Generation of a Single-Cycle Replicable Rift Valley Fever Vaccine
Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is an arbovirus that causes severe disease in humans and livestock in sub-Saharan African countries. The virus carries a tripartite, single-stranded, and negative–sense RNA genome, designated as L, M, and S RNAs. RVFV spread can be prevented by the effective vaccination of animals and humans. Although the MP-12 strain of RVFV is a live attenuated vaccine candidate, MP-12 showed neuroinvasiveness and neurovirulence in young mice and immunodeficiency mice. Hence, there is a concern for the use of MP-12 to certain individuals, especially those that ...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Analytic Vaccinology: Antibody-Driven Design of a Human Cytomegalovirus Subunit Vaccine
Identification of the most relevant protective antigens has represented a considerable obstacle for the development of subunit vaccines against viral infections, including human cytomegalovirus (HCMV) infection. This chapter describes the method of analytic vaccinology, centered on the clonal analysis of human B cell response to HCMV, which represents an essential tool for assessing the impact of individual viral antigens in the antiviral antibody response. By providing key information on the immunogenicity and protective properties of the antibodies elicited by viral proteins, the analytic vaccinology method guides the se...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Development of Rabies Virus-Like Particles for Vaccine Applications: Production, Characterization, and Protection Studies
Rabies is a viral infection of the central nervous system for which vaccination is the only treatment possible. Besides preexposure, vaccination is highly recommended for people living in endemic areas, veterinarians, and laboratory workers. Our group has developed rabies virus-like particles (RV-VLPs) with immunogenic features expressed in mammalian cells for vaccine applications. In this chapter the methods to obtain and characterize a stable HEK293 cell line expressing RV-VLPs are detailed. Further, analytical ultracentrifugation steps to purify the obtained VLPs are developed, as well as western blot, dynamic light sca...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Reverse Genetics Approaches to Control Arenavirus
Several arenavirus cause hemorrhagic fever disease in humans and pose a significant public health problem in their endemic regions. To date, no licensed vaccines are available to combat human arenavirus infections, and anti-arenaviral drug therapy is limited to an off-label use of ribavirin that is only partially effective. The development of arenavirus reverse genetics approaches provides investigators with a novel and powerful approach for the investigation of the arenavirus molecular and cell biology. The use of cell-based minigenome systems has allowed examining the cis- and trans-acting factors involved in arenavirus ...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses
There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycl...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Generation and Characterization of a Chimeric Tick-Borne Encephalitis Virus Attenuated Strain ChinTBEV
Tick-borne encephalitis (TBE), caused by TBE virus (TBEV), is one of the most serious human viral diseases endemic in Europe and East Asia. No effective treatment for TBEV infection exists and the primary preventive measure is vaccination. Although several inactivated vaccines have been licensed, the development of novel and more effective vaccines remains a high priority especially in disease-endemic countries. Here we describe a universal vaccine design approach to construct a live chimeric recombinant TBEV attenuated strain ChinTBEV based on the infectious full-length cDNA clone of Japanese encephalitis virus using stan...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant
Given periodic outbreaks of fatal human infections caused by coronaviruses, development of an optimal coronavirus vaccine platform capable of rapid production is an ongoing priority. This chapter describes the use of an insect cell expression system for rapid production of a recombinant vaccine against severe acute respiratory syndrome coronavirus (SARS). Detailed methods are presented for expression, purification, and release testing of SARS recombinant spike protein antigen, followed by adjuvant formulation and animal testing. The methods herein described for rapid development of a highly protective SARS vaccine are equa...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Assessment of Functional Norovirus Antibody Responses by Blocking Assay in Mice
Norovirus (NoV)-specific serum antibodies bind to NoV-derived virus-like particles (VLPs) and block the binding of VLPs to the host cell attachment factors/receptors, histo-blood group antigens (HBGAs). Blocking antibodies in human sera have been associated with a protection from NoV infection and disease. Studies of experimental NoV VLP-based vaccines measure blocking antibodies in animal sera instead of a traditional virus neutralization assay. This chapter describes the methodology for analyzing blocking antibodies from NoV GII.4 VLP-immunized mouse sera. Protocol for obtaining mouse NoV GII.4-specific immune sera is de...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Designing Efficacious Vesicular Stomatitis Virus-Vectored Vaccines Against Ebola Virus
Infection with the Ebola virus (EBOV) causes an aggressive hemorrhagic disease in humans and nonhuman primates. Traditional approaches, such as vaccination with inactivated virion preparations, have had limited efficacy, whereas immunization with live-attenuated EBOV is not feasible due to the highly lethal nature of the pathogen. This has necessitated the development of other approaches towards an effective EBOV vaccine. Over the past decade, recombinant viruses expressing the EBOV glycoprotein (GP) have constituted the most promising platforms, as evidenced by their ability to protect naïve nonhuman primates from a ...
