Flowjo facs12/14/2023 It also enables the comparison of Ca 2+ influx kinetics in more than one lymphocyte subsets of the same sample simultaneously. Thus the inhibition of Kv1.3 channels does not seem to be specific enough in peripheral human RA lymphocytes, since anti-inflammatory Th2 cells are also affected to a noteworthy extent ( 7).įlow cytometry is a suitable method for the sequential determination of cyt in millions of stimulated lymphocytes over a time period. However, our earlier trial on human samples from patients with RA showed that Th2 and particularly CD8 cells are inhibited more dominantly than Th1 and CD4 cells. In addition, no clinical side-effects or signs of toxicity were identified during the trial. The animals treated with the specific Kv1.3 channel blockers showed significantly less joint deviations and significant improvement in radiological and histopathological findings ( 6). also evaluated the therapeutic effects of Kv1.3 inhibitors in a rat model of RA. Consequently, Kv1.3 inhibitors primarily suppress the activation and proliferation of the autoantigen-specific TEM cells, while sparing other classes of T cells ( 5). According to Beeton et al., disease-associated autoreactive T cells are mainly CCR7− CD45RA− effector memory T cells (TEM cells) with elevated Kv1.3 channel expression, in comparison with naive and central memory T cells (TCM cells) that express low levels of Kv1.3 channels. This observation offers a unique opportunity for targeted pharmacological intervention with improved specificity from existing drugs against disease-causing autoreactive lymphocytes. Specific inhibition of these channels results in diminished Ca 2+ influx and a lower level of lymphocyte activation and proliferation ( 3, 4). Voltage-gated Kv1.3 and calcium-dependent IKCa1 K + channels play a pivotal role in the maintenance of cyt, since they sustain the electrochemical potential gradient needed for further Ca 2+ entry by the efflux of K + from the cytoplasm ( 1, 2). This is followed by further Ca 2+ entry from the extracellular space through the store-operated calcium release activated calcium (CRAC) channels. The engagement of the TCR/CD3 complex upon antigen presentation leads to the activation of several transmembrane signaling pathways which result in Ca 2+ release from intracellular stores. The transient increase of the cytoplasmic free calcium level ( cyt) in T lymphocytes is an indispensable part of this process. The short-term activation of T lymphocytes, especially that of autoreactive T cells, plays a key role in initiating and maintaining most autoimmune reactions, including that observed in rheumatoid arthritis (RA). © 2013 International Society for Advancement of Cytometry The function fitting algorithm applied by FacsKin is suitable to provide a common basis for evaluating and comparing flow cytometry kinetic data. With the algorithm of function fitting instead of smoothing, more statistically significant differences of potassium channel inhibition between the two RA groups could be demonstrated. While initially Th1 cells are less sensitive to the inhibition of Kv1.3 and IKCa1 channels in RA, their sensitivity increases along with the duration of the disease. Th2 cells of patients with established RA react slower to activating stimuli, whereas CD8 cells show a faster reaction than in patients with recently diagnosed RA. We assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2, and CD8 cells applying an approach based on smoothing of median fluorescence values (FlowJo) and an algorithm based on function fitting (FacsKin). We took peripheral blood samples from nine patients with recently diagnosed and six patients with established RA. We aimed to compare peripheral T lymphocyte calcium influx kinetics upon activation in patients with recently diagnosed and established RA, and to demonstrate the differences in analysis of kinetic flow cytometry data when using two different algorithms. Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation. The transient increase of the cytoplasmic free calcium level in T lymphocytes plays a key role in initiating and maintaining the autoimmune reaction in rheumatoid arthritis (RA).
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