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The Fine Balance of Chemokines During Disease: Trafficking, Inflammation, and Homeostasis
The action of chemokines (or “chemotactic cytokines”) is recognized as an integral part of inflammatory and regulatory processes. Leukocyte mobilization during physiological conditions, trafficking of various cell types during pathological conditions, cell activation, and angiogenesis are among the target functions exerted by chemokines upon signaling via their specific receptors. Current research is focused in analyzing changes in chemokine/chemokine receptor patterns during various diseases with the aim to modulate pathological trafficking of cells, or to attract particular cell types to specific tissues. Thi...
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news

Studying Extracellular Signaling Utilizing a Glycoproteomic Approach: Lectin Blot Surveys, a First and Important Step
Successful innovative proteomic analysis is highly dependent on molecular biology techniques at the ­surveying and validation stage. This is because mass spectrometry (MS) analyses of complex samples are limited by their dynamic range for detection—so careful front-end sample preparation, fractionation, and enrichment are crucial to find biologically relevant signals in an extremely complex extracellular environment. Here, we share a very useful and simple front-end surveying methodology—lectin blotting—for proteomic analysis of glycosylation patterns—the most abundant posttranslational modifica...
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news

Live Cell Imaging of Chemotactic Dendritic Cell Migration in Explanted Mouse Ear Preparations
We present methods for the preparation of mouse ear sheets and their use in multidimensional confocal imaging experiments to monitor and analyze the directional migration of fluorescently labelled DCs through the dermis and into afferent lymphatic vessels. The assay provides a more physiological approach to study leukocyte migration than in vitro three-dimensional (3D) or 2-dimensional (2D) migration assays such as collagen gels and transwell assays. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news

Using Fluorescent Chemokine Uptake to Detect Chemokine Receptors by Fluorescent Activated Cell Sorting
Fluorescent activated cell sorting (FACS) is a powerful technique that allows rapid quantitative cell-by-cell analysis of the expression of multiple distinct proteins and the isolation of specific cell types from complex cellular mixtures. FACS-based detection of chemokine receptors is typically achieved using chemokine receptor-specific antibodies. However, this approach has some limitations, and, in our experience, many anti-chemokine receptor antibodies have poor signal-to-noise ratios and thus low sensitivity and reliability in FACS. Moreover, antibodies against some chemokine receptors are not commercially available, ...
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news

Epigenetic Regulation of Chemokine/Chemokine Receptor Expression
Epigenetic regulation of gene expression is an important event for normal cellular homeostasis. Gene expression may be “switched” on or “turned” off via epigenetic means through adjustments in DNA architecture. These structural alterations result from changes to the DNA methylation status in addition to histone posttranslational modifications such as acetylation and methylation. Drugs which can alter the status of these epigenetic markers are currently undergoing clinical trials in a wide variety of diseases, including cancer. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news

Visualizing Chemokine-Dependent T Cell Activation and Migration in Response to Central Nervous System Infection
In response to central nervous system (CNS) injury and infection, astrocytes, neurons, and CNS vasculature express several chemokines, including CCL21. Quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemical methods can quantify mRNA and protein expression. However, these methods do not quantify chemokine bioavailability and bioactivity, variables modified by many environmental factors including composition of the extracellular matrix (ECM). Here we illustrate how two-photon microscopy and carboxyfluorescein succinimidyl ester (CFSE or CFDA SE) labeling of T cells coupled with flow cytometry ca...
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news

Inflammatory Macrophage Migration in Experimental Autoimmune Encephalomyelitis
We describe a flow cytometric technique to monitor the migration of macrophages during EAE development. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - May 3, 2013 Category: Biochemistry Source Type: news

Photoreceptor Degeneration in Mice: Adeno-Associated Viral Vector-Mediated Delivery of Erythropoietin
The exogenous delivery of erythropoietin (EPO) and EPO derivatives (EPO-Ds) represents a valuable strategy to protect the retina from degeneration. In this chapter we describe a method to deliver EPO and the EPO derivative S100E in the light-damage model of induced retinal degeneration using adeno-­associated viral (AAV) vectors and to evaluate the functional and morphological protection of the retina from light damage. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 6, 2013 Category: Biochemistry Source Type: news

