Springer protocols feed by Immunology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.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...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news
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
Analysis of Post-transcriptional Gene Regulation of Nod-Like Receptors via the 3 & prime;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 & prime; 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 ...
Source: Springer protocols feed by Immunology - January 6, 2016 Category: Allergy & Immunology Source Type: news
Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures
Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood–brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes. ...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Measuring the Uptake and Transactivation Function of HIV-1 Tat Protein in a Trans-cellular Cocultivation Setup
HIV-1 Tat protein is secreted from infected cells and is endocytosed by uninfected bystander cells. Subsequently, Tat is translocated to the nucleus and binds to promoters of host cell genes, increasing the production of inflammatory host cytokines and chemokines. This inflammatory activation of uninfected cells by HIV-1 Tat protein contributes to the overall inflammatory burden in the central nervous system (CNS) that leads to the development of HIV-associated neurocognitive disorders (HAND). Here we describe methods to evaluate the uptake and transcriptional impact of HIV-1 Tat on uninfected cells by using a trans-cellul...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Protocol for Detection of HIV-Tat Protein in Cerebrospinal Fluid by a Sandwich Enzyme-Linked Immunosorbent Assay
The human immunodeficiency virus (HIV) transactivator of transcription (Tat) is a virally produced protein that is required for efficient viral replication. Once formed inside an infected cell, Tat is secreted into the extracellular space where it has pathophysiological consequences on cells it interacts with. Tat has been demonstrated to be neurotoxic and is produced even under the pressures of anti-retroviral therapy; therefore Tat is suspected to contribute to the development of HIV-associated neurocognitive disorders. In this chapter, we describe a sandwich enzyme-linked immunosorbent assay protocol for the detection o...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot
HIV-1 Tat is efficiently secreted by HIV-1-infected or Tat-transfected cells. Accordingly, Tat concentrations in the nanomolar range have been measured in the sera of HIV-1-infected patients, and this protein acts as a viral toxin on bystander cells. Nevertheless, assaying Tat concentration in media or sera is not that straightforward because extracellular Tat is unstable and particularly sensitive to oxidation. Moreover, most anti-Tat antibodies display limited affinity. Here, we describe methods to quantify extracellular Tat using a sandwich ELISA or Western blotting when Tat is secreted by suspension or adherent cells, ...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Proteomic Characterization of Exosomes from HIV-1-Infected Cells
Proteomics has increasingly become an invaluable tool to characterize proteomes from various subcellular compartments. Here, we describe a quantitative proteomics method using the technique of Stable Isotope Labeling by Amino acids in Cell culture (SILAC) to analyze the effects of HIV infection on host exosomal proteomes. The procedure, described below, involves differential isotope labeling of cells, exosome purification, mass spectrometric quantification, and various bioinformatic analyses/verifications. Although this chapter focuses on analyzing the effects of HIV-1 infection on the exosomal proteome, the protocol can e...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
The Proteomic Characterization of Plasma or Serum from HIV-Infected Patients
Proteomics holds great promise for uncovering disease-related markers and mechanisms in human disorders. Recent advances have led to efficient, sensitive, and reproducible methods to quantitate the proteome in biological samples. Here we describe the techniques for processing, running, and analyzing samples from HIV-infected plasma or serum through quantitative mass spectroscopy. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Analysis of ABCA1 and Cholesterol Efflux in HIV-Infected Cells
Cholesterol is an essential component of the cellular membranes and, by extension, of the HIV envelope membrane, which is derived from the host cell plasma membrane. Depletion of the cellular cholesterol has an inhibitory effect on HIV assembly, reduces infectivity of the produced virions, and makes the cell less susceptible to HIV infection. It is not surprising that the virus has evolved to gain access to cellular proteins regulating cholesterol metabolism. One of the key mechanisms used by HIV to maintain high levels of cholesterol in infected cells is Nef-mediated inhibition of cholesterol efflux and the cholesterol tr...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Improved Methods to Detect Low Levels of HIV Using Antibody-Based Technologies
Persistence of latent virus represents a major barrier to eradicating HIV even in the current antiretroviral therapy era. A critical limitation to eliminating these viral reservoirs is the lack of reliable methods to detect, quantify, and characterize cells harboring low levels of virus. However, recent work of several laboratories indicates that PCR and viral amplification based technologies underestimate or overestimate the size of the reservoirs. Thus, new technologies and methodologies to detect, quantify, and characterize these viral reservoirs are necessary to monitor and eradicate HIV. Recent developments in imaging...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
LGIT In Vitro Latency Model in Primary and T Cell Lines to Test HIV-1 Reactivation Compounds
Persistent latent HIV-1 reservoirs pose a major barrier for combinatorial antiretroviral therapy (cART) to achieve eradication of the virus. A variety of mechanisms likely contribute to HIV-1 persistence, including establishment of post-integration latency in resting CD4+ T-lymphocytes, the proliferation of these latently infected cells, and the induced or spontaneous reactivation of latent virus. To elucidate the mechanisms of latency and to investigate therapeutic strategies for reactivating and purging the latent reservoir, investigators have developed in vitro models of HIV-1 latency using primary CD4+ T-lymphocytes an...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
