Normal activity of inflammatory and anti-inflammatory mechanism of immune system is crucial for successful regulation of inflammation caused by infections and injuries. Inflammation is normally regulated by the right proportion of inflammatory and anti-inflammatory cytokines, so excessive systemic inflammation and sepsis are excluded. Secretion of inflammatory and anti-inflammatory cytokines is regulated by humoral system (cortisol) through hypothalamic-pituitary-adrenal axis and nervous system through vagus nerve. In the last 20 years it has been reported about many different discoveries by laboratories around the world, which lead to understanding of the mechanism and meaning of the newly discovered cholinergic anti-inflammatory pathway, which is supervised by vagus nerve. Cholinergic anti-inflammatory pathway is based on activation of vagus nerve which transducts the signal to splenic nerve, which secretes noradrenaline, the activator of specific subset of T lymphocytes in spleen. T lymphocytes then start secreting acetylcholine, which binds to α7 nicotinic receptors on macrophages and prevents the secretion of inflammatory cytokines through different molecular mechanisms. Determination of alpha-7 nicotinic receptor mRNA expression in human peripheral blood could present a simple method to determine the activity of human cholinergic anti-inflammatory response during different diseases. We have suggested methodological protocol to establish routine measurement of alpha-7 nicotinic receptor mRNA expression, which includes isolation of PBMC from human peripheral blood, isolation of whole RNA and two-level RT-PCR. This could become a useful methodological tool for clinical researches and in time also a routine diagnostic biomarker for determining the condition of cholinergic anti-inflammatory response. Findings about protocols for isolation of PBMC and RT-PCR were compared to the methodology of isolation of PMBC, which was used for routine measurement of alpha-7 nicotinic receptor mRNA expression in peripheral blood of different patients, described in different researches. Newly discovered and rarely used method for isolation of specific cells of PBMC is immunomagnetic negative selection with EasySep, which is based on labelling blood cells with monoclonal antibodies and suspension of magnetic particles. This method seems to be very useful, because it sufficiently and quickly enables isolating of specific subsets of lymphocytes and monocytes. With the suggested method and qPCR the differences between alpha-7 nicotinic receptor mRNA expression in patients with different forms and intensities of inflammation and healthy people would like to be found.
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