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Methods to Evaluate Novel Hepatitis C Virus Vaccines
In conclusion, this chapter describes a method to design HCV vaccines and also a protocol to assess their efficacy. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Application of Droplet Digital PCR to Validate Rift Valley Fever Vaccines
Droplet Digital™ polymerase chain reaction (ddPCR™) is a promising technique that quantitates the absolute concentration of nucleic acids in a given sample. This technique utilizes water-in-oil emulsion technology, a system developed by Bio-Rad Laboratories that partitions a single sample into thousands of nanoliter-sized droplets and counts nucleic acid molecules encapsulated in each individual particle as one PCR reaction. This chapter discusses the applications and methodologies of ddPCR for development of Rift Valley fever (RVF) vaccine, using an example that measures RNA copy numbers of a live-attenuated M...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Proteomic Monitoring of B Cell Immunity
Immune monitoring is critical in settings of infection, autoimmunity, and cancer, but our understanding of the diversity of the antibody immune repertoire has been limited to selected target antigens and epitopes. Development of new vaccines requires monitoring of B cell immunity and identification of candidate antigens. As vaccines become more complex, novel techniques are required for monitoring the diversity of the B cell immune response. Since antibodies recognize both linear and conformational protein and glycoprotein epitopes, recent advances in proteomic and glycomic technologies for rapid display of antigenic struc...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Systems Vaccinology: Applications, Trends, and Perspectives
The strategies employed in vaccinology have improved since the seminal work of Edward Jenner in the eighteenth century. Stimulated by failure to develop vaccines for cancers and chronic infectious diseases as well as an emergence of a multitude of new technologies not available earlier, vaccinology has moved from a largely experimental art to a new phase of innovation. Currently, immune reactions can be predicted and modeled before they occur and formulations can be optimized in advance for genetic background, age, sex, lifestyle, environmental factors, and microbiome. A multitude of scientific insights and technological a...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Reverse Vaccinology: The Pathway from Genomes and Epitope Predictions to Tailored Recombinant Vaccines
In this chapter, we review the computational approaches that have led to a new generation of vaccines in recent years. There are many alternative routes to develop vaccines based on the technology of reverse vaccinology. We focus here on bacterial infectious diseases, describing the general workflow from bioinformatic predictions of antigens and epitopes down to examples where such predictions have been used successfully for vaccine development. (Source: Springer protocols feed by Immunology)
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Practical Approaches to Forced Degradation Studies of Vaccines
During the early stages of vaccine development, forced degradation studies are conducted to provide information about the degradation properties of vaccine formulations. In addition to supporting the development of analytical methods for the detection of degradation products, these stress studies are used to identify optimal long-term storage conditions and are part of the regulatory requirements for the submission of stability data. In this chapter, we provide detailed methods for forced degradation analysis under thermal, light, and mechanical stress conditions. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Development of Antibody-Based Vaccines Targeting the Tumor Vasculature
A functional vasculature is essential for tumor progression and malignant cell metastasis. Endothelial cells lining blood vessels in the tumor are exposed to a unique microenvironment, which in turn induces expression of specific proteins designated as tumor endothelial markers (TEMs). TEMs either localized at the plasma membrane or secreted into the extracellular matrix are accessible for antibody targeting, which can be either infused or generated de novo via vaccination. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Preconditioning Vaccine Sites for mRNA-Transfected Dendritic Cell Therapy and Antitumor Efficacy
