Telomere length is an important indicator of proliferative cell history and potential. Decreasing telomere length in the cells of an immune system can indicate immune aging in immune-mediated and chronic inflammatory diseases. Quantitative fluorescent in situ hybridization (Q-FISH) of a labeled (CTA[Formula: see text] peptide nucleic acid probe onto fixed metaphase cells followed by digital image microscopy allows the evaluation of telomere length in the arms of individual chