Bifunctional Antibody Fragment-Based Fusion Proteins for the Targeted Elimination of Pathogenic T-Cell Subsets
Pathogenic effector T cells are key contributors to autoimmune diseases such as systemic lupus erythematosus (SLE). General inhibition of T cells using, e.g., methotrexate, prednisolone, or TNF blockers, has prominent therapeutic effects frequently at the cost of severe long-term side effects and toxicity. Therefore, targeted strategies that can selectively inhibit or eliminate pathogenic T cells are sought after as a new approach to safely block perpetual inflammatory T-cell responses and inhibit concomitant progressive tissue destruction. Of particular interest in this respect is the use of the so-called single-chain fra...
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

TLC Immunostaining for Detection of “Antiphospholipid” Antibodies
Thin-layer chromatography (TLC) is a nonquantitative technique, which has been employed in the detection of antiphospholipid (aPL) antibodies. Antiphospholipid syndrome (APS) is the most frequently acquired thrombophilia, characterized by thrombosis and obstetric manifestations associated to an autoimmune trait, represented by the positivity of antiphospholipid (aPL) antibodies. Immunoassays for anticardiolipin (aCL) and anti-β2 glycoprotein I (aβ2GPI) antibodies and clotting tests for lupus anticoagulant (LA) represent the standard tests for the routine detection of aPL. The term “seronegative APS” h...
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Induced Murine Models of Systemic Lupus Erythematosus
Induced mouse models of systemic lupus erythematosus (SLE) have been developed to complement the spontaneous models. This chapter describes the methods used in the pristane-induced model and the chronic graft-versus-host disease (cGVHD) model, both of which have been extensively used. We will also outline the specific mechanisms of systemic autoimmunity that can be best characterized using each of these models. (Source: Springer protocols feed by Immunology)
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Measuring Interferon Alpha and Other Cytokines in SLE
We describe a functional assay to measure type I IFN activity in SLE plasma and have also measured the response of peripheral blood cells to that cytokine family. This method can be scaled to assess IFN functional activity, as well as activity and cellular response to other cytokines, in relation to cellular and serologic parameters relevant to SLE. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

Detection of SLE Antigens in Neutrophil Extracellular Traps (NETs)
Neutrophils are sentinel cells of the innate immune system with a primary role of clearing extracellular pathogens. The release of weblike structures decorated with granular proteins called neutrophil extracellular traps (NETs) has recently been implicated in the pathogenesis of inflammatory and autoimmune diseases. Indeed, NETs may represent an important source of autoantigens and immunostimulatory proteins in systemic lupus erythematosus (SLE). In this chapter, we describe protocols to isolate human peripheral neutrophils, to generate and isolate NETs, and to detect SLE antigens in NETs using immunofluorescence and immun...
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Detection and Characterization of Autoantibodies Against Modified Self-Proteins in SLE Sera After Exposure to Reactive Oxygen and Nitrogen Species
There are over 120 types of autoantibodies found in the blood of SLE patients against cellular and extracellular components in both their native and posttranslationally modified forms. In recent years, these autoantibodies have provoked interest as initiators of pathology and as biomarkers of disease activity. Often, the host antigens employed in lab-based and commercially developed immunoassays use non-human antigen or non-modified host antigen as a probe for autoantibodies. Here, we describe methods to posttranslationally modify host antigens, which better represent the antigen recognized by autoantibodies in vivo. This ...
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

Generation of Self-Peptides to Treat Systemic Lupus Erythematosus
Synthetic peptides are attracting increasing attention as therapeutics. Despite their potential, however, only a few selected peptides have been able to enter in clinical trials for chronic autoimmune diseases and systemic lupus erythematosus (SLE) in particular. Here, we describe and discuss a series of assays, which may help in characterizing valuable candidate peptides that were applied in our laboratory to develop the lupus P140 peptide program. The different steps of selection include the choice of the initial autoantigen, the design, synthesis and purification of peptides, their preliminary screen by measuring cytoki...
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Measurement of Malondialdehyde, Glutathione, and Glutathione Peroxidase in SLE Patients
Oxidative stress contributes to chronic inflammation of tissues and plays a central role in immunomodulation, which may lead to autoimmune diseases such as systemic lupus erythematosus (SLE) and antiphospholipid syndrome. Markers of oxidative damage include malondialdehyde (MDA), antioxidant scavengers as glutathione (GSH), and glutathione peroxidase (GSH Px), which all correlate well with SLE disease activity. Amelioration of some clinical manifestations of SLE may be expected by targeting lipid peroxidation with dietary or pharmacological antioxidants. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

