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, 21 Mar 2010 13:39:38 +0100 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info 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 info http://www.medworm.com/rss/comments.php?id=3127307 Thu, 10 Dec 2009 00:00:00 +0100 3127307 http://www.medworm.com/index.php?rid=3127306&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_12 We describe here a model system that overcomes this problem. This system allows the study of many aspects of NK cell receptor function with complete control over the variables that may affect activity such as cis versus trans ligand engagement, homotypic interactions, multiple target types, receptor number, receptor-ligand affinity, and signaling adaptor molecule expression. Although we give examples only for 2B4, Ly49C, and CD48, any NK cell receptors could be studied using these methods. Since many NK cell receptors such as 2B4, CD48, and the Ly49 family can be expressed in T cells, this model system allows the study of not only NK cells but also T cells with NK cell receptors. A standardized system for determining the regulation of NK cell receptor signaling can be important for underst... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3127306 Thu, 10 Dec 2009 00:00:00 +0100 3127306 http://www.medworm.com/index.php?rid=3127305&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_11 Cytokines stimulate biological responses by activating intracellular signaling pathways. We have been adapting flow cytometric techniques to measure the levels of expression and activation of signaling molecules within mixed populations containing NK cells and to characterize their differences within NK cell subpopulations. Approaches for evaluating the total levels of the signal transducers and activators of transcription STAT1 and STAT4, of STAT1 in cells expressing IFNγ, and of the type 1 interferon (type 1 IFN) activation by phosphorylation, i.e., induction of pSTAT1 and pSTAT4, have been developed. The results of experiments using these techniques have demonstrated that an unusual feature of NK cells is high basal expression of STAT4 but reduced STAT1 levels. The condition predi... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3127305 Thu, 10 Dec 2009 00:00:00 +0100 3127305 http://www.medworm.com/index.php?rid=3127304&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-362-6_10 This chapter describes a method by which activating receptor-mediated calcium signaling can be measured in individual murine NK cells using a flow cytometer fitted with a UV laser. One major advantage of this method is that the calcium response of the minority NK cell population and even smaller NK cell subpopulations can be measured simultaneously from a mixture of freshly prepared total splenocytes without resorting to prior cell sorting or expansion in culture. Briefly, cells are harvested and stained to mark the populations of interest, then loaded with indo-1 AM dye and analyzed on the flow cytometer. After an appropriate baseline is established, the cells are treated with a biotinylated antibody to activating receptors, which are subsequently cross-linked by addition of streptavidin.... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3127304 Thu, 10 Dec 2009 00:00:00 +0100 3127304 http://www.medworm.com/index.php?rid=3090016&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_25 Immunoenzyme procedures take on many forms, including, simply, antibody coupled to enzyme. These direct techniques require the labeling of all the primary antibodies and can produce more background. A more economical method uses a secondary antibody or one that has the primary antibody as its antigen. Labeling this secondary antibody with enzyme provides detection for many primary antibodies directed against different antigens of interest. A more sensitive approach involves the use of antibodies directed against enzyme connected to same-species primary antibodies by a secondary linking antibody. This “all immunologic” technique is more sensitive and can result in less background than the covalently labeled methods. Finally, an immune polymer consisting of several secondary anti... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090016 Wed, 01 Jul 2009 00:00:00 +0100 3090016 http://www.medworm.com/index.php?rid=3090015&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_24 The identification of antigenic substances with antibodies can only occur through the use of a reporter molecule. One way of doing this is through the use of enzymes. Enzymes act upon a substrate and that substrate, or a molecule affected by that substrate, in turn becomes detectable by a variety of methods. There are many enzymes available for this purpose. The most common is peroxidase. Another widely used enzyme is alkaline phosphatase. Each enzyme has a few chromogenic substrate solutions with which it can react to change a color visualized through the use of selected instruments, including the microscope. Antibodies can be labeled with an enzyme directly, or secondary antibodies can be labeled with the enzyme and employed in an indirect technique. Also, immunoglobulin labeled polymers... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090015 Wed, 01 Jul 2009 00:00:00 +0100 3090015 http://www.medworm.com/index.php?rid=3090014&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_23 Laser capture microdissection (LCM) is a technical approach for obtaining microscopic samples as small as individual cells from tissues for molecular analysis. While the principles and details of the operation of LCM instruments, the technical requirements for obtaining identified cells for LCM “picking”, all share the common feature of using a laser in combination with a microscope to microdissect and remove cells from tissue slices (or cultured cells) mounted on a glass slide. The use of LCM is becoming widespread in pathology laboratories and is increasingly being used for gene expression studies in cell biology. The approach is particularly powerful when used in conjunction with immunostaining techniques to obtain enriched RNA samples from cells that have been collected by ... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090014 Wed, 01 Jul 2009 00:00:00 +0100 3090014 http://www.medworm.com/index.php?rid=3090013&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_22 This chapter reviews several techniques which combine the use of laser microbeams with antibodies to study molecular and cellular biology. An overview of the basic properties of lasers and their integration with microscopes and computers is provided. Biophysical applications, such as fluorescence recovery after photobleaching to measure molecular mobility and fluorescence resonance energy transfer to measure molecular distances, as well as ablative applications for the selective inactivation of proteins or the selective killing of cells are described. Other techniques, such as optical trapping, that do not rely on the interaction of the laser with the targeting antibody, are also discussed. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090013 Wed, 01 Jul 2009 00:00:00 +0100 3090013 http://www.medworm.com/index.php?rid=3090012&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_21 Born out of the need to overcome an imaging problem in the 1950s, confocal microscopes today allow researchers to go beyond simple imaging and ask biochemical questions. This chapter provides background information on the development of modern confocal microscopes, their uses and applications. Sample preparation and observation are also discussed. Information is also provided about more advanced applications such as FRAP, FRET and 2-photon imaging. The requirements for setting up a confocal laboratory and the instrumentation needs are also discussed. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090012 Wed, 01 Jul 2009 00:00:00 +0100 3090012 http://www.medworm.com/index.php?rid=3090011&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_20 In an age of digital imaging, photographic film still provides a viable and effective means for recording fluorescence images by photomicrography. To maximize the quality of results that are obtained, a photographic emulsion with sufficient sensitivity for the low light level characteristic of Immunofluorescence must be selected, exposures adjusted for reciprocity failure, and modern, high numerical aperture objective lenses employed to produce the brightest possible image. Mounting media that reduce the effects of photobleaching on fluorochromes also help to maintain image brightness, and so reduce exposure times. Digital scanning of film-based micrographs provides the convenience of utilizing image processing software to adjust image density and contrast, and to produce quality prints. (... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090011 Wed, 01 Jul 2009 00:00:00 +0100 3090011 http://www.medworm.com/index.php?rid=3090010&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_19 The ability to determine the expression dynamics of individual genes “in situ” by visualizing the precise spatial and temporal distribution of their products in whole mounts by histochemical and immunocytochemical reactions has revolutionized our understanding of cellular processes. Drosophila developmental genetics was one of the fields that benefited most from these technologies, and a variety of fluorescent methods were specifically designed for investigating the localization of developmentally important proteins and cell markers during embryonic and post embryonic stages of this model organism. In this chapter we present detailed protocols for fluorescence immunocytochemistry of whole mount embryos, imaginal discs, pupal retinas, and salivary glands of Drosophila melanogast... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090010 Wed, 01 Jul 2009 00:00:00 +0100 3090010 http://www.medworm.com/index.php?rid=3090009&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_18 This chapter deals with the detection of antigens located in the interior of isolated cells, in which antibodies can detect their antigens only after fixation and permeabilization of cell structure. Unlike surface antigens, cell structure must be preserved first with a fixative, and the lipid barrier of the plasma membrane must be permeabilized using solvents or detergents. Fluorescence detection enhances the sensitivity of detection of small objects in the cytoplasm of cells because the fluorochrome acts as a point source of light. Because many fixatives preserve the native conformation of fixed antigens, it is important to select antibody reagents that can react with undenatured antigen. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090009 Wed, 01 Jul 2009 00:00:00 +0100 3090009 http://www.medworm.com/index.php?rid=3090008&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_17 This chapter deals with the detection of antigens that are accessible on the surface of isolated living cells using fluorochrome labels. By incubating live cells at 4°C, to prevent endocytosis of bound molecules, the attached antibody can remain on the cell surface, and either be observed in the live state or subsequently fixed. This method yields the greatest sensitivity and best morphologic preservation for detection of surface molecules using antibodies for microscopy. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090008 Wed, 01 Jul 2009 00:00:00 +0100 3090008 http://www.medworm.com/index.php?rid=3090007&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_16 In the direct immunofluorescent labeling technique, fluorochrome-labeled antibodies are used as probes for particular antigens or biomolecules. Cells, usually after appropriate fixation, are incubated with the antibodies to which fluorochromes have been directly conjugated. Following incubation, excess antibody is washed off with PBS and the cells are mounted on coverslips with antifade mounting medium. Immunofluorescent labeled cells are analyzed using a conventional fluorescence microscope or by confocal microscopy. Direct labeling has two major advantages: it requires only a single incubation with the labeled reagent, decreasing the number of steps in the staining procedure; and more importantly, provides minimal nonspecific staining and less background. Additionally, the direct labelin... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090007 Wed, 01 Jul 2009 00:00:00 +0100 3090007 http://www.medworm.com/index.php?rid=3090006&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_15 Immunofluorescence microscopy provides a sensitive means by which antigens can be localized within tissues or individual cells. For the most effective use of this technique the researcher can draw upon basic information on factors that affect the brightness of the fluorescence image, and how well that image can be distinguished from background fluorescence or interfering fluorescence signals. A wide variety of fluorochromes are available, with emitting wavelengths that range from the blue-violet end of the visible spectrum to the infrared. Individual fluorochromes are characterized by their extinction coefficients, quantum yields, susceptibility to photobleaching, the wavelengths at which they maximally absorb excitatory and emit fluorescent light, and how far apart those wavelength maxima... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090006 Wed, 01 Jul 2009 00:00:00 +0100 3090006 http://www.medworm.com/index.php?rid=3090005&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_9 In order to detect intracellular antigens, cells must first be permeabilized especially after fixation with cross-linking agents such as formaldehyde and glutaraldehyde. Permeabilization provides access to intracellular or intraorganellar antigens. Two general types of reagents are commonly used: organic solvents, such as methanol and acetone, and detergents such as saponin, Triton X-100 and Tween-20. The organic solvents dissolve lipids from cell membranes making them permeable to antibodies. Because the organic solvents also coagulate proteins, they can be used to fix and permeabilize cells at the same time. Saponin interacts with membrane cholesterol, selectively removing it and leaving holes in the membrane. The disadvantage of detergents such as Triton X-100 and Tween-20 is that they ... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090005 Wed, 01 Jul 2009 00:00:00 +0100 3090005 http://www.medworm.com/index.php?rid=3090004&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_8 Fixation is one of the most critical steps in immunostaining. The object of fixation is to achieve good morphological preservation, while at the same time preserving antigenicity. Tissue blocks, sections, cell cultures or smears are usually immersed in a fixative solution, while in other situations, whole body perfusion of experimental animals is preferable. Fixation can be accomplished by either chemical or physical methods. The chemical methods include cross-linking agents such as formaldehyde, glutaraldehyde and succinimide esters as well as solvents such as acetone and methanol, which precipitate proteins. Of the physical methods, freezing tissue and air drying are most widely used. This chapter deals with the chemical fixation methods most commonly used for light microscopy. (Source: ... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090004 Wed, 01 Jul 2009 00:00:00 +0100 3090004 http://www.medworm.com/index.php?rid=3090003&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_14 The development of heat-induced antigen (epitope) retrieval (HIER) technologies has led to dramatic improvements in our ability to detect antigens in formalin fixed, archival tissues. Paradoxically, wet heat treatment at temperatures greater than 95°C in appropriate buffer solutions can reconstitute the antigenicity of many proteins that have been rendered nonreactive during the fixation and paraffin embedding process, which heretofore could only be identified in fresh or frozen tissues. The reason for this effect is unclear, but it has been suggested that the vigorous heat treatment partially reverses or disrupts the aldehyde cross-links occurring in proteins during formalin fixation and restores the original conformation of antigenic epitopes. The great success of antigen/epitope ret... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090003 Wed, 01 Jul 2009 00:00:00 +0100 3090003 http://www.medworm.com/index.php?rid=3090002&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_13 In order to test tissue specimens with antibody, they first have to be preserved in fixative, embedded in paraffin, and sectioned very thinly onto glass microscope slides. Any piece of tissue, immediately after excision, must be placed into an adequate volume of fixative. Fixatives vary, but the standard one is 10% buffered formalin. After an optimum fixation time (for formalin, about 16 h), the sample must be embedded in paraffin and sectioned on a microtome. Paraffin-embedded sections placed on positively charged slides (either coated or commercially prepared) are then ready for various pretreatment steps. First, the paraffin must be replaced with water through a series of rehydration steps. Then, depending on the antigen to be tested, the section can be proteolytically digested with enz... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090002 Wed, 01 Jul 2009 00:00:00 +0100 3090002 http://www.medworm.com/index.php?rid=3090001&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_12 Live cells are often studied, in vitro, bathed in nutrient growth media. It is sometimes necessary to study individual compounds produced by these cells and antibodies work well for this purpose. These cells must first be concentrated and fixed before testing. There are a couple of ways to study cells in culture using antibodies. One is to fix the cells in place as they adhere to a solid surface and then test them as though they were cells on a slide. Another is to retrieve them and pellet the cells, fixing them in a test tube and then embedding and sectioning them as though they were a solid tissue. Fixatives can be mild to moderate depending on the antigens to be studied. Sectioned cells can be tested following mild pretreatment steps. Cells fixed in the culture dish can be tested follow... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090001 Wed, 01 Jul 2009 00:00:00 +0100 3090001 http://www.medworm.com/index.php?rid=3090000&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_11 Individual cells often need to be examined with antibodies apart from the surrounding tissue. They may be cells in fluid, cells encased in mucus from a swab, or cells directly extracted from a piece of tissue. Cells can be viewed on a glass slide as cell smears produced from a cell enriched source, introduced as a touch preparation from a piece of wet tissue, concentrated on a slide by the use of a cytocentrifuge, or applied directly to a slide from a solid medium such as a cotton swab. These cell preparations can then be optimally fixed in weakly or nondenaturing solutions such as acetone or those that are alcohol based. They can also be postfixed in formalin if desired. Incubation in buffers containing 0.25% Triton X-100 and 5% dimethylsulfoxide (DMSO) allow for easier antibody penetrati... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3090000 Wed, 01 Jul 2009 00:00:00 +0100 3090000 http://www.medworm.com/index.php?rid=3089999&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_10 The analysis of frozen tissue by antibodies can be accomplished by the quick freezing of a small tissue sample in liquid nitrogen. Super-cooled isopentane can also be used to further the preservation process. Freezing preserves the available proteins in a near-native state for their identification by antibodies raised against naturally folded proteins. The tissues are sectioned onto charged glass slides where they can be optimally fixed in weakly or non-denaturing solutions such as acetone or those that are alcohol-based. Following mild pretreatment steps to allow for antibody use with low background, (the endogenous peroxidase enzyme or oxidative compounds quenched in a hydrogen peroxide solution and available charged sites blocked by incubation in a normal serum solution) the sections ar... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3089999 Wed, 01 Jul 2009 00:00:00 +0100 3089999 http://www.medworm.com/index.php?rid=3089998&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_7 Using the characteristic of a high-affinity complex between avidin and biotin, biotinylated antibodies have wide applications in various immunochemical assays, especially where signal amplification is required. A method is described here for the biotinylation of immunoglobulins. The procedure utilizes water-soluble succinimidyl ester of biotin that reacts with primary amines of the lysine residues or the amino terminus on the antibody to form amide bonds. The method is simple and specific and results in stable conjugates retaining full immunologic activity. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3089998 Wed, 01 Jul 2009 00:00:00 +0100 3089998 http://www.medworm.com/index.php?rid=3089997&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_6 Immunolocalization of antigen via fluorescence requires that fluorochromes be linked either to the primary antibody (direct method) or to a second antibody (indirect method) to provide a fluorescent signal to mark the site of antibody-antigen binding. Of these two methods, the indirect technique is generally more useful and practical. Fluorochromes can be covalently conjugated to antibodies through reactions with thiol or amine groups. Typically, fluorochromes containing isothiocyanate, succinimidyl ester, or sulfonyl chloride reactive groups are conjugated to amines on the antibody molecules. Provided are step-by-step instructions for conjugating isothiocyanate derivates of fluorescein and sulfonyl chloride derivatives of rhodamine to the amine groups of antibodies. (Source: Springer prot... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3089997 Wed, 01 Jul 2009 00:00:00 +0100 3089997 http://www.medworm.com/index.php?rid=3089996&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_5 Affinity chromatography relies on the reversible interaction between a protein and a specific ligand immobilized in a chromatographic matrix. The sample is applied under conditions that favor specific binding to the ligand as the result of electrostatic and hydrophobic interactions, van der Waals’ forces and/or hydrogen bonding. After washing away the unbound material the bound protein is recovered by changing the buffer conditions to those that favor desorption. The technique has been used not only to isolate antigen-specific antibodies but also to remove specific contaminants from biological samples. Methods are described for the purification of immunoglobulins, namely IgG, IgG fragments and subclasses, using the high affinity of protein A and protein G coupled to agarose. In the S... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3089996 Wed, 01 Jul 2009 00:00:00 +0100 3089996 http://www.medworm.com/index.php?rid=3089995&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_4 Ion exchange chromatography techniques are the focus of this chapter and they showcase the power of this method for the purification of proteins and monoclonal antibodies. The technique is powerful and can separate biomolecules that have minor differences in their net charge, e.g., two protein molecules differing by a single charged amino acid. Given the amphoteric character of proteins the pH of the solution is important in the determination of the type of ion exchanger used. Immunoglobulins, although they can be purified by either cation or anion exchange chromatography, are most frequently purified by anion exchange with DEAE resins. The purification of the rabbit IgG fraction from serum using a DEAE column is detailed as well as the purification of IgG from ascitic fluid using FPLC, fr... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3089995 Wed, 01 Jul 2009 00:00:00 +0100 3089995 http://www.medworm.com/index.php?rid=3089994&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_3 Antibodies can be purified by a variety of methods based on their unique physical and chemical properties such as size, solubility, charge, hydrophobicity and binding affinity. This chapter focuses on ammonium sulfate precipitation as a convenient first step in antibody purification in that, it allows the concentration of the starting material and the precipitation of the desired protein. The principle of ammonium sulfate precipitation lies in “salting out” proteins from the solution. The proteins are prevented to form hydrogen bonds with water and the salt facilitates their interaction with each other forming aggregates that afterward precipitate out of solution. Gel filtration or size- exclusion chromatography is also discussed in this chapter. Gel filtration is based on the ... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3089994 Wed, 01 Jul 2009 00:00:00 +0100 3089994 http://www.medworm.com/index.php?rid=3089993&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_2 Antibodies are a powerful and essential tool in scientific laboratories being used in an array of applications such as immuno-histochemistry, immunobloting, immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). The different sources for antibodies include polyclonal antisera from immunized animals and monoclonal antibodies from cells in culture or from ascites in animals. Both polyclonal and monoclonal antibodies have their advantages, and or disadvantages, but in general the production of monoclonal antibodies is more time consuming and requires tissue culture facilities and skills. The use of either monoclonal or polyclonal antibodies in some of the applications may require that the antibody is in a purified form. They can be purified by a variety of methods described in th... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3089993 Wed, 01 Jul 2009 00:00:00 +0100 3089993 http://www.medworm.com/index.php?rid=3089992&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-324-0_1 Immunohistochemistry is widely used to identify, in situ, various components of cells and tissues in both normal and pathological conditions and is an exceptionally powerful method to demonstrate the localization of cellular components. Immunoglobulins (antibodies) are glycoproteins and are divided into five major classes. IgG, which composes approximately 75% of the immunoglobulins in human serum, is most commonly used for immunostaining. Two types of detection systems, fluorescent and enzyme based are used for immunostaining. The choice of detection system depends on the type of sample and the availability of fluorescent or bight field microscopes as well as the type of information the investigator would like to obtain. This chapter provides an overview of antibody characteristics, and t... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=3089992 Wed, 01 Jul 2009 00:00:00 +0100 3089992 http://www.medworm.com/index.php?rid=2390510&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_11 The following method describes the generation of a recombinant vaccinia virus expressing a costimulatory molecule (human CD80 or B7.1). (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2390510 Tue, 01 Jul 2008 04:00:00 +0100 2390510 http://www.medworm.com/index.php?rid=2359270&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_9 Subsets of T cells can be distinguished on basis of their cytokine production and secretion profile. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2359270 Tue, 01 Jul 2008 04:00:00 +0100 2359270 http://www.medworm.com/index.php?rid=2359269&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_8 The following method describes the identification of candidate immunogenic peptides through their ability to recall an immune T-cell activation from peripheral blood mononuclear cells (PBMCs) of individuals with defined HLA–peptide restrictions that have been previously exposed to the antigen. Isolated PBMCs are plated out at a concentration of 1×106 cells/ml in a 200 μl medium and incubated overnight to reduce cytokine gene expression due to cell manipulation. After starving, cells are either directly stimulated with individual peptides or not stimulated and incubated from 3 to 12 h according to experimental conditions. Quantitative real-time PCR (qrt-PCR) is performed on reverse-transcribed complementary DNA (cDNA) from total RNA that is isolated from peptide-cul... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2359269 Tue, 01 Jul 2008 04:00:00 +0100 2359269 http://www.medworm.com/index.php?rid=2359268&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_7 We describe here an example of LDA experiment assessing antigen-specific T cell proliferation of microcultures in the presence or absence of adjuvant and illustrate how to estimate the frequencies of precursor T cells using an online tool that we made publicly available. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2359268 Tue, 01 Jul 2008 04:00:00 +0100 2359268 http://www.medworm.com/index.php?rid=2359267&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_6 Peripheral blood T lymphocytes are a pool of cells with extremely different characteristics and, therefore, it may be difficult to obtain clear-cut results and to attribute a certain function to a defined T cell population in several experimental settings. The availability of a population of human T lymphocytes deriving from the same progenitor with a unique phenotype and function (clone) may therefore be of help. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2359267 Tue, 01 Jul 2008 04:00:00 +0100 2359267 http://www.medworm.com/index.php?rid=2359266&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_5 Since its development, flow cytometry gave a relevant contribution to the field of Immunology. Its unique potential to analyse multiple parameters at the single cell level allowed the identification of unknown cell subsets with specific roles in immunoregulation as well as in the pathogenesis of several diseases. More recently, with the advent of new equipments and fluorochromes, the possibility exists to analyse simultaneously a large number (up to 19) of parameters in a single cell. This strategy, defined polychromatic flow cytometry (PFC), has been widely utilised in the last years for the fine analysis of immune cell phenotypes, including antigen-specific T lymphocytes, B cell subsets, and the intracellular phosphoproteome, among others. A huge amount of data can be generated by such a... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2359266 Tue, 01 Jul 2008 04:00:00 +0100 2359266 http://www.medworm.com/index.php?rid=2359265&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_4 Flow cytometry is combined with highly specific fluorophore-conjugated antibodies that will only bind to the activated forms of molecules. The advances in flow cytometry enable to perform quantitative multiplexed analysis of single cells within heterogeneous populations stained with specific antibodies for phenotyping in conjunction with antibodies to phosphorylated, i.e., activated molecules within signaling pathways. By reactivating signaling pathways in vitro it is possible to collect data on the responsive state of complex cell populations such as immune cells. In this protocol, peripheral blood mononuclear cells (PBMC) are stimulated with cytokines for the indicated time in a 37°C/CO2 incubator, fixed immediately with paraformaldehyde to freeze signaling, permeabilized with methan... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2359265 Tue, 01 Jul 2008 04:00:00 +0100 2359265 http://www.medworm.com/index.php?rid=2359264&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_3 RNA interference is a phenomenon in which specific, endogenous genes are silenced by mRNA degradation. This technology is highly regarded as a potential therapeutic due to its high efficacy and low toxicity. However, the difficulty of delivering RNAi to target cells has impeded the development of RNAi-based therapies. One method to overcome this barrier is the use of a nonpathogenic bacteria vector, Escherichia coli, to deliver RNAi to target cells with high efficacy. In transkingdom interference RNAi (tkRNAi) delivery, E. coli were engineered to transcribe short RNA (shRNA) from a plasmid (TRIP) containing the invasin gene Inv and the listeriolysin O gene Hly. tkRNAi is successful in eliciting efficient gene silencing in vitro and in vivo. (Source: Springer protocols feed by Immuno... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2359264 Tue, 01 Jul 2008 04:00:00 +0100 2359264 http://www.medworm.com/index.php?rid=2359263&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_2 Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC–peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC–peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called “B cell helper assays” that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell r... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2359263 Tue, 01 Jul 2008 04:00:00 +0100 2359263 http://www.medworm.com/index.php?rid=2359262&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_11 The following method describes the generation of a recombinant vaccinia virus expressing a costimulatory molecule (human CD80 or B7.1). (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2359262 Tue, 01 Jul 2008 04:00:00 +0100 2359262 http://www.medworm.com/index.php?rid=2359261&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_10 We present here four methods to isolate the TCR in a native form and details to analyse it by BN-PAGE. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2359261 Tue, 01 Jul 2008 04:00:00 +0100 2359261 http://www.medworm.com/index.php?rid=2359260&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_1 Over the last decade, our understanding of the cellular immune system has been greatly advanced through the development of methods to identify antigen-specific T cells directly ex vivo. The major reagents and techniques used for this purpose are (i) tetramerised MHC:peptide complexes (tetramers) which bind to specific T-cell receptors (TCR) and (ii) assays that detect T cells which synthesise cytokines in response to cognate stimulation (intracellular cytokine staining (ICS)). Here, we provide a detailed description of the procedure for generating and using class I MHC:peptide tetramers to label peptide-specific T cells and for carrying out ICS to measure antigen-specific T lymphocytes. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2359260 Tue, 01 Jul 2008 04:00:00 +0100 2359260 http://www.medworm.com/index.php?rid=2007181&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_9 Subsets of T cells can be distinguished on basis of their cytokine production and secretion profile. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2007181 Tue, 01 Jul 2008 04:00:00 +0100 2007181 http://www.medworm.com/index.php?rid=2007180&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_8 The following method describes the identification of candidate immunogenic peptides through their ability to recall an immune T-cell activation from peripheral blood mononuclear cells (PBMCs) of individuals with defined HLA–peptide restrictions that have been previously exposed to the antigen. Isolated PBMCs are plated out at a concentration of 1×106 cells/ml in a 200 μl medium and incubated overnight to reduce cytokine gene expression due to cell manipulation. After starving, cells are either directly stimulated with individual peptides or not stimulated and incubated from 3 to 12 h according to experimental conditions. Quantitative real-time PCR (qrt-PCR) is performed on reverse-transcribed complementary DNA (cDNA) from total RNA that is isolated from peptide-cul... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2007180 Tue, 01 Jul 2008 04:00:00 +0100 2007180 http://www.medworm.com/index.php?rid=2007179&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_7 We describe here an example of LDA experiment assessing antigen-specific T cell proliferation of microcultures in the presence or absence of adjuvant and illustrate how to estimate the frequencies of precursor T cells using an online tool that we made publicly available. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2007179 Tue, 01 Jul 2008 04:00:00 +0100 2007179 http://www.medworm.com/index.php?rid=2007178&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_6 Peripheral blood T lymphocytes are a pool of cells with extremely different characteristics and, therefore, it may be difficult to obtain clear-cut results and to attribute a certain function to a defined T cell population in several experimental settings. The availability of a population of human T lymphocytes deriving from the same progenitor with a unique phenotype and function (clone) may therefore be of help. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2007178 Tue, 01 Jul 2008 04:00:00 +0100 2007178 http://www.medworm.com/index.php?rid=2007177&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_5 Since its development, flow cytometry gave a relevant contribution to the field of Immunology. Its unique potential to analyse multiple parameters at the single cell level allowed the identification of unknown cell subsets with specific roles in immunoregulation as well as in the pathogenesis of several diseases. More recently, with the advent of new equipments and fluorochromes, the possibility exists to analyse simultaneously a large number (up to 19) of parameters in a single cell. This strategy, defined polychromatic flow cytometry (PFC), has been widely utilised in the last years for the fine analysis of immune cell phenotypes, including antigen-specific T lymphocytes, B cell subsets, and the intracellular phosphoproteome, among others. A huge amount of data can be generated by such a... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2007177 Tue, 01 Jul 2008 04:00:00 +0100 2007177 http://www.medworm.com/index.php?rid=2007176&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_4 Flow cytometry is combined with highly specific fluorophore-conjugated antibodies that will only bind to the activated forms of molecules. The advances in flow cytometry enable to perform quantitative multiplexed analysis of single cells within heterogeneous populations stained with specific antibodies for phenotyping in conjunction with antibodies to phosphorylated, i.e., activated molecules within signaling pathways. By reactivating signaling pathways in vitro it is possible to collect data on the responsive state of complex cell populations such as immune cells. In this protocol, peripheral blood mononuclear cells (PBMC) are stimulated with cytokines for the indicated time in a 37°C/CO2 incubator, fixed immediately with paraformaldehyde to freeze signaling, permeabilized with methan... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2007176 Tue, 01 Jul 2008 04:00:00 +0100 2007176 http://www.medworm.com/index.php?rid=2007175&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_3 RNA interference is a phenomenon in which specific, endogenous genes are silenced by mRNA degradation. This technology is highly regarded as a potential therapeutic due to its high efficacy and low toxicity. However, the difficulty of delivering RNAi to target cells has impeded the development of RNAi-based therapies. One method to overcome this barrier is the use of a nonpathogenic bacteria vector, Escherichia coli, to deliver RNAi to target cells with high efficacy. In transkingdom interference RNAi (tkRNAi) delivery, E. coli were engineered to transcribe short RNA (shRNA) from a plasmid (TRIP) containing the invasin gene Inv and the listeriolysin O gene Hly. tkRNAi is successful in eliciting efficient gene silencing in vitro and in vivo. (Source: Springer protocols feed by Immuno... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2007175 Tue, 01 Jul 2008 04:00:00 +0100 2007175 http://www.medworm.com/index.php?rid=2007174&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_2 Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC–peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC–peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called “B cell helper assays” that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell r... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2007174 Tue, 01 Jul 2008 04:00:00 +0100 2007174 http://www.medworm.com/index.php?rid=2007173&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_11 The following method describes the generation of a recombinant vaccinia virus expressing a costimulatory molecule (human CD80 or B7.1). (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2007173 Tue, 01 Jul 2008 04:00:00 +0100 2007173 http://www.medworm.com/index.php?rid=2007172&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_10 We present here four methods to isolate the TCR in a native form and details to analyse it by BN-PAGE. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2007172 Tue, 01 Jul 2008 04:00:00 +0100 2007172 http://www.medworm.com/index.php?rid=2007171&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-527-9_1 Over the last decade, our understanding of the cellular immune system has been greatly advanced through the development of methods to identify antigen-specific T cells directly ex vivo. The major reagents and techniques used for this purpose are (i) tetramerised MHC:peptide complexes (tetramers) which bind to specific T-cell receptors (TCR) and (ii) assays that detect T cells which synthesise cytokines in response to cognate stimulation (intracellular cytokine staining (ICS)). Here, we provide a detailed description of the procedure for generating and using class I MHC:peptide tetramers to label peptide-specific T cells and for carrying out ICS to measure antigen-specific T lymphocytes. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=2007171 Tue, 01 Jul 2008 04:00:00 +0100 2007171 http://www.medworm.com/index.php?rid=1539004&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_18 We describe here a method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to determine the location and number of methyl groups added at any site in the rRNA. The method is particularly suited to studying in vitro methylation of RNA transcripts by resistance methyltransferases such as Erm. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1539004 Sat, 01 Dec 2007 05:00:00 +0100 1539004 http://www.medworm.com/index.php?rid=1539003&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_12 This chapter is orgTreatment of Gram-negative bacterial infections is complicated by innate and acquired drug resistance resulting in a limited number of effective antibiotics. Several Gram-negative bacteria, for which current therapies are ineffective, have recently been identified as potential bioterror agents. These findings highlight the need for new antibiotics, specifically antibiotics that act on new drug targets to circumvent drug resistance. Potential targets in Gram-negative bacteria include enzymes involved in the biosynthesis of lipopolysaccharides (LPS) that form outer membranes of these organisms. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the committed step in the biosynthesis of the lipid A portion of LPS. Therefore, inhibitors of this e... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1539003 Sat, 01 Dec 2007 05:00:00 +0100 1539003 http://www.medworm.com/index.php?rid=1539002&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_15 Infections caused by multidrug-resistant Gram-negative pathogens play a major role in the morbidity and mortality of hospitalized patients. The rise of resistance to current antibiotic therapies has made the discovery of new agents urgent. One of the major antibiotic resistance mechanisms utilized by more than 15 species of Gram-negative bacterial cells is the Resistance Nodulation Division (RND) efflux pump, which eliminates several classes of antibiotics such as penicillins and cephalosporin macrolides aminoglycosides, fluoroquinolonesx and tetracyclines. Here we describe a multistep process to identify compounds that inhibit the RND-type efflux pumps. This involves measuring the inhibition of accumulation of ethidium bromide in E. coli or Haemophilus influenzae cells and confirming that... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1539002 Sat, 01 Dec 2007 05:00:00 +0100 1539002 http://www.medworm.com/index.php?rid=1539001&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_7 In Escherichia coli, the molecular chaperone HSP70 (DnaK) is necessary for 30S and 50S ribosomal subunit assembly at temperatures above 37°C. Inhibitors of DnaK should therefore hinder ribosome biogenesis, in addition to all of the other DnaK-dependent cellular functions. An easily testable phenotype of DnaK is described here based on α-complementation of β-galactosidase. This protein fragment complementation requires a functional DnaK in vivo, offering a suitable method for screening for DnaK inhibitors. Subsequently, it will be of great importance to check whether inhibitors of bacterial DnaK selected in this way have an effect (inhibitory or stimulatory) on the activities of eukaryotic HSP70 and HSC70 chaperones, because of the universal conservation in all biota of these... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1539001 Sat, 01 Dec 2007 05:00:00 +0100 1539001 http://www.medworm.com/index.php?rid=1539000&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_4 RNA polymerase is essential to the viability of bacteria in all phases of growth and development and is a proven chemotherapeutic target as the cellular target of the rifamycin class of antibiotics. However, despite the characterization of multiple different classes of natural products that selectively target bacterial RNA polymerase, and the identification of a limited number of synthetic compound inhibitors, only agents of the rifamycin class have been developed and approved for human clinical use as antibiotics. Herein we describe a scintillation proximity assay (SPA) for identifying and characterizing inhibitors of bacterial RNA polymerases and that is applicable to de novo drug discovery programs through application of automated high-throughput screening methods. In addition, we descr... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1539000 Sat, 01 Dec 2007 05:00:00 +0100 1539000 http://www.medworm.com/index.php?rid=1538999&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_20 Aminoglycoside antibiotics are highly potent, wide-spectrum bactericidals (1, 2). Bacterial resistance to aminoglycosides, however, is a major problem in the clinical use of aminoglycosides. Enzymatic modification of aminoglycosides is the most frequent resistance mode among several resistance mechanisms employed by resistant pathogens (1,3). Three families of aminoglycoside modifying enzymes, O-phosphotransferases, N-acetyltransferases, and N-nucleotidyltransferases, are known to have more than 50 enzymes (1,3,4). In this chapter, determination of enzymatic activity of a single enzyme from each family in the presence and absence of an inhibitor is described. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538999 Sat, 01 Dec 2007 05:00:00 +0100 1538999 http://www.medworm.com/index.php?rid=1538998&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_6 The inhibition of bacterial ribosomal subunit formation is a novel target for translational inhibitors. Inhibition of subunit biogenesis has been shown to be equivalent to the inhibition of protein biosynthesis for many antibiotics. This chapter describes three methods for examining the inhibition of subunit formation in growing bacterial cells. The first method permits the determination of the IC50 value for inhibition of assembly and protein synthesis. The second is a pulse and chase labeling procedure to measure the kinetics of subunit formation. The third procedure allows an examination of ribosome reformation after antibiotic removal as a part of the post-antibiotic effect. Together these procedures give a description of the relative inhibitory effects of an antibiotic on translation ... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538998 Sat, 01 Dec 2007 05:00:00 +0100 1538998 http://www.medworm.com/index.php?rid=1538997&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_3 The need for new drugs to treat infections caused by antibiotic-resistant bacterial strains has prompted many studies to identify novel targets in pathogenic bacteria. Among the three DNA polymerases expressed by bacteria, one of these, designated pol III, is responsible for DNA replication and growth of bacteria and, therefore, warrants consideration as a drug target. However, the pol III enzymes of Gram-positive and Gram-negative species are quite different, and the Gram-positive enzyme pol IIIC has been more extensively studied as a drug target than the Gram-negative enzyme pol IIIE. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538997 Sat, 01 Dec 2007 05:00:00 +0100 1538997 http://www.medworm.com/index.php?rid=1538996&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_1 In conclusion, the results of such a strategy underscore the utility of large genomic databases for in silico systematic drug target identification in the post-genomic era. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538996 Sat, 01 Dec 2007 05:00:00 +0100 1538996 http://www.medworm.com/index.php?rid=1538995&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_17 We describe a model system that instead is based on the interaction between a test compound and a response regulator in a homogeneous phase reaction. In this system, response regulator-DNA complex formation and its inhibition by a test compound are measured by fluorescence polarization. The model system should be readily adaptable to drug discovery based on other bacterial two-component s transduction systems. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538995 Sat, 01 Dec 2007 05:00:00 +0100 1538995 http://www.medworm.com/index.php?rid=1538994&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_9 The formation of peptide bonds is the central chemical reaction during protein synthesis and is catalyzed by the peptidyl transferase center residing in the large ribosomal subunit. This active site is composed of universally conserved rRNA nucleosides. The peptidyl transferase center is by far the most frequently used target site of natural antibiotics in the cell. Here we describe a novel, simple, and convenient method to assess peptide bond formation which we named SPARK. The basic principle of SPARK is the use of two reaction substrates that closely resemble the natural tRNA substrates (one is biotinylated and the other carries a tritium label) that become covalently connected during transpeptidation. Formation of this peptide bond then allows capture and direct quantification of the r... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538994 Sat, 01 Dec 2007 05:00:00 +0100 1538994 http://www.medworm.com/index.php?rid=1538993&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_11 Key enzymes that assemble the bacterial cell wall are also the target of the Β-lactam class of antibiotics. The covalent binding of labeled penicillin to these proteins has been used in numerous studies in drug discovery, antibiotic mechanisms of action and resistance, and cell wall physiology. Methods to label and measure penicillin binding proteins in two prototypical organisms, a Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus), are described. The methods discussed include identifying penicillin-binding proteins in both intact cells (in vivo measurements) and isolated cell membranes. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538993 Sat, 01 Dec 2007 05:00:00 +0100 1538993 http://www.medworm.com/index.php?rid=1538992&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_2 DNA gyrase and DNA topoisomerase (topo) IV are the bacterial targets of coumarin and quinolone antimicrobial agents. Widespread resistance to clinically important antibiotics such as beta-lactams and macrolides has stimulated the development of novel gyrase and topo IV inhibitors especially against Streptococcus pneumoniae and other Gram-positive pathogens. Here, we describe how gyrase and topo IV activities are measured and how inhibitors of these enzymes may be assayed, focusing as a paradigm on DNA supercoiling by S. pneumoniae gyrase, DNA decatenation by S. pneumoniae topo IV, and DNA cleavage by both enzymes. These approaches provide mechanistic insight on inhibitor action and allow identification of dual gyrase/topo IV targeting agents that can minimize the emergence of bacterial res... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538992 Sat, 01 Dec 2007 05:00:00 +0100 1538992 http://www.medworm.com/index.php?rid=1538991&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_8 While bacterial protein synthesis is the target of about half of the known antibiotics, the great structural-functional complexity of the translational machinery still offers remarkable opportunities for identifying novel and specific inhibitors of unexploited targets. We designed a knowledge-based in vitro translation assay to identify inhibitors selectively targeting the bacterial or the yeast translational apparatus, preferentially blocking the early steps of protein synthesis. Using a natural-like, “universal” model mRNA and cell-free extracts prepared from Eschericha coli, Saccharomyces cerevisiae, and HeLa cells, we were able to translate, with comparable yields in the three systems, the immunogenic peptide encoded by this “universal” mRNA. The immuno-enzymati... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538991 Sat, 01 Dec 2007 05:00:00 +0100 1538991 http://www.medworm.com/index.php?rid=1538990&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_14 This chapter describes reliable flow cytometric methods for assessment of two important physiologic characteristics of bacteria, membrane potential and membrane permeability, which can provide indications of the effects of antimicrobial agents on microorganisms. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538990 Sat, 01 Dec 2007 05:00:00 +0100 1538990 http://www.medworm.com/index.php?rid=1538989&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_5 Aminoacyl-tRNA synthetases (aa-RS) attracted interest as potential targets for new antibacterial compounds. Most organisms express 20 aa-RSs: one for each amino acid. Aa-RSs are essential proteins in all living organisms. When one aa-RS is inhibited, the corresponding tRNA is not charged and is therefore unavailable for translation. This leads to protein synthesis inhibition, which in turn causes cell growth arrest. Consequently, each compound that inhibits any of the aa-RS could be a potential antibacterial agent. Only one aa-RS inhibitor, the Ile-RS inhibitor mupirocin, is currently marketed as an antibacterial agent. We focused on phenylalanyl (Phe)-tRNA synthetase (Phe-RS), but the described methods are not restricted to Phe-RS and might be adapted to other aa-RS. (Source: Springer pro... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538989 Sat, 01 Dec 2007 05:00:00 +0100 1538989 http://www.medworm.com/index.php?rid=1538988&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_19 The ability, either innate or acquired, to produce β-lactamases, enzymes capable of hydrolyzing the endocyclic peptide bond in β-lactam antibiotics, would appear to be a primary contributor to the ever-increasing incidences of resistance to this class of antibiotics. To date, four distinct classes, A, B, C, and D, of β-lactamases have been identified. Of these, enzymes in classes A, C, and D utilize a serine residue as a nucleophile in their catalytic mechanism while class B members are Zn+2-dependent for their function. Efforts have been and still continue to be made toward the development of potent inhibitors of these enzymes as a means to ensure the efficacy of β-lactam antibiotics in clinical medicine. This chapter concerns procedures for the evaluation of the catal... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538988 Sat, 01 Dec 2007 05:00:00 +0100 1538988 http://www.medworm.com/index.php?rid=1538987&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_13 Widespread resistance to antibiotics in current clinical use is increasing at an alarming rate. Novel approaches in antimicrobial therapy will be required in the near future to maintain control of infectious diseases. An enormous array of small cationic peptides exists in nature as part of the innate defense systems of organisms ranging from bacteria to humans. For most naturally occurring linear peptides, such as magainins and cecropins, a common feature is their capacity to form an amphipathic α-helix (with polar and nonpolar groups on opposite faces of the helix), a structural feature believed to be important in their antimicrobial function as membrane-lytic agents. A massive effort over the past two decades has resulted in a better understanding of the molecular mechanism of anti... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538987 Sat, 01 Dec 2007 05:00:00 +0100 1538987 http://www.medworm.com/index.php?rid=1538986&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_16 Fatty acid biosynthesis is one of the relatively newer targets in antibacterial drug discovery. The presence of distinct fatty acid synthases (FAS) in mammals and bacteria and the fact that most bacterial FAS enzymes are essential for viability make this a very attractive antimicrobial drug target. The enzyme β-ketoacyl ACP synthase (KASIII or FabH) is the key enzyme that initiates fatty acid biosynthesis in a type II dissociated FAS. This enzyme catalyzes the condensation of acyl CoA and malonyl ACP (acyl carrier protein) to form a β-ketoacyl ACP product, which is further processed to form mature fatty acids that are involved in various essential cellular processes and structures like phospholipid biosynthesis, cell wall formation, etc. Herein we describe a new assay for the Myc... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538986 Sat, 01 Dec 2007 05:00:00 +0100 1538986 http://www.medworm.com/index.php?rid=1538985&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-246-5_10 The emergence of bacterial pathogens resistant to current antibiotics has caused an urgent demand for new treatments. Peptide deformylase (PDF) has become an exciting target for designing novel antibiotics. To facilitate the screening of PDF inhibitors, three robust, coupled assays have been developed. The first method couples the PDF reaction with that of formate dehydrogenase. Formate dehydrogenase oxidizes formate into CO2 with a concomitant reduction of NAD+ to NADH, which can be monitored spectrophotometrically. The second method involves Aeromonas aminopeptidase (AAP) as the coupling enzyme and an artificial substrate, f-Met-Leu-p-nitroanilide. The sequential action of PDF and AAP releases p-nitroanilide as a highly chromogenic product. In the third method, f-Met-Lys-7-amino-4-methyl... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1538985 Sat, 01 Dec 2007 05:00:00 +0100 1538985 http://www.medworm.com/index.php?rid=1539009&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-570-1_1 Forward genetics has led to many “breakthrough” discoveries, and with the mouse genome almost fully sequenced, the creation of phenotypes through random germline mutagenesis has become an efficient means by which to find the function of yet undescribed genes. In this chapter, we will provide a practical guideline for performing germline mutagenesis in mice. In particular, we will focus on the application of this technology to identify genes that are essential to innate immune defense. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1539009 Mon, 19 Nov 2007 05:00:00 +0100 1539009 http://www.medworm.com/index.php?rid=1539008&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-570-1_25 Innate immunity is an ancient and conserved defense mechanism. The worm Caenorhabditis elegans provides a useful tool for studying the function of the innate immune system at the molecular and cellular levels within the context of a whole organism. The powerful genetics of the worm, combined with efficacy of gene knockdown by RNA interference (RNAi), offer complementary tools for analyzing the contribution of individual genes to innate immunity. It is important, however, to exclude pleiotropic effects that confound results. In this chapter, we will describe the procedures for performing both forward and reverse genetic screens and will discuss a number of techniques developed to resolve confounding effects, thus enhancing the power of this system. (Source: Springer protocols feed by Immuno... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1539008 Mon, 19 Nov 2007 05:00:00 +0100 1539008 http://www.medworm.com/index.php?rid=1539007&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-570-1_8 Our understanding of virus control by natural killer (NK) cells relies mainly on in vitro observations. The significance of these findings for virus control in vivo is not yet fully understood. Complexity is added by the fact that many viruses, particularly herpesviruses, are equipped with sets of genes that, dependent on the genetic background of the host, modify the NK cell response. The advent of recombinant DNA technology and mutagenesis procedures for BAC-cloned viral genomes has made it possible not only to screen for viral proteins with such functions but also to assess their biological relevance. Mutant viruses with gene defects reveal the efficacy and complexity of NK cell control. Here, we describe procedures to assess the NK cell response to mouse cytomegalovirus (MCMV), a promi... Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1539007 Mon, 19 Nov 2007 05:00:00 +0100 1539007 http://www.medworm.com/index.php?rid=1539006&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-570-1_14 We describe here (1) fractionation methods suitable for purifying mouse or rat peritoneal mast cells and for purifying human mast cells of various origins, and (2) conditions for generating pure cultured mast cell populations from mouse, rat, and human tissues. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1539006 Mon, 19 Nov 2007 05:00:00 +0100 1539006 http://www.medworm.com/index.php?rid=1539005&cid=s_37124_3_f&fid=37124&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-570-1_22 Invertebrates lack an adaptive immune system and rely on innate immunity to resist pathogens. The response of Drosophila melanogaster to bacterial and fungal infections involves two signaling pathways, Toll and Imd, both of which activate members of the nuclear factor (NF)-κB family of transcription factors, leading to antimicrobial peptide (AMP) gene expression. In this chapter, we present the current methods used in our laboratory to monitor the activity of both signaling pathways. (Source: Springer protocols feed by Immunology) Springer protocols feed by Immunology info http://www.medworm.com/rss/comments.php?id=1539005 Mon, 19 Nov 2007 05:00:00 +0100 1539005