In Vivo Angiogenic Activity of Erythropoietin
The role of erythropoietin (Epo) has been demonstrated in tissues outside the hematopoietic system, including the cardiovascular system, where Epo promotes various effects in endothelial cells. Here, we have demonstrated the angiogenic capacity of recombinant human Epo (rhuEpo) in vivo, by means of the chick embryo chorioallantoic membrane (CAM) assay, a well-established in vivo assay to study angiogenesis and antiangiogenesis. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 6, 2013 Category: Biochemistry Source Type: news

ARA29 in a Rat Model of Inflammatory Pain
Chronic pain affects as many as one in five people. A proportion of patients with symptoms of neuropathic ­pain do not have clinical signs of any obvious tissue or nerve injury. Such patients include those with diffuse limb pain, back pain, and complex regional pain syndrome type 1. These patients remain a clinical enigma. However, through the development of the neuritis model, it has become apparent that local nerve inflammation in the absence of gross pathology (i.e., axonal degeneration and demyelination) may underlie part of the mechanisms of pain. In this chapter, we describe a method to induce the neuritis model....
Source: Springer protocols feed by Biochemistry - March 6, 2013 Category: Biochemistry Source Type: news

Intra-epidermal Nerve Fibers Density and Nociception in EPO-Treated Type 1 Diabetic Rats with Peripheral Neuropathy
Small-diameter nerve fibers, which subserve nociception, can be affected early in peripheral neuropathies, although their injury may not be detectable by routine neurophysiologic tests. On the other hand, skin biopsy has proved to be a reliable tool to examine nonmyelinated nerve fibers, as assessed by the quantification of intra-epidermal nerve fiber (IENF) density not only along with the degenerative process but, noteworthy, IENF density could be very helpful in evaluating drug efficacy such as erythropoietin (EPO) treatment. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 6, 2013 Category: Biochemistry Source Type: news

Assessment of Allodynia Relief by Tissue-Protective Molecules in a Rat Model of Nerve Injury-Induced Neuropathic Pain
Neuropathic pain following nerve injury is a chronic disease characterized by allodynia and hyperalgesia of either mechanical or thermal origin. The mechanism underlying this disease is poorly understood leading to pharmacologic and physiotherapeutic control that is often insufficient. In this chapter, we describe a method to induce nerve injury in rats to create a robust animal model for studying neuropathic pain. Additionally we describe a method to follow up on animals in the process of testing treatments for efficacy in alleviating allodynia by testing for both mechanical and thermal allodynia with reproducible results...
Source: Springer protocols feed by Biochemistry - March 6, 2013 Category: Biochemistry Source Type: news

Deciphering the Intracellular Signaling of Erythropoietin in Neuronal Cells
The search for potential drugs to treat neurodegenerative diseases has been intense in the last two decades. Among many candidates, erythropoietin (EPO) was identified as a potent protectant of neurons suffering from various adverse conditions. A wide array of literature indicates that endogenous or exogenous recombinant human erythropoietin and its variants activate cell signaling that initiates survival-promoting events in neurons and neuronal cells. This chapter gives an overview of the pro-survival signaling induced by endogenous and exogenous erythropoietin in vitro and in vivo and provides methods to further investig...
Source: Springer protocols feed by Biochemistry - March 6, 2013 Category: Biochemistry Source Type: news

Therapeutic Efficacy of Erythropoietin in Experimental Autoimmune Encephalomyelitis in Mice, a Model of Multiple Sclerosis
Erythropoietin (EPO) has neuroprotective effects in many models of damage and disease of the nervous system where neuroinflammation plays a substantial role, including experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Since the first pioneering studies, in which EPO was shown to protect rats with acute EAE mainly by inhibiting inflammation, many other studies have pointed out other mechanisms of protection, including oligodendrogenesis and inhibition of axonal damage. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 6, 2013 Category: Biochemistry Source Type: news

Oligonucleotide Recombination Enabled Site-Specific Mutagenesis in Bacteria
Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligonucleotide recombination is one type of recombineering that uses ssDNA oligonucleotides to direct chromosomal mutations. Oligo recombination occurs without addition of any exogenous functions, making this approach potentially useful in many different bacteria. Here we describe the basic technique for constructing a site-specific genomic mutation in Pseudomonas syringae. (Source: Springer protoc...
Source: Springer protocols feed by Biochemistry - February 21, 2013 Category: Biochemistry Source Type: news