Messenger RNA (mRNA)-transfected dendritic cell (DC) vaccines have been shown to be a powerful modality for eliciting antitumor immune responses in mice and humans; however, their application has not been fully optimized since many of the factors that contribute to their efficacy remain poorly understood. Work stemming from our laboratory has recently demonstrated that preconditioning the vaccine site with a recall antigen prior to the administration of a dendritic cell vaccine creates systemic recall responses and resultantly enhances dendritic cell migration to the lymph nodes with improved antitumor efficacy. This chapt...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Peptide-Based Cancer Vaccine Strategies and Clinical Results
Active cancer immunotherapy is an exciting and developing field in oncology research. Peptide vaccines, the use of isolated immunogenic tumor-associated antigen (TAA) epitopes to generate an anticancer immune response, are an attractive option as they are easily produced and administered with minimal toxicity. Multiple TAA-derived peptides have been identified and evaluated with various vaccine strategies currently in clinical testing. Research suggests that utilizing vaccines in patients with minimal-residual disease may be a more effective strategy compared to targeting patients with widely metastatic disease as it avoid...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

T-Cell Epitope Discovery for Therapeutic Cancer Vaccines
The success of recent immune checkpoint blockade trials in solid tumors has demonstrated the tremendous potential of immune-mediated treatment strategies for cancer therapy. These immune therapies activate preexisting cytotoxic CD8+ T cells (CTL) to selectively target and eradicate malignant cells. In vitro models suggest that these therapies may be more effective in combination with priming of CTL using cancer vaccines. CTL-mediated tumor targeting is achieved by its recognition of tumor antigenic epitopes presented on human leukocyte antigen (HLA) class I molecules by tumor cells. Discovering CTL-antigenic epitopes is th...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Dendritic Cell Vaccines
Exploitation of the patient’s own immune system to induce antitumor immune responses using dendritic cell (DC) immunotherapy has been established in early clinical trials as a safe and promising therapeutic approach for cancer. However, their limited success in larger clinical trials highlights the need to optimize DC vaccine preparations. This chapter describes the methodologies utilized for the preparation of the DC vaccine most commonly used in clinical trials. Optional variations at different stages in DC vaccine preparation, based on the nature of antigen, delivery of antigen, maturation stimuli, and mode of adm...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Cancer Vaccines: A Brief Overview
Vaccine approaches for cancer differ from traditional vaccine approaches for infectious disease in tending to focus on clearing active disease rather than preventing disease. In this review, we provide a brief overview of different types of vaccines and adjuvants that have been investigated for the purpose of controlling cancer burdens in patients, some of which are approved for clinical use or in late-stage clinical trials, such as the personalized dendritic cell vaccine sipuleucel-T (Provenge) and the recombinant viral prostate cancer vaccine PSA-TRICOM (Prostvac-VF). Vaccines against human viruses implicated in the deve...
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Principles of Vaccination
While many of the currently available vaccines have been developed empirically, with limited understanding on how they activate the immune system and elicit protective immunity, the recent progress in basic sciences like immunology, microbiology, genetics, and molecular biology has fostered our understanding on the interaction of microorganisms with the human immune system. In consequence, modern vaccine development strongly builds on the precise knowledge of the biology of microbial pathogens, their interaction with the human immune system, as well as their capacity to counteract and evade innate and adaptive immune mecha...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Future Challenges for Vaccinologists
Vaccination is one of the cheapest health-care interventions that have saved more lives than any other drugs or therapies. Due to successful immunization programs we rarely hear about some of the common diseases of the early twentieth century including small pox and polio. Vaccination programs have also helped to increase food production notably poultry, cattle, and milk production due to lower incidence of infectious diseases in farm animals. Though vaccination programs have eradicated several diseases and increased the quality of life there are several diseases that have no effective vaccines. Currently there are no vacc...
Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Clinical Impact of Vaccine Development
The discovery and development of immunization has been a singular improvement in the health of mankind. This chapter reviews currently available vaccines, their historical development, and impact on public health. Specific mention is made in regard to the challenges and pursuit of a vaccine for the human immunodeficiency virus as well as the unfounded link between autism and measles vaccination. (Source: Springer protocols feed by Immunology)
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Erratum
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Source: Springer protocols feed by Immunology - April 8, 2016 Category: Allergy & Immunology Source Type: news

Investigating the Role of Toll-Like Receptors in Mouse Models of Gastric Cancer
Gastric cancer (GC) is the second most lethal cancer world-wide, and the poor overall 5-year survival rate of
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

The Use of MiRNA Antagonists in the Alleviation of Inflammatory Disorders
Toll-like receptors (TLR), a family of pattern-recognition receptors (PRRs) stimulated by pathogen-associated molecular patterns (PAMPs), generate antigen-triggered innate and adaptive immune responses. Recent studies have indicated that several small, regulatory RNAs, called microRNAs (miRNas), are induced by TLR activation in immune cells and that many microRNAs can control the inflammatory process and response to infection by positively or negatively regulating TLR signaling. Among these miRNAs, aberrant microRNA-155 (miR-155) has been implicated in diverse immune processes including the pathogenesis of several autoimmu...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Delineating the Role of Toll-Like Receptors in the Neuro-inflammation Model EAE
Experimental autoimmune encephalomyelitis (EAE) is the most relevant and commonly used animal model to study autoimmune demyelinating diseases like Multiple Sclerosis (MS). In EAE, the activation of CD4+ T-cells is considered to be the main trigger leading to inflammation and central nervous system (CNS) demyelination. Toll-like receptors (TLRs) are the most important and first class of pattern recognition receptors (PRRs) in innate immune system and play critical roles in initiating inflammatory responses and promoting adaptive immune responses due to their ability to recognize a wide range of pathogen associated molecula...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Investigating the Role of Toll-Like Receptors in Models of Arthritis
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation leading to tissue destruction and progressive loss of joint function. Here we describe two methods that can be used to assess the contribution of toll-like receptors (TLRs), and their potential ligands, to RA pathogenesis. We focus on the antigen-induced model of murine arthritis and human synovial tissue explant models. Both enable detection of TLR, and TLR ligand, expression, as well as investigation of the effect of inhibition of these molecules. Each offers a unique insight into disease; with murine models allowi...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Allergens and Activation of the Toll-Like Receptor Response
Pattern recognition receptors (PRRs) provide a crucial function in the detection of exogenous and endogenous danger signals. The Toll-like receptors (TLRs) were the first family of PRRs to be discovered and have been extensively studied since. Whilst TLRs remain the best characterized family of PRRs there is still much to be learnt about their mode of activation and the mechanisms of signal transduction they employ. Much of our understanding of these processes has been gathered through the use of cell based signaling assays utilizing specific gene-reporters or cytokine secretion based readouts. More recently it has become ...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Methods to Investigate the Role of Toll-Like Receptors in Allergic Contact Dermatitis
Allergic contact disease is a common inflammatory skin disease resulting from hyperresponsiveness to harmless nonprotein substances such as metals, fragrances, or rubber. Recent research has highlighted a prominent role of Toll-like receptors, particularly TLR4 in contact allergen-induced innate immune activation that crucially contributes to the pathogenesis of this disease. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Understanding the Role of Cellular Molecular Clocks in Controlling the Innate Immune Response
