Methods in molecular biology
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Macrophages play a key role in the innate immune response and help to direct the acquired immune response. Early in the innate immune response, they produce reactive oxygen species and pro-inflammatory cytokines and chemokines to drive inflammation and are referred to as "classically activated" or "killer" macrophages (M1). During the resolution phase of inflammation, they switch to what is known as an "alternatively activated" phenotype or "healer" macrophage (M2) and contribute to debris scavenging, angiogenesis, and wound healing. ⋯ M1 macrophages also produce relatively higher levels of pro-inflammatory IL-12 and lower levels of anti-inflammatory IL-10 relative to M2 macrophages. In this chapter, we describe in vitro derivation of polarized bone marrow macrophages and methods to analyze the resulting phenotype including Q-PCR, Western blotting, and enzyme assays to determine argI and iNOS expression and activity, as well as production of IL-12p40 and IL-10 and determination of IL-12/IL-10 ratios. Production of iNOS, NO, IL-12p40, and IL-10 are measured after treatment with LPS.
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The various biochemical cascades that follow primary brain injury result in secondary brain injury which can adversely affect the clinical outcome. Over the last few years it has been well established that molecules like erythropoietin (Epo) have a neuroprotective role in experimental traumatic brain injury (TBI). Epo is shown to produce this effect by modulating multiple cellular processes, including apoptosis, inflammation, and regulation of cerebral blood flow. ⋯ Peptides that mimic a portion of the Epo molecule, including Helix B surface peptide and Epotris, have also been developed to isolate the neuroprotective activities. The TBI model in rodents most commonly used to study the effect of Epo and these derivatives in TBI is controlled cortical impact injury, which is a model of focal contusion following a high velocity impact to the parietal cortex. Following TBI, rodents are given Epo or an Epo derivative vs. placebo and the outcome is evaluated in terms of physiological parameters (cerebral blood flow, intracranial pressure, cerebral perfusion pressure), behavioral parameters (motor and memory), and histological parameters (contusion volumes, hippocampus cell counts).
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Peptide microarray technology can be used to identify substrates for recombinant kinases, to measure kinase activity and changes thereof in cell lysates and lysates from fresh frozen (tumor) tissue. The effect of kinase inhibitors on the kinase activities in relevant tissues can be investigated as well. The method for performing experiments on dynamic peptide microarrays with real-time readout is described, as well as the influence of assay parameters and suggestions for optimization of experiments.
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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. ⋯ The signaling pathways involved in EPO are multiple; some are well known whereas others are still under intense investigation and few are observed in very specific cell types. It is important to note that neuronal signaling events triggered by EPO are still incomplete and require further research. Therefore, excellent review articles that explore specific EPO-signaling events are referenced.
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Hypertrophic scar (HTS) represents the dermal equivalent of fibroproliferative disorders that occur after injury involving the deep dermis while superficial wounds to the skin heal with minimal or no scarring. HTS is characterized by progressive deposition of collagen that occurs with high frequency in adult dermal wounds following traumatic or thermal injury. Increased levels of transforming growth factor-β1 (TGF-β1), decreased expression of small leucine-rich proteoglycans (SLRPs), and/or fibroblast subtypes may influence the development of HTS. ⋯ Studying the characteristics of superficial dermal injuries that heal with minimal scarring will help us identify therapeutic approaches for tissue engineering and wound healing. In addition, our ability to develop novel therapies for HTS is hampered by limitations in the available animal models used to study this disorder in vivo. We also describe a nude mouse model of transplanted human skin that develops a hypertrophic proliferative scar consistent morphologically and histologically with human HTS, which can be used to test novel treatment options for these dermal fibrotic conditions.