, microRNAs (miRNAs) and other noncoding RNAs (ncRNAs) have emerged as elements with critically high importance in posttranscriptional control of cellular and, more recently, viral processes. Endogenously produced by a component of the miRNA-guided RNA silencing machinery known as Dicer, miRNAs are known to control messenger RNA (mRNA) translation through recognition of specific binding sites usually located in their 3′ untranslated region. Recent evidences indicate that the host miRNA pathway may represent an adapted antiviral defense mechanism that can act either by direct miRNA-mediated modulation of viral gene expression or through recognition and inactivation of structured viral RNA species by the protein components of the RNA silencing machinery such as... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3351365 Thu, 11 Mar 2010 13:39:06 +0100 3351365 http://www.medworm.com/index.php?rid=3351364&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_2 The use of small interfering RNAs (siRNAs) in human therapy may be hindered by the recruitment of nonspecific effects such as the activation of innate immune responses. Recently, several innate immune receptors have been implicated in the detection of siRNAs. This chapter provides a brief overview of the current knowledge of siRNA-induced innate immunity, as well as protocols for the rapid identification of siRNAs with innate immune stimulatory activity. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3351364 Thu, 11 Mar 2010 13:39:06 +0100 3351364 http://www.medworm.com/index.php?rid=3351363&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_1 RNA interference (RNAi) is a natural process that occurs in many organisms ranging from plants to mammals. In this process, double-stranded RNA or hairpin RNA is cleaved by a RNaseIII-type enzyme called Dicer into small interfering RNA duplex. This then directs sequence-specific, homology-dependent, posttranscriptional gene silencing by binding to its complementary RNA and triggering its elimination through degradation or by inducing translational inhibition. In plants, worms, and insects, RNAi is a strong antiviral defense mechanism. Although, at present, it is unclear whether RNA silencing naturally restricts viral infection in vertebrates, there are signs that this is certainly the case. In a relatively short period, RNAi has progressed to become an important experimental tool both in v... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3351363 Thu, 11 Mar 2010 13:39:05 +0100 3351363 http://www.medworm.com/index.php?rid=3351362&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-588-0_10 Chemical synthesis has been a major endeavor to create active siRNAs. The downregulation of mRNA by 21-mer double-stranded siRNAs can be improved by using modified nucleotides, especially 2′-O-alkylated ones. Besides the commercially available 2¢-O-methyl ribosides, 2′-alkyl groups bearing positive charges are especially promising candidates. We have shown that in a proper formulation they are superior to unmodified siRNAs. This may be due to enhanced stability and most probably to a better uptake into the cells. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3351362 Thu, 11 Mar 2010 13:39:05 +0100 3351362 http://www.medworm.com/index.php?rid=3284936&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_9 Activation of cells of the innate immunity such as macrophages and dendritic cells is critical to mount an adaptive immune response. Recent advances on the understanding of innate immune receptors such as the Toll-like receptors (TLR) and NOD-like receptors (NLR) and the demonstration that microbial products activate specific receptors. This discovery represented a major advance and provided tools to test novel adjuvants in vitro to investigate activation on innate immune cells. Here the isolation and culture of murine macrophages is described, and the use of macrophages derived from gene-deficient mice is proposed to define receptor usage. Novel adjuvants may be tested for their capacity to induce cytokines, chemokines and the expression of costimulatory molecules. The basic methods to as... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284936 Fri, 19 Feb 2010 13:39:06 +0100 3284936 http://www.medworm.com/index.php?rid=3284935&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_8 Efficient Vacciness against intracellular microbes or tumors will be based on innovative adjuvants able to induce efficient activation of dendritic cells. Indeed, natural or synthetic products activating Toll-like receptors (TLR) on dendritic cells (DCs) are currently in development for this purpose. Herein, we describe in vitro assays on human cells which might be useful for the preclinical screening and assessment of potential DC activators. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284935 Fri, 19 Feb 2010 13:39:06 +0100 3284935 http://www.medworm.com/index.php?rid=3284934&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_7 Recombinant proteins are increasingly being used as a novel approach for antigens in Vacciness. These genetically engineered antigens are poorly immunogenic and require a delivery system and adjuvant to elicit their effect at targeted site of action. A delivery system transports the antigen to site of action and an adjuvant activates the cells via interaction with cell receptors and enhances the potency of the antigen. Micro/nanoparticles made from biodegradable and biocompatible polyesters, polylactide-co-glycolides (PLG), have been extensively used as an adjuvant and delivery system. This chapter discusses the applications of PLG micro/nanoparticles as delivery systems and adjuvant for antigens. PLG microparticles are prepared by a solvent evaporation method while nanoparticles are prepa... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284934 Fri, 19 Feb 2010 13:39:06 +0100 3284934 http://www.medworm.com/index.php?rid=3284933&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_6 Many preclinical and clinical results indicate that liposomal systems can serve as effective adjuvants to subunit Vacciness by enabling the formulation and delivery of Vaccines antigens and immunopotentiators. The adjuvant effect of liposomes usually depends on both the composition of the lipid vesicles and their physical association with the Vaccines antigen. This chapter describes methods for the preparation and characterization of sterile small, mostly unilamellar, lipid vesicles and for their association with Vaccines antigens. It gives also some recommendations for the optimization of liposomal Vacciness in preclinical testing. The most common immunopotentiators used in liposomal adjuvants are also described. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284933 Fri, 19 Feb 2010 13:39:06 +0100 3284933 http://www.medworm.com/index.php?rid=3284932&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_5 Quality control and quality assurance procedures are discussed for the agreed benchmark standard Freund’s complete adjuvant (FCA). In addition, the use of the incomplete adjuvant (FIA) in the preparation of antisera is discussed. A major problem is the use of a safe and suitable mineral oil in FCA and FIA; manufacturers should provide infra-red spectra and gas liquid chromatography analyses. A range of safety tests, toxicity, pyrogenicity and endotoxin assays and advice on practical procedures for the use of these adjuvants are described. