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Microglia are multifunctional immune cells of the brain executing various functions and rapidly responding to pathological insults. Brain injury, hypoxia, infection or aberrant protein accumulation may lead to chronic infl ammation with a progressive shift in microglia function towards infl ammatory phenotype and accumulation of immune cells. Under pathological conditions, the interplay of extrinsic signals directs microglia towards neuroprotective or detrimental phenotype. Molecular mechanisms of initiation, progression and termination of microglia-initiated infl ammatory responses in the brain, in particular gene networks and signaling pathways are poorly understood. Characterization of the global transcriptome of microglia exposed to infl ammatory or cytoprotective signals and analysis of signalling pathways revealed differences in expression of genes encoding cytokines/ chemokines and transcription regulators. Identifi cation of signalling pathways contributing to discrete microglia phenotypes and discovery of transcription regulators which may serve as “master switches” for induction of an infl ammatory phenotype, will allow to target specifi c functions of microglia. Therapeutic approaches targeting signal transduction in microglia will be discussed. A greater understanding of microglia functions coupled with advances in pharmacology and gene therapy will support development of functionally “engineered” microglia able to convey neuroprotection.
Adenosine 5'-triphosphate (ATP) and adenosine are the crucial endogenous signaling molecules in immunity and inflammation. In this study we identified the source of extracellular adenosine in human B lymphoblasts, and evaluate the ATP release and metabolism. We observed that the B cells continuously released substantial quantities of ATP (35 pmol/106 cells) when subjected to slow motion in the incubation medium. The adenosine level in the B cell incubation medium was very low, and increased (5-fold) upon inhibition of adenosine deaminase activity with 10 µM of 2-deoxycoformycin (DCF). Inclusion of an inhibitor of equilibrate nucleoside transport (nitrobenzylthioinosine) in the incubation medium in the presence of DCF resulted in the elevation of adenosine level by 9-fold. Inhibition of ecto-ATPase activity with 100 µM of ARL67156 was associated with a 2-fold increase of the extracellular ATP level and a 3-fold decrease of adenosine concentration in the cell culture media. Inclusion of ,ß-methyleneadenosine 5'-diphosphate, a selective inhibitor of ecto-5'-nucleotidase in the incubation medium resulted in a significant decrease (7-fold) the adenosine concentration. In conclusion, our results indicate that ATP released from the B cell is the primary source of peripheral adenosine, and that the activities of ecto enzymes and the efficiency of Ado uptake through the nucleoside transporters determine the Ado level on the B cell surface.
The paper describes the immunological response in the matter of percentage of T cells (receptor CD5+) and subpopulations (Th with receptor CD4+, Tc/Ts with receptor CD8+, T with receptor CD25+) and B cells with receptor CD19+, as well as the percentage of apoptotic granulocytes and lymphocytes, in rabbits experimentally infected with the Hagenow strain of the RHD virus. The material chosen for the experiment is special, as among all strains of RHD virus, there are only two strains which carry the variable haemagglutination capacity of red cells. The results of the study show that the Hagenow strain gives an untypical picture of T and B lymphocytes, whereas the results in inducing apoptosis seems to corespond with previous data, confirming the inclusion of apoptosis from 4 h p.i. and the intensity of the phenomenon being higher in granulocytes.
Increased susceptibility to infections can be a consequence of altered function of immune cells including neutrophils. The goal of the study was to evaluate the process of neutrophil activation via Ca2+-mediated signal. The study was performed on isolated peripheral blood neutrophils obtained from 41 children with recurrent infections (21 girls, 20 boys) 3-10 years old with more than five episodes of respiratory tract infection (RI) per year and from a control group of 30 healthy children age and sex matched, free from allergic, immune and hematological disorders. Neutrophils were activated by bacterial peptide fMLP, opsonized zymosan (OZ), and phorbol myristat acetate (PMA). The kinetics of the intracellular Ca2+ concentration i[Ca2+] was assessed by flow cytometry (Coulter Epics XL) with the use of Fluo3 and Fura Red fluorescent dyes. Data were collected in histograms displaying Fluo3 fluorescence vs. time and Fura Red fluorescence vs. time and the mean channels of fluorescence intensity were used for calculations. fMLP and OZ-induced Ca2+ mobilization lasted shorter in the RI group (P<0.05). The peak influx of free Ca2+ and i[Ca2+] in the resting state after stimulation with fMLP were lower in the patients (P<0.05). In the RI group stimulation with OZ was delayed compared with that in the control group (P<0.01). In response to PMA, i[Ca2+] decreased faster. The kinetic slopes of i[Ca2+] in both groups examined differed statistically at all points measured. A decrease in i[Ca2+] after PMA stimulation was greater (P<0.01) and lasted longer in the RI group. We conclude that increased sensitivity to infections in RI children may be related to the disturbance in neutrophil activation that is mediated by changes in i[Ca2+] with the subsequent production of free oxygen radicals. Such a disturbance may be inheritable or secondary to infection and antibiotic therapy.
Development of neuropathic pain is accompanied by many changes in immune and glial cells. These changes correspond to activation of immune and glial cells that have been shown to influence the opioid effectiveness and can be modulated by minocycline (a potent inhibitor of microglial activation). In earlier study we have demonstrated that function of opioidergic neurons may be modulated by the immune system. These changes have been shown to be responsible for the efficacy of opioids. The aim of our study was to examine the effect of the minocycline-triggered inhibition of microglia activation on the injury-induced changes and the efficacy of mu and delta opioid receptor ligands in a rat model of neuropathic pain (chronic constriction injury to the sciatic nerve). In cell culture studies, we examined the influence of opioids (morphine, DAMGO, DPDPE, deltorphin II) on activated primary cultured rat microglia by using MTT and/or NO assays. All experiments were performed according to the IASP recommendations and were approved by a local Bioethics Committee. On the spinal cord level the injury to the sciatic nerve induced an up-regulation of IL-1beta, IL-6 expression, CX3CR1 and C1q (marker of microglia, macrophage and leukocyte activation). Chronic administration of minocycline not only diminished neuropathic pain-related behavior and C1q-positive cell activation, but also attenuate the changes in proinflammatory factors like IL1beta, IL-6 and CX3CR1 in the spinal cord and DRG. In in vivo experiments, the analgesic effects of mu-opioid (morphine and DAMGO), but not delta-opioid (DPDPE, deltorphin II) receptor ligands were lower in the rats under neuropathic pain. Moreover, the analgesic effects of morphine and DAMGO, but not DPDPE and deltorphin II were significantly potentiated by minocycline chronic administration. Our in vitro findings that non-stimulated microglia cells respond differently to opioids in comparisons with stimulated cells as measured by MTT and/or NO assays, corresponded well with the results of in vivo studies. Our study underlined that inhibition of microglial activation could differently influence analgesic effects of mu- but not delta-opioid ligands in injury-induced pathologies, which may influence the effect of various opioid drugs used in chronic pain therapy.
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