Evaluating a Particular Circulating MicroRNA Species from an SLE Patient Using Stem-Loop qRT-PCR
Systemic lupus erythematosus (SLE) is a complex autoimmune disease, and correct judgment of SLE activity is very important in guiding precise clinical treatment. Circulating microRNAs (miRNAs) could serve as potential biomarkers of disease activity or status in SLE, and here we describe a modified qRT-PCR method for detecting them. Stem loop has become one of the most powerful methods for determining miRNA expression because it is highly sensitive and accurate and requires only small amount of sample. In this chapter, we focus on a stem-loop reverse transcription-bound SYBR green qRT-PCR protocol for evaluating a particula...
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

Laboratory Tests for the Antiphospholipid Syndrome
Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by recurrent vascular thrombosis (VT) and/or pregnancy morbidity (PM) in the presence of persistent antiphospholipid antibodies (aPL), detected by lupus anticoagulant (LA), anticardiolipin (aCL) antibody, and/or anti‐β2 glycoprotein I (aβ2GPI) antibody assays. These aPL, considered to be diagnostic markers and pathogenic drivers of APS, are a heterogeneous group of antibodies directed against anionic phospholipids, phospholipid-binding plasma proteins, and phospholipid–protein complexes. Although APS is currently considered as a s...
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Microarray Technology for Analysis of MicroRNA Expression in Renal Biopsies of Lupus Nephritis Patients
This article offers a technological overview of microarray technology for analysis of microRNA gene expression in kidney biopsies from SLE patients. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

Meta-analysis as a Diagnostic Tool for Predicting Disease Onset and/or Activity in Systemic Lupus Erythematosus
Systemic lupus erythematous (SLE) is a relatively rare disorder with prevalence rates between 5 and 50 per 100,000 population. This means performing any epidemiological analysis in a specific research center is difficult, due to the low number of cases within any one location. There is a need for biomarkers and diagnostic aids to monitor SLE disease activity and severity prior, during, and after treatment. Many specialist lupus clinics worldwide have published trials following in detail small numbers of patients that have been monitored for a disease biomarker, e.g., an autoantibody against a self-molecule in prospective a...
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

Isolation, Polarization, and Expansion of CD4+ Helper T Cell Lines and Clones Using Magnetic Beads
Autoreactive CD4+ helper T cells specific for a range of nucleoprotein-derived autoantigens are an important feature of systemic lupus erythematosus, driving B cell differentiation and autoantibody production and contributing to the inflammatory lesions caused by immune complex deposition. Several peptide epitopes from nucleoprotein antigens have been identified and offer a means selectively to manipulate T cell responses by skewing toward a profile of cytokines that is less pro-inflammatory. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

Pathology of Systemic Lupus Erythematosus: The Challenges Ahead
Many studies have explored the pathology of systemic lupus erythematosus (SLE), an autoimmune rheumatic disorder with a striking female predominance. Numerous autoimmune phenomena are present in this disease, which ultimately result in organ damage. However, the specific cellular and humoral mechanisms underlying the immune dysfunction are not yet fully understood. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news

The Lupus Biomarker Odyssey: One Experience
The last decade has witnessed an explosion in efforts to discover and validate lupus biomarkers. The currently steep trajectory of this progress is unprecedented. However, advances in the lupus biomarker field remain fewer and slower than physicians, patients, and pharmaceutical companies have hoped for. This chapter will review the challenges confronted by physicians and scientists in pursuit of lupus biomarkers and will present our experience on this path and specific efforts to surmount some of the obstacles in this endeavor. A comprehensive review of the current landscape in lupus biomarker research has recently been p...
Source: Springer protocols feed by Immunology - February 7, 2014 Category: Allergy & Immunology Source Type: news