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284932 Fri, 19 Feb 2010 13:39:06 +0100 3284932 http://www.medworm.com/index.php?rid=3284931&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_4 Important new knowledge about the effect of aluminum adjuvants on the immune response in terms of their impact on cytokine profiles, uptake by antigen-presenting cells (APC), and surface marker expression has been published in recent years. However, although the knowledge about these adjuvants is thus more comprehensive now than ever before, the user is often still confined to a more empirical approach when confronted with practical issues when it comes to the handling and use of these adjuvants. In this chapter we have given focus to the user’s perspective, discussing practicalities like dosage, temperature stability, relevant monographs, and preparation with antigen. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284931 Fri, 19 Feb 2010 13:39:06 +0100 3284931 http://www.medworm.com/index.php?rid=3284930&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_3 To ensure the safe administration of Vacciness to humans, Vacciness (just like any new chemical entity) are evaluated in a series of nonclinical safety assessment studies that aim at identifying the potential toxicities associated with their administration. The nonclinical safety assessment of Vacciness, however, is only part of a testing battery performed prior to human administration, which includes (1) the evaluation of the Vaccines in efficacy and immunogenicity studies in animal models, (2) a quality control testing program, and (3) toxicology (nonclinical safety assessment) testing in relevant animal models. Although each of these evaluations plays a critical role in ensuring Vaccines safety, the nonclinical safety assessment is the most relevant to the evaluation in human clinical t... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284930 Fri, 19 Feb 2010 13:39:06 +0100 3284930 http://www.medworm.com/index.php?rid=3284929&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_2 Recent knowledge on Vaccines-induced immunity led to the development of Vaccines Adjuvant Systems specially designed and adapted to Vaccines needs. AS04 is such a tailored Adjuvant System developed by GlaxoSmithKline Biologicals. This chapter focuses on the methods that were used during the preclinical evaluation of AS04. AS04 consists of the combination of aluminum salts and 3'-O-deacylated monophosphoryl lipid A (MPL), a detoxified lipid A derivative with retained immunostimulatory capa- city. MPL also induces considerably less pro-inflammatory cytokines, as compared to the parent LPS molecule. Preclinical evaluation of AS04 allowed the determination of the optimal size of MPL particles. The added value of MPL in AS04-based formulations was evidenced by higher Vaccines-elicited antibody ... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284929 Fri, 19 Feb 2010 13:39:06 +0100 3284929 http://www.medworm.com/index.php?rid=3284928&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_1 In recent times Vaccines adjuvants, or immunopotentiators, received abundant attention in the media as critical ingredients of current and future Vacciness. Indeed, Vaccines adjuvants are recognized to make the difference between competing Vacciness based on identical antigens. Moreover, it is recognized that Vacciness designed for certain indications require a matching combination of selected antigen(s) together with a critical immunopotentiator that selectively drives the required immune pathway with minimal adverse reactions. Recently, the mechanistic actions of some immunopotentiators have become clearer as a result of research focused on innate immunity receptors. These insights enable more rational adjuvant and Vaccines design, which, ideally, is based on predictable immunophenotypes... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284928 Fri, 19 Feb 2010 13:39:06 +0100 3284928 http://www.medworm.com/index.php?rid=3284927&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_19 The activity of several potent adjuvants, including incomplete Freund’s adjuvant, CpG oligodeoxynucleotides, and alum, has been shown to be due at least in part to the induction of cytokines, including type I interferons (IFNs), IFN-?, interleukin-2 (IL-2), and IL-12, that play key roles in the regulation of innate and adaptive immunity. The relatively short half-life of recombinant homologues of cytokines has limited their use as Vaccines adjuvants. These difficulties have been overcome by encapsulation into liposomes and the use of cytokine expression vectors co-administered with DNA Vacciness. Although a number of cytokines including IFN-a, IFN-?, IL-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, and Flt-3 ligand have been shown to potentiate the immune response to vaccination in various ... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284927 Fri, 19 Feb 2010 13:39:06 +0100 3284927 http://www.medworm.com/index.php?rid=3284926&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_18 Mucosal vaccination offers the advantage of blocking pathogens at the portal of entry, improving patient’s compliance, facilitating Vaccines delivery, and decreasing the risk of unwanted spread of infectious agents via contaminated syringes. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284926 Fri, 19 Feb 2010 13:39:06 +0100 3284926 http://www.medworm.com/index.php?rid=3284925&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_17 To develop novel adjuvants for use in humans, the efficacy/toxicity (E/T) ratio of experimental products in large animal species can be investigated. The test model included two intramuscular immunizations in pigs at 3 weeks interval and analysis of immune responses and local reactions 1 week after the second injection. The antigen used to determine adjuvant activity was a well-defined, purified, viral glycoprotein that without adjuvant induces low immune responses and no detectable local reactions. Efficacy was determined by measuring ELISA and virus-neutralizing antibody titres. Toxicity was determined by necropsy and estimating size and severity of local reactions to each treatment. The persistence of the side effects was deduced from the difference in the local reaction 4 weeks after t... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284925 Fri, 19 Feb 2010 13:39:05 +0100 3284925 http://www.medworm.com/index.php?rid=3284924&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_16 Large numbers and functionally competent T cells are required to protect from diseases for which antibody-based Vacciness have consistently failed (1), which is the case for many chronic viral infections and solid tumors. Therefore, therapeutic Vacciness aim at the induction of strong antigen-specific T-cell responses. Novel adjuvants have considerably improved the capacity of synthetic Vacciness to activate T cells, but more research is necessary to identify optimal compositions of potent Vaccines formulations. Consequently, there is a great need to develop accurate methods for the efficient identification of antigen-specific T cells and the assessment of their functional characteristics directly ex vivo. In this regard, hundreds of clinical vaccination trials have been implemented during... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284924 Fri, 19 Feb 2010 13:39:05 +0100 3284924 http://www.medworm.com/index.php?rid=3284923&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_15 Adjuvants constitute a critical component in Vaccines development in terms of both stimulating and directing immune responses of a suitable profile to promote protection against a diverse range of disease targets. In the past, the field of adjuvant research was mainly dominated by empirical testing and serendipity. However, there is a strong need to develop new generations of adjuvants based on rational design, as well as a requirement to characterise and comprehend their mechanism(s) of action. Adjuvant development can be characterised as an iterative process where potential candidates are repeatedly tested in vitro and in vivo for immunogenicity and optimised in terms of formulation and delivery. Novel lead candidates of adjuvants with a suitable immunological profile relative to specifi... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284923 Fri, 19 Feb 2010 13:39:05 +0100 3284923 http://www.medworm.com/index.php?rid=3284922&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_14 Functional antibody assays can broadly be divided into three categories: neutralisation, serum bactericidal antibody (SBA) and opsonophagocytic assays (OPA). These biological assays are generally more complex than antibody-binding counterparts. They invariably involve multiple biological components, many of which are difficult or impossible to standardise. The aim of this chapter is to provide working examples of these assays and highlight the key issues to be addressed to ensure they are able to provide reliable data. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284922 Fri, 19 Feb 2010 13:39:05 +0100 3284922 http://www.medworm.com/index.php?rid=3284921&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_13 Antibody titre is a measure of the presence and amount of antibodies specific to an antigen that are present in the blood. In particular, the titre of an antibody sample is a measure of the antibody concentration determined under a defined set of conditions, with the antibody concentration being commonly established by enzyme-linked immunosorbent assay, also called ELISA, or a variation of this technology. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284921 Fri, 19 Feb 2010 13:39:05 +0100 3284921 http://www.medworm.com/index.php?rid=3284920&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_12 The reasons why certain Vaccines adjuvants and/or delivery systems are more or less effective at inducing immune responses or promoting the preferential induction of particular types of response are unknown. While Vaccines antigen discovery has benefited from a systematic approach, our limited understanding of the interactions of adjuvants with cells of the immune system has hampered rational adjuvant discovery and handicapped the development of new and more effective Vacciness. It is well accepted that the component parts of the immune system do not work in isolation and their interactions occur in distinct and specialised micro- and macro-anatomical locations. Consequently, significant obstacles to the systematic investigation of adjuvant effects have been the complexity of the physiolog... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284920 Fri, 19 Feb 2010 13:39:05 +0100 3284920 http://www.medworm.com/index.php?rid=3284919&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_11 NKT cells are a distinct lineage of T lymphocytes that are usually identified by the co-expression of the semi-invariant CD1d-restricted aß TCR and the NK1.1 allelic marker of NK lineage receptors in the C57BL/6 mice and related strains. NKT cells can be subdivided based on CD4/CD8 expression and on tissue of origin. NKT cells express significantly the TCR gene products Va24 JaQ in humans, the homolog of mouse Va14 Ja18, paired with Vß11, the homolog of mouse Vß8.2. NKT cells are most frequent in liver (up to 30% of T cells in mice and approximately 4% of hepatic T cells in human), bone marrow, and thymus and represent a smaller proportion of T cells in other tissues including spleen, lymph nodes, blood, and lung. NKT cells recognize a broad array of glycolipids... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284919 Fri, 19 Feb 2010 13:39:05 +0100 3284919 http://www.medworm.com/index.php?rid=3284918&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-585-9_10 Stimulation of B cells not only through the B cell antigen receptor (BCR) but also through Toll-like receptors (TLRs) can drive activation, proliferation, and differentiation of B cells to result in antigen-specific antibody secretion. In addition, B cells are co-stimulated by specific antigen and the presence of a TLR ligand such as for TLR9, which selectively enhances the development of antigen-specific antibodies and endows B cells with strong antigen-presenting capabilities to T cells. These effects promote antigen-specific immune responses and account for the strong adjuvant effect of TLR9 ligands. Several studies have described the activation of human or murine B cells by TLR ligands or other adjuvants. However, there are no reports summarizing the various different effects adjuvants... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3284918 Fri, 19 Feb 2010 13:39:04 +0100 3284918 http://www.medworm.com/index.php?rid=3213507&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_25 This chapter describes the basic methodology to assay the activity of antimicrobial peptides (AMPs) on Leishmania, a human protozoan parasite. The protocols included can be methodologically divided into two major blocks. The first one addresses the basic technology for growth of the different stages of Leishmania, assessment of leishmanicidal activity, and monitoring of plasma membrane permeabilization. The second block encompasses the monitoring of bioenergetic parameters of the parasite, visualization of structural damage by transmission electron microscopy, or those methods more closely related to the involvement of intracellular AMP targets, as subcellular localization of the peptide and induction of parasite apoptosis. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213507 Fri, 22 Jan 2010 00:00:00 +0100 3213507 http://www.medworm.com/index.php?rid=3213506&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_24 Widespread antibiotic resistance is a major incentive for the investigation of novel ways to treat or prevent infections. Much effort has been put into the discovery of peptides in nature accompanied by manipulation of natural peptides to improve activity and decrease toxicity. The ever increasing knowledge about bacteria and the discovery of quorum sensing have presented itself as another mechanism to disrupt the infection process. We have shown that the natural quorum sensing (QS) peptide, competence-stimulating peptide (CSP), used by the caries causing bacteria Streptococcus mutans when used in higher than normally present concentrations can actually contribute to cell death in S. mutans. Using an analogue of this quorum sensing peptide (KBI-3221), we have shown it to be beneficial at d... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213506 Fri, 22 Jan 2010 00:00:00 +0100 3213506 http://www.medworm.com/index.php?rid=3213505&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_23 To more accurately assess the activity and role of epithelial cell-derived antimicrobial peptides in their native settings, it is essential to perform assays at the surfaces under relevant conditions. In order to carry this out, we utilize three-dimensional cultures of airway and gingival epithelium, which are grown at an air–liquid interface. Under these conditions, the cultures can be subjected to challenge with a variety of factors known to cause an increase in antimicrobial peptide gene expression. The functional relevance of this induction can then be assessed by quantifying antibacterial activity either directly on the surface of the cells or using the fluid secreted onto the apical surface of the cultures. The relative contribution of the peptides can also be measured by pre-i... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213505 Fri, 22 Jan 2010 00:00:00 +0100 3213505 http://www.medworm.com/index.php?rid=3213504&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_22 Over the past decade, the emergence of bacterial strains resistant to conventional antibiotics has necessitated the discovery and development of new antimicrobial therapies. This chapter describes a skin infection model that is based on the use of excised skin derived from the domestic pig. The model conditions mimic the environment of human skin and efficiently support the growth of clinically relevant bacterial and fungal species, thus making it useful for evaluating the activity of antimicrobial peptides and other antibiotics as well as their respective formulations. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213504 Fri, 22 Jan 2010 00:00:00 +0100 3213504 http://www.medworm.com/index.php?rid=3213503&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_21 It is widely accepted that cationic antimicrobial peptides possess potent microbicidal properties. Recent studies show that in addition to their antimicrobial action, these peptides can exhibit anti-inflammatory activity. The purpose of this chapter is to describe in vivo ear inflammation models that can be used for evaluating the anti-inflammatory activity of antimicrobial peptides. The models are based on different mechanisms of inflammation development and include irritant dermatitis (a model induced by a single application of 12-o-tetradecanoylphorbol acetate [TPA]) and allergic dermatitis, or delayed type hypersensitivity reaction (a model induced by repetitive application of oxazolone). (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213503 Fri, 22 Jan 2010 00:00:00 +0100 3213503 http://www.medworm.com/index.php?rid=3213502&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_20 One of the most significant advances in medical history is the discovery and development of antibiotics, which in the middle of last century was flourishing and appeared to be the ultimate solution to the treatment of life-threatening human bacterial diseases. However, lately there has been a huge decline in the rate of discovery of new antimicrobial intervention strategies in parallel with an increasing incidence of multidrug-resistant pathogens; if these circumstances do not change we will continue to approach the end of the antibiotic era. Facing this dark future, scientists are considering new strategies for intervention tailored around the appropriate (selective) stimulation of the host’s immune system, and particularly rapid acting innate immunity, as an alternative to direct t... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213502 Fri, 22 Jan 2010 00:00:00 +0100 3213502 http://www.medworm.com/index.php?rid=3213501&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_19 Host defense peptides (HDPs) are relatively small, mostly cationic, amphipathic, and of variable length, sequence, and structure. The majority of these peptides exhibit broad-spectrum antimicrobial activity and often activity against viruses and some cancer cell lines. In addition, HDPs also provide a range of immunomodulatory activities related to innate immunity defense, inflammation, and wound healing. The development of these multi-faceted molecules and their bioactivities into clinically important therapeutics is being pursued using a number of different approaches. Here we review the role of HDPs in nature and application of this role to the development of novel therapeutics. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213501 Fri, 22 Jan 2010 00:00:00 +0100 3213501 http://www.medworm.com/index.php?rid=3213500&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_18 This chapter describes a computer-based method for analyzing the quantitative structure–activity relationships (QSAR) of antimicrobial peptides. Quantitative or qualitative activity measurements and known peptide sequences are used as input for the analysis. The analysis steps consist of the preprocessing which specifically deals with dilution series from an assay with luminescent bacteria, transformation of quantitative activity values into activity classes, a feature extraction method using molecular descriptors for amino acids, feature selection methods, visualization strategies, the classifier model design for discrimination of active and inactive peptides, and the in silico design of promising new peptide candidates. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213500 Fri, 22 Jan 2010 00:00:00 +0100 3213500 http://www.medworm.com/index.php?rid=3213499&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_17 Recent advances in molecular dynamics (MD) simulation methods and in available computational resources have allowed for more reliable simulations of biological phenomena. From all-atom MD simulations, we are now able to visualize in detail the interactions between antimicrobial peptides (AMPs) and a variety of membrane mimics. This helps us to understand the molecular mechanisms of antimicrobial activity and toxicity. This chapter describes how to set up and conduct molecular dynamics simulations of AMPs and membrane mimics. Details are given for the construction of systems of interest for studying AMPs, which can include simulations of peptides in water, micelles, or lipid bilayers. Explanations of the parameters needed for running a simulation are provided as well. (Source: Springer prot... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213499 Fri, 22 Jan 2010 00:00:00 +0100 3213499 http://www.medworm.com/index.php?rid=3213498&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_16 Due to the increasing resistance of microbial pathogens to the available drugs, the identification of new antimicrobial agents with a new mechanism of action is urgently needed. In this context, cationic antimicrobial peptides (AMPs) are considered promising candidates. Although there is evidence that, in contrast to conventional antibiotics, microbial membranes are the principal target of a large number of AMPs, thus making it difficult for the pathogen to acquire resistance, their mode(s) of action is not yet completely clear. Intense research is currently devoted to understand the effect(s) of AMPs on intact cells, either at sub-lethal or at lethal peptide concentrations, and fluorescence/electron microscopy techniques represent a valid tool to get insight into the damage caused by thes... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213498 Fri, 22 Jan 2010 00:00:00 +0100 3213498 http://www.medworm.com/index.php?rid=3213497&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_15 Atomic force microscopy (AFM) has been extensively used to image the three-dimensional surface morphology of a broad range of biological samples, including Gram-negative bacteria, imaged in the presence of antimicrobial peptides (AMPs). Although this technique provides high molecular resolution, it only requires minimum sample treatment and can even be performed in liquid and at varying temperatures while keeping the bacterial cells viable. In this chapter, we describe an easy, fast, and yet effective method for preparing AMP-treated Gram-negative bacteria samples for AFM imaging. The results obtained using this method show a series of morphological changes of Gram-negative bacteria upon treatment with selected AMPs, thus providing vivid insights into the mechanisms of how AMPs perturb and... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213497 Fri, 22 Jan 2010 00:00:00 +0100 3213497 http://www.medworm.com/index.php?rid=3213496&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_14 Solid-state NMR and other biophysical investigations have revealed many mechanistic details about the interactions of antimicrobial peptides with membranes. These studies have shaped our view on how these peptides cause the killing of bacteria, fungi, or tumour cells and how they permeabilize model membranes. As a result, we better understand the biological activities of these peptides and we are now able to design new and better sequences. Here we present some of the tools that have allowed these solid-state NMR investigations, including detailed protocols on how to reconstitute the peptides into oriented or non-oriented membranes as well as simple set-up procedures for 2H as well as proton-decoupled 31P or 15N solid-state NMR measurements. Static and magic angle spinning experiments are ... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213496 Fri, 22 Jan 2010 00:00:00 +0100 3213496 http://www.medworm.com/index.php?rid=3213495&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_13 Membrane-active peptides or protein segments play an important role in many biological processes at the cellular interface to the environment. They are involved, e.g., in cellular fusion or host defense, where they can cause not only merging but also the destabilization of cell membranes. Many factors determine how these typically amphipathic peptides interact with the lipid bilayer. For example, the peptide orientation in the membrane determines which parts of the peptide are exposed to the hydrophobic bilayer interior or to the polar lipid/water interface. As another example, oligomerization is required for many activities such as pore formation. Peptides have been often classified according to a single characteristic mode of interaction with the bilayer, but over the years a more versat... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213495 Fri, 22 Jan 2010 00:00:00 +0100 3213495 http://www.medworm.com/index.php?rid=3213494&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_12 Interactions with bacterial membranes are integral to the mechanisms of action of all antimicrobial peptides (AMPs), regardless of their final cellular targets. Here, we describe in detail two biophysical techniques that can be used to measure the membrane activities of AMPs and antimicrobial peptidomimetics: (1) a calcein leakage assay to investigate interactions between AMPs/peptidomimetics with large unilamellar vesicles and (2) a potential-sensitive dye-based depolarization assay to investigate interactions with the membranes of live bacteria. By comparing the membrane interactions of AMPs and their mimics, these techniques can provide insights into their extent of mimicry and their antimicrobial mechanisms. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213494 Fri, 22 Jan 2010 00:00:00 +0100 3213494 http://www.medworm.com/index.php?rid=3213493&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_11 Measurement of binding and activity of antimicrobial and cytolytic amphipathic peptides on membranes is essential to understanding their function and cell specificity. The use of model systems has provided a wealth of information on the interactions of amphipathic peptides with membranes. Binding of peptides to membranes can be monitored by measuring Förster resonance energy transfer from a Trp residue on the peptide to a lipid fluorophore incorporated in the membrane. Especially for peptides that perturb or disrupt the membrane, it is advantageous to perform these measurements as a function of time, rather than in steady state. The activity of these amphipathic peptides toward model membranes is usually measured by dye efflux kinetics. One of those methods, based on self-quenching of... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213493 Fri, 22 Jan 2010 00:00:00 +0100 3213493 http://www.medworm.com/index.php?rid=3213492&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_10 An expanding body of evidence is rendering manifest that many cationic antimicrobial peptides are endowed with different properties and activities, well beyond their direct action on microbes. One of the most interesting and potentially important research avenue on the alternative use of antimicrobial peptides grounds on their affinity toward lipopolysaccharide (LPS), the endotoxin, responsible for the systemic inflammatory response syndrome (SIRS) and related, often fatal, disorders that can follow Gram-negative infections. Indeed, not only do several antimicrobial peptides, such as cathelicidins, display an ability to strongly bind LPS and break its aggregates, but they have also been demonstrated to suppress LPS-induced pro-inflammatory responses in vitro and to protect from sepsis in a... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213492 Fri, 22 Jan 2010 00:00:00 +0100 3213492 http://www.medworm.com/index.php?rid=3213491&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_9 The SPOT technique provides a fast, cost-efficient, and highly parallel method to synthesize peptide arrays on cellulose. Peptides synthesized on cellulose can be easily cleaved from the support and used directly in a screening assay for antimicrobial activity. Depending on the equipment, the synthesis and the screening can be performed in a medium- or high-throughput manner. High-sensitivity screening is achieved using a bacterial strain (e.g., Pseudomonas aeruginosa H1001) in which a luminescence-encoding gene cassette has been introduced. The intensity of light produced is directly dependent on the energy level of the bacteria. This screening supports the development of new drugs against multidrug-resistant bacteria. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213491 Fri, 22 Jan 2010 00:00:00 +0100 3213491 http://www.medworm.com/index.php?rid=3213490&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_8 Developing new lead structures for drugs against multiresistant bacteria is an urgent need for modern medicine. Antimicrobial peptides are a class of drugs that can be used to discover such structures. In order to support development of this research, a fast, easy, and inexpensive method to synthesize peptides is necessary. The SPOT synthesis has the potential to produce the required peptide arrays, synthesizing up to 8,000 peptides, peptide mixtures, or other organic compounds on cellulose or other planar surfaces in a positionally addressable and multiple manner. Protocols for the preparation of cellulose membranes and the SPOT synthesis as well as cleavage of peptides from the support are described. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213490 Fri, 22 Jan 2010 00:00:00 +0100 3213490 http://www.medworm.com/index.php?rid=3213489&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_7 One promising strategy to combat the proliferation of bacteria resistance toward current antibiotics is the development of peptide-based drug. Among these compounds is a group of small cyclic peptides rich in arginine (Arg) and tryptophan (Trp) residues with selective toxicity toward Gram-negative bacteria. The small size of these peptides with improved toxicity toward Gram-negative bacteria makes them an interesting candidate to understand the forces responsible for their selectivity and paves the way to develop new therapeutics with potent activity toward multi-resistant Gram-negative bacteria. To reach this goal, isothermal titration calorimetry (ITC) is a useful technique which may provide the complete set of thermodynamic parameters of the interaction of peptides with lipid bilayers m... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213489 Fri, 22 Jan 2010 00:00:00 +0100 3213489 http://www.medworm.com/index.php?rid=3213488&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_6 Antimicrobial peptides are ubiquitous in nature where they play important roles in host defense and microbial control. More than 1,000 naturally occurring peptides have been described so far and those considered for pharmaceutical development have all been further optimized by rational approaches. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213488 Fri, 22 Jan 2010 00:00:00 +0100 3213488 http://www.medworm.com/index.php?rid=3213487&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_5 We describe antimicrobial peptide production in E. coli based on forcing antimicrobial peptides into inclusion bodies, which is affective for the production of large quantities of antimicrobial peptides. Chemical reagents for cleaving peptide bond between antimicrobial peptides and fusion proteins such as cyanogen bromide and diluted acid are selective and provide antimicrobial peptides for biological studies in short time. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213487 Fri, 22 Jan 2010 00:00:00 +0100 3213487 http://www.medworm.com/index.php?rid=3213486&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_4 Recombinant expression of α-defensins can be obtained at efficient levels in Escherichia coli. Amplified α-defensin or pro-α-defensin coding cDNA sequences are cloned directionally between EcoRI and SalI sites of the pET-28a expression vector and expressed in E. coli BL21 RIS cells. Cells growing exponentially in nutrient-rich liquid medium are induced to express the recombinant protein by addition of 50 mM isopropyl β-d-1-thiogalactopyranoside for 3–6 h. After bacterial cells collected by centrifugation are lysed in 6 M guanidine-HCl under non-reducing conditions, the expressed defensin fused to its 6xHis-34 amino acid N-terminal fusion partner is purified by affinity chromatography on nickel-NTA columns. A Met codon introduced at the N terminus... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213486 Fri, 22 Jan 2010 00:00:00 +0100 3213486 http://www.medworm.com/index.php?rid=3213485&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_3 We describe a bioassay-based method suitable for finding antibacterial lantibiotics from actinomycete strains and provide selected procedures for characterizing newly discovered lantibiotics for their antibacterial properties. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213485 Fri, 22 Jan 2010 00:00:00 +0100 3213485 http://www.medworm.com/index.php?rid=3213484&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_2 Human skin is a rich source of human antimicrobial peptides. Its cellular source is the keratinocyte, which terminally differentiates in the uppermost parts of the skin, eventually forming the stratum corneum, the horny layer. The easy availability of human stratum corneum makes it possible to identify and characterize human antimicrobial peptides with a biochemical approach. Moreover, the availability of lesional scales of patients with psoriasis, an inflammatory skin disease, allows the identification of human-inducible peptide antibiotics, which are absent in healthy skin. With this strategy, the beta-defensins hBD-2 and hBD-3, RNase-7 as well as psoriasin/S100A7 have been discovered as human antimicrobial peptides and proteins. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213484 Fri, 22 Jan 2010 00:00:00 +0100 3213484 http://www.medworm.com/index.php?rid=3213483&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-594-1_1 Skin secretions from many species of anurans (frogs and toads) are a rich source of peptides with broad-spectrum antimicrobial activities that may be developed into agents with therapeutic potential, particularly for topical applications. This chapter describes the use of norepinephrine (injection or immersion) to stimulate peptide release from granular glands in the skin in procedures that do not appear to cause distress to the animals. The peptide components in the secretions are separated using reversed-phase HPLC on octadecylsilyl-silica (C18) columns after partial purification on Sep-Pak C18 cartridges. Peptides with antimicrobial activity are then identified by demonstration of their abilities to inhibit growth of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aur... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3213483 Fri, 22 Jan 2010 00:00:00 +0100 3213483 http://www.medworm.com/index.php?rid=3127328&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_32 Being exposed to food products, pathogens and harmless commensal bacteria, the mucosal immune system faces a constant challenge. Therefore, maintenance of a homeostatic balance is required to achieve tolerance to harmless bacteria and their products and to induce potent immunity to infection with pathogenic bacteria. Until recently, the literature on mucosal natural killer (NK) cells residing in the intestinal lamina propria was scarce and phenotype and function of gut mucosal NK cells did not receive much attention. Recently, data have become available identifying two distinct subsets of mucosal NKp46+ lymphocytes based on the expression of the orphan transcription factor RORγt. In many ways, the RORγt− subset resembled “classical” NK cells in that it was dev... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127328 Thu, 10 Dec 2009 00:00:00 +0100 3127328 http://www.medworm.com/index.php?rid=3127327&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_31 The term uterine natural killer (uNK) cell is applied in mice to an abundant but transient NK cell population that undergoes unique, terminal differentiation within embryo implantation sites during endometrial decidualization and pregnancy. In mice, decidualization is induced by attachment and implantation of hatched, blastocyst-stage embryos. Within each implantation site, uNK cells proliferate and rapidly differentiate into highly restricted regions called decidua basalis and the mesometrial lymphoid aggregate of pregnancy (MLAp). uNK cells begin to die within healthy decidua basalis by day 8 of the 19–20 day pregnancy of mice. By gestation day 12, uNK cell numbers have peaked and most uNK cells show in situ nuclear fragmentation indicative of disintegration. Morphological stu... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127327 Thu, 10 Dec 2009 00:00:00 +0100 3127327 http://www.medworm.com/index.php?rid=3127326&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_30 Natural killer (NK) cells account for 70% of the leukocytes in the mucosal lining of the uterus (the decidua) in the first trimester of pregnancy. They are CD56superbright granulated cells expressing a repertoire of Killer-cell Immunoglobulin-like Receptors (KIR) skewed towards recognising HLA-C, which is the only classical class I MHC found on placental trophoblast cells. The function of decidual NK cells is not yet known, but there is evidence to suggest that they are involved in mediating trophoblast invasion into the decidua and modifying maternal spiral arteries to increase blood flow to the placenta. In order to characterise decidual NK cells and to understand their interactions with other cells at the maternal–foetal interface, it is useful to be able to isolate these cells. H... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127326 Thu, 10 Dec 2009 00:00:00 +0100 3127326 http://www.medworm.com/index.php?rid=3127325&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_2 The in vitro culture system outlined in this chapter allows for the delineation of events that occur during the development of CD34+ hematopoietic precursor cells into mature KIR+ human NK cells. This system can also be utilized to study the effects of gene overexpression or knockdown on the process of NK cell differentiation through retroviral transduction and long-term culture. The necessary soluble factors and contact-dependent conditions for in vitro human NK cell development have been worked out in our laboratory over the past 16 years. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127325 Thu, 10 Dec 2009 00:00:00 +0100 3127325 http://www.medworm.com/index.php?rid=3127324&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_29 This chapter describes a protocol to assess activation of human NK cells following in vitro stimulation with malaria-infected red blood cells. Activation is assessed by flow cytometry, staining for cell surface expression of CD69 and accumulation of intracellular IFN-γ. Procedures are described for in vitro propagation and purification of Plasmodium falciparum parasites, separation of peripheral blood mononuclear cells from heparinised blood by density centrifugation, in vitro culture of PBMC and for staining and analysis of PBMC by flow cytometry. Some examples of typical FACS plots are shown. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127324 Thu, 10 Dec 2009 00:00:00 +0100 3127324 http://www.medworm.com/index.php?rid=3127323&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_28 Here we describe methods for the in vivo study of antiviral NK cell responses using the mouse Orthopoxvirus ectromelia virus as a model, the agent of mousepox. The methods include those specific for the preparation and use of ectromelia virus such as the production of virus stocks in tissue culture and in live mice, the purification of virus stocks, the titration of virus stocks and virus loads in organs, and the infection of mice. The chapter also includes methods for the specific study of NK cell responses in infected mice such as the preparation of organs (lymph nodes, spleen, and liver) for analysis, the study of NK cell responses by flow cytometry, the adoptive transfer of NK cells, the measurement of NK cell cytolytic activity ex vivo and in vivo, and the determination of NK cell pro... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127323 Thu, 10 Dec 2009 00:00:00 +0100 3127323 http://www.medworm.com/index.php?rid=3127322&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_27 Genetically distinct inbred strains of mice that differ in their susceptibility to mouse cytomegalovirus (MCMV) are invaluable for dissecting complex host–pathogen interactions. Their study has allowed the identification of host-resistance loci, including several activating NK cell receptors of major histocompatibility complex (MHC) class I. In this chapter, we provide a practical guide to the genetic mapping and functional characterization of NK cell receptors that control innate immunity against MCMV via specific recognition of infected cells. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127322 Thu, 10 Dec 2009 00:00:00 +0100 3127322 http://www.medworm.com/index.php?rid=3127321&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_26 We present here all of the techniques used to systematically determine if a gene possesses these types of control elements. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127321 Thu, 10 Dec 2009 00:00:00 +0100 3127321 http://www.medworm.com/index.php?rid=3127320&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_25 We describe here a simple and reliable multiplex PCR-SSP (sequence-specific priming) method for relatively rapid and inexpensive genotyping of 15 KIR genes using standard agarose gel electrophoresis. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127320 Thu, 10 Dec 2009 00:00:00 +0100 3127320 http://www.medworm.com/index.php?rid=3127319&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_24 We present an antibody panel allowing simultaneous assessment of the four major inhibitory KIRs and NKG2A. We further provide guidance on how to apply standard operating procedures to multi-color flow cytometry experiments. Finally, we discuss possibilities as well as limitations with the application of multi-color flow cytometry techniques to future studies of human NK cells. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127319 Thu, 10 Dec 2009 00:00:00 +0100 3127319 http://www.medworm.com/index.php?rid=3127318&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_23 Natural killer (NK) cells are a subset of lymphocytes that contribute to innate immunity through cytokine secretion and target cell lysis. NK cell function is regulated by a multiplicity of activating and inhibitory receptors. The advance in instrumentation for multi-color flow cytometry and the generation of specific mAbs for different epitopes related to phenotypic and functional parameters have facilitated our understanding of NK cell responses. Here, we provide protocols for flow cytometric evaluation of degranulation and cytokine production by human NK cells from peripheral blood at the single-cell level. In addition to offering insight into the regulation of human NK cell responses, these techniques are applicable to the assessment of various clinical conditions, including the diagno... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127318 Thu, 10 Dec 2009 00:00:00 +0100 3127318 http://www.medworm.com/index.php?rid=3127317&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_22 Natural killer (NK) cells were discovered in the 1970 s and named after their naturally occurring cytotoxic activity against tumor cells. It has recently become clear that NK cells are not just killers and that malignancy is unlikely to be the selective pressure driving the evolution of NK cells. Indeed, NK cells secrete a host of cytokines and chemokines that contribute to tissue remodeling at the feto-maternal interface and to both innate and adaptive immunity during infection. Moreover, in certain conditions, they cannot deliver functions cell autonomously, as they require priming from other cells, namely dendritic cells. Nevertheless, natural cytotoxicity is still considered an important parameter used to evaluate NK cell biology, both in the clinic and in the research lab. In thi... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127317 Thu, 10 Dec 2009 00:00:00 +0100 3127317 http://www.medworm.com/index.php?rid=3127316&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_21 Certain receptors on natural killer (NK) cells, which are specific for MHC class I (MHC-I) molecules, do not only interact with ligand expressed on opposing cell membranes (in trans) but also interact with those on the same cell membrane (in cis). Cis interactions have been demonstrated for only a small number of cell surface receptors. However, this has not been tested systematically, raising the possibility that additional receptors may be able to bind ligand expressed in cis. Here we describe a number of approaches to evaluate trans and cis binding of the Ly49A NK cell receptor to its H-2Dd ligand. These procedures should facilitate the investigation of cis/trans interactions of other receptor–ligand pairs and simplify the analysis of NK cell receptor variants. (Source: Springer p... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127316 Thu, 10 Dec 2009 00:00:00 +0100 3127316 http://www.medworm.com/index.php?rid=3127315&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_20 Ly49 receptors in rodents, like KIR in humans, play an integral role in the regulation of NK cell activity. Some inhibitory Ly49 are known to interact with specific MHC I alleles to maintain tolerance to self tissues, and NK activation is triggered upon the loss of inhibitory signals due to pathological downregulation of self MHC I. Although a virally encoded ligand has been identified that can trigger NK cytotoxicity through an activating Ly49, some activating Ly49 also recognize MHC I and the role of most activating receptors in NK effector function remains poorly defined. As many Ly49 remain orphan receptors, we describe methods to unambiguously discern receptor–ligand pairs. Additionally, we describe a method for the mutagenesis of Ly49 and MHC ligands that can be used to define ... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127315 Thu, 10 Dec 2009 00:00:00 +0100 3127315 http://www.medworm.com/index.php?rid=3127314&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_1 Accumulating data indicate that human natural killer (NK) cells undergo terminal maturation in secondary lymphoid tissues (SLTs) including lymph nodes (LNs) and tonsils. In addition, recent studies have revealed that maturing NK cells progress through at least five functionally discrete stages of development within SLTs. These discoveries provide unique possibilities for researchers to investigate the natural processes governing human NK cell development, as they exist in vivo, through analysis of NK cell maturational intermediates found in situ. Herein we describe a detailed, yet simple, four-step protocol for the viable enrichment and purification of human NK cell developmental intermediates from LNs and tonsils. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127314 Thu, 10 Dec 2009 00:00:00 +0100 3127314 http://www.medworm.com/index.php?rid=3127313&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_19 NK cell responses are regulated by a balance of inhibitory and activating signals, reflecting the net effect of interactions between receptors and ligands on target and effector cell surfaces. The identification of ligands for orphan NK cell receptors is key to enhancing our understanding of NK cell biology. Here we describe a strategy (protocol) for the identification of ligands for orphan NK cell receptors using signaling reporter cells in combination with a virus rescue system. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127313 Thu, 10 Dec 2009 00:00:00 +0100 3127313 http://www.medworm.com/index.php?rid=3127312&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_18 We present here (i) a detailed protocol for the production and purification of soluble recombinant NK cell receptors tagged with human IgG1-Fc (thus termed receptor-Fc chimera or receptor-Ig fusion protein) and (ii) a protocol for cell staining with these recombinant receptor-Fc chimeras. As these recombinant proteins are produced in eukaryotic cells, we further discuss the glycosylation pattern of these receptors that might interfere with their ligand-binding phenotype. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127312 Thu, 10 Dec 2009 00:00:00 +0100 3127312 http://www.medworm.com/index.php?rid=3127311&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_17 Different cellular immune responses are modulated by the cross talk between activating and inhibitory signaling pathways initiated via different cell surface receptors. Similarly, the killing of NK cells is controled by multiple activating and inhibitory surface receptors. In humans, the major NK triggering receptors, identified so far, include NKp80, 2B4 NKG2D, and CD16 and the natural cytotoxic receptors (collectively named NCRs) include NKp46, NKp44, and NKp30. The two major families of MHC-specific inhibitory receptors identified in humans are the Ig superfamily (KIR and LIR) and the C-type lectin (CD94/NKG2A) receptor superfamily. The different inhibitory receptors show diverse specificity and discriminate between different class I MHC proteins. Much is known about the function and ex... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127311 Thu, 10 Dec 2009 00:00:00 +0100 3127311 http://www.medworm.com/index.php?rid=3127310&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_16 Natural killer (NK) cells are lymphocytes that provide an important line of defense against viruses and tumors. Technical hurdles in genetic modifications of primary NK cell ex vivo had limited our studies of protein function(s) in NK cell differentiation, acquisition of self-tolerance, and induction of anti-tumor responses. We used VSV-G-pseudotyped, EGFP-expressing lentiviral vectors to develop an efficient gene transfer system to modify gene expression in primary murine NK cells with or without prior IL-2 activation. Lentiviral vector transduction did not impair NK cellular viability, phenotype, or functions. We also demonstrated the use of this system in modifying differentiating NK cells derived from lentiviral-transduced murine hematopoietic progenitor cells. Furthermore, the same tr... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127310 Thu, 10 Dec 2009 00:00:00 +0100 3127310 http://www.medworm.com/index.php?rid=3127309&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_15 Natural killer (NK) cell lines are difficult to transfect using standard techniques, which limits the ability to establish long-term knockdown of proteins with short-hairpin (sh)RNAs. We have developed a method to stably knockdown protein expression in human NK-like lines by introducing shRNAs in retroviral vectors. After a single transduction with retrovirus, shRNA-containing cells can be selected with drug treatment or sorted for enhanced green fluorescent protein (EGFP) expression. With this method, protein expression can be stably decreased to less than 10% of wild-type levels. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127309 Thu, 10 Dec 2009 00:00:00 +0100 3127309 http://www.medworm.com/index.php?rid=3127308&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_14 Natural killer (NK) cells play a vital role in the control of cancer and microbial infections. A major hinderance in studying NK cells is the resistance of these cells to gene transfer. Considering over-expression and gene knockdown studies are crucial tools to study the biology of cells, technologies suitable for transfering genes into NK cells are invaluable. Among various technologies available for gene transfer, lentiviral-mediated transduction has been successful in introducing genes into NK cells. We have standardized methods of lentiviral infection in human and mouse NK cell lines. We obtain transduction efficiencies of 15% in the NK-92 cell line and 30–40% in LNK, YT, and DERL7 cell lines. This method allows efficient and stable introduction of genes and shRNAs into NK cell l... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127308 Thu, 10 Dec 2009 00:00:00 +0100 3127308 http://www.medworm.com/index.php?rid=3127307&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_13 Human NK-like cell lines are difficult to transfect using standard mammalian expression vectors and conventional transfection protocols, but they are susceptible to retroviral transduction as a means to introduce cDNAs. Our laboratory has exploited this technique to study a number of receptors in human NK cell lines. The method utilizes a bicistronic retroviral vector that co-expresses either drug resistance or enhanced green fluorescent protein (EGFP) in parallel with the gene of interest. After a single infection with recombinant retrovirus, transduced NK cells can be sorted for expression of EGFP or the transduced cell surface marker. Alternatively, cells expressing the transduced cDNAs can be selected for by treatment with neomycin, puromycin, or hygromycin. Using this method, the sort... Springer protocols feed by Immunology news http://www.medworm.com/rss/comments.php?id=3127307 Thu, 10 Dec 2009 00:00:00 +0100 3127307