The importance of the 24-h daily cycle, termed circadian, on immune function has been highlighted by a number of recent studies. Immune parameters such as the response to bacterial challenge or immune cell trafficking change with time of day and disruption of circadian rhythms has been linked to inflammatory pathologies. We are beginning to uncover that the key proteins that comprise the molecular clock, most notably BMAL1, CLOCK, and REV-ERBα, also control fundamental aspects of the immune response. Given the ubiquitous nature of the molecular clock in controlling many other types of physiologies such as metabolism ...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Toll-Like Receptor Signalling and the Control of Intestinal Barrier Function
Epithelial barrier function and innate immunity are fundamental to the pathogenesis of inflammatory and infectious disease. Along with plasma membranes, epithelial cells are the primary cellular determinant of epithelial barrier function. The mechanism by which polarized epithelia form a permeability barrier is of fundamental importance to the prevention of many infectious and inflammatory diseases. Moreover, epithelial cells express Toll-like receptors (TLRs) which upon recognition of conserved microbial factors such as lipopolysaccharide (LPS) induce epithelial responses including epithelial cell proliferation, secretion...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Analysis of TLR-Induced Metabolic Changes in Dendritic Cells Using the Seahorse XFe96 Extracellular Flux Analyzer
Engagement of Toll-like receptors (TLRs) on dendritic cells (DCs) triggers the expression of a large set of genes involved in DC activation and maturation, which allow them to act efficiently as antigen-presenting cells. Recently, it has become clear that TLR signalling in DCs also results in dramatic metabolic changes that are integral to their changed biology. Here, we describe a detailed protocol on how DC metabolism can be studied after TLR stimulation using the 96-well format Extracellular Flux (XFe96) Analyzer from Seahorse Bioscience, a machine that allows one to simultaneously assess rates of oxidative phosphorylat...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Toll-Like Receptor-Dependent Immune Complex Activation of B Cells and Dendritic Cells
High titers of autoantibodies reactive with DNA/RNA molecular complexes are characteristic of autoimmune disorders such as systemic lupus erythematosus (SLE). In vitro and in vivo studies have implicated the endosomal Toll-like receptor 9 (TLR9) and Toll-like receptor 7 (TLR7) in the activation of the corresponding autoantibody producing B cells. Importantly, TLR9/TLR7-deficiency results in the inability of autoreactive B cells to proliferate in response to DNA/RNA-associated autoantigens in vitro, and in marked changes in the autoantibody repertoire of autoimmune-prone mice. Uptake of DNA/RNA-associated autoantigen immune...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Analysis by Flow Cytometry of B-Cell Activation and Antibody Responses Induced by Toll-Like Receptors
Toll-like receptors (TLRs) are expressed in B lymphocytes and contribute to B-cell activation, antibody responses, and their maturation. TLR stimulation of mouse B cells induces class switch DNA recombination (CSR) to isotypes specified by cytokines, and also induces formation of IgM+ as well as class-switched plasma cells. B-cell receptor (BCR) signaling, while on its own inducing limited B-cell proliferation and no CSR, can enhance CSR driven by TLRs. Particular synergistic or antagonistic interactions among TLR pathways, BCR, and cytokine signaling can have important consequences for B-cell activation, CSR, and plasma c...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

TLR Function in Murine CD4+ T Lymphocytes and Their Role in Inflammation
Toll-like receptor (TLR) signaling represents an evolutionary-conserved mechanism allowing for the rapid detection of broad molecular patterns that are common to different groups of pathogens. TLRs are traditionally associated with cells of the innate immune response where ligation of a TLR alone can lead to cellular activation and the initialization of an immune response. Cells of adaptive immunity, namely different classes of T and B lymphocytes, are also known to express a variety of TLRs. Conversely, the functional and signaling outcomes of TLRs are decidedly different in cells of the adaptive immune response. T lympho...
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Analysis of Post-transcriptional Gene Regulation of Nod-Like Receptors via the 3′UTR
Innate immune signaling is the front line of defense against pathogens, leading to an appropriate response of immune cells upon activation of their pattern recognition receptors (PRRs) by microbial products, such as Toll-like receptors (TLRs). Apart from transcriptional control, gene expression in the innate immune system is also highly regulated at the post-transcriptional level. miRNA or RNA-binding protein can bind to the 3′ untranslated region (UTR) of target mRNAs and affect their mRNA stability and translation efficiency, which ultimately affects the amount of protein that is produced. In recent years, a new gr...
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Determining the Function of Long Noncoding RNA in Innate Immunity
The advent of deep sequencing technologies has provided us with an unprecedented view of the human genome. Over 85 % of the genome is actively transcribed, yet we do not know the function of the vast majority of these RNA transcripts. Long noncoding RNAs (lncRNA) represent the largest group of RNA genes transcribed in the cell and currently there is limited experimental data supporting the functions of a very small proportion of these transcripts. lncRNA are expressed in a highly cell type specific manner and our interests involve understanding the role they play in innate immune signaling networks. In this chapter I will ...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Simple Methods to Investigate MicroRNA Induction in Response to Toll-Like Receptors
In this chapter, we describe simple methods to investigate microRNA (miRNA) induction in response to lipopolysaccharide, the ligand for Toll-Like Receptor-4 activation. In brief, we demonstrate how to investigate global miRNA induction and/or repression in bone marrow-derived macrophages using TaqMan MicroRNA Arrays, followed by methods to measure individual miRNAs and target mRNA expression. Moreover, we explain step-by-step instructions on how to modulate endogenous miRNA expression through the use of miRNA inhibitors and mimics as well as highlight how miRNA modulation can be used to confirm mRNA targeting via Luciferas...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Co-transcriptomic Analysis by RNA Sequencing to Simultaneously Measure Regulated Gene Expression in Host and Bacterial Pathogen
Intramacrophage pathogens subvert antimicrobial defence pathways using various mechanisms, including the targeting of host TLR-mediated transcriptional responses. Conversely, TLR-inducible host defence mechanisms subject intramacrophage pathogens to stress, thus altering pathogen gene expression programs. Important biological insights can thus be gained through the analysis of gene expression changes in both the host and the pathogen during an infection. Traditionally, research methods have involved the use of qPCR, microarrays and/or RNA sequencing to identify transcriptional changes in either the host or the pathogen. He...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Measuring Monomer-to-Filament Transition of MAVS as an In Vitro Activity Assay for RIG-I-Like Receptors
During viral infection, the innate immune RIG-I like receptors (RLRs) recognize viral double stranded RNA (dsRNA) and trigger filament assembly of the adaptor protein Mitochondrial Anti-viral Signaling protein (MAVS). The MAVS filament then activates anti-viral signaling events including the up-regulation of type I interferon expression. In recent years, much insight has been gained into how RLRs recognize dsRNA, but the precise mechanism of how activated RLRs stimulate MAVS filament formation remains less understood. In this chapter, we describe an in vitro reconstitution assay that we have previously developed to study t...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Flow Cytometry-Based Bead-Binding Assay for Measuring Receptor Ligand Specificity
In this chapter we describe a fluorescent bead-binding assay, which is an efficient and feasible method to measure interaction between ligands and receptors on cells. In principle, any ligand can be coated on fluorescent beads either directly or via antibodies. Binding between ligand-coated beads and cells can be measured by flow cytometry, which results in an easily quantifiable readout. Furthermore, it allows measuring of binding by specific cell subsets within a mixed cell population. Overall, this method is a convenient and easily standardized assay for measuring binding. (Source: Springer protocols feed by Immunology)
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Detection of Interaction Between Toll-Like Receptors and Other Transmembrane Proteins by Co-immunoprecipitation Assay
Toll-like receptors are type I membrane proteins and bind other membrane proteins often via a specific interaction between transmembrane domains. The co-immunoprecipitation assay is a widely used biochemical technique for assessing interactions among proteins in cell lysates or tissue extracts. By isolating a native protein complex with a specific antibody against a protein of interest, followed by western blotting with an antibody for a binding partner, the co-immunoprecipitation assay can be used to confirm a putative interaction between two proteins. The co-immunoprecipitation assay can also be combined with a proteomic...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Methods for Delivering DNA to Intracellular Receptors
We describe here optimized procedures for both electroporation and chemical transfection, including the centrifugation of chemical transfection reagent onto cells, which greatly increases the speed and strength of responses. Appropriate choice of DNA and use of these methods allow study of either the cytosolic DNA responses in isolation or the simultaneous stimulation of cytosolic receptors and the CpG DNA receptor toll-like receptor 9 (TLR9) in the endosomes. (Source: Springer protocols feed by Immunology)
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Assessing the Inhibitory Activity of Oligonucleotides on TLR7 Sensing
Aberrant sensing of self-nucleic acids by Toll-like receptor (TLR) 7, 8, or 9 is associated with several autoimmune disorders, including systemic lupus erythematosus (SLE), rheumatoid arthritis, psoriasis, or systemic sclerosis. In recent years, several classes of synthetic oligonucleotides have been shown to antagonize sensing of immunostimulatory nucleic acids by TLR7/8/9, indicating that these molecules could have therapeutic applications in such autoimmune diseases. Conversely, synthetic oligonucleotides used in therapeutic technologies such as antisense and microRNA inhibitors also have the potential to inhibit TLR7/8...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Using Confocal Microscopy to Investigate Intracellular Trafficking of Toll-Like Receptors
Toll-like receptors (TLR) survey the extracellular space, cytoplasm, and endosomal compartments for signs of infection or tissue injury. Over the past decade, it has become evident that TLR activation and signal transduction can be regulated by subcellular compartmentalization of both the receptors and their downstream signaling components. Immunofluorescence and/or overexpression of fluorescently “tagged”’ proteins teamed with confocal microscopy presents a powerful technique for studying the spatial organization of TLRs, their signaling mediators, and the dynamic processes they activate. This chapter de...
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Toll-Like Receptor Interactions Measured by Microscopic and Flow Cytometric FRET
Protein–protein interactions regulate biological networks. The most proximal events that initiate signal transduction frequently are receptor dimerization or conformational changes in receptor complexes. Toll-like receptors (TLRs) are transmembrane receptors that are activated by a number of exogenous and endogenous ligands. Most TLRs can respond to multiple ligands and the different TLRs recognize structurally diverse molecules ranging from proteins, sugars, lipids, and nucleic acids. TLRs can be expressed on the plasma membrane or in endosomal compartments and ligand recognition thus proceeds in different microenvi...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Bioinformatic Analysis of Toll-Like Receptor Sequences and Structures
Continual advancements in computing power and sophistication, coupled with rapid increases in protein sequence and structural information, have made bioinformatic tools an invaluable resource for the molecular and structural biologist. With the degree of sequence information continuing to expand at an almost exponential rate, it is essential that scientists today have a basic understanding of how to utilise, manipulate and analyse this information for the benefit of their own experiments. In the context of Toll-Interleukin I Receptor domain containing proteins, we describe here a series of the more common and user-friendly...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Toll-Like Receptors: Ligands, Cell-Based Models, and Readouts for Receptor Action
This chapter details Toll-like receptors (TLRs) and the tools available to study their biology in vitro. Key parameters to consider before exploring TLR action such as receptor localization, signaling pathways, nature of ligands and cellular expression are introduced. Cellular models (i.e., host cells and readouts) based on the use of cell lines, primary cells, or whole blood are presented. The use of modified TLRs to circumvent some technical problems is also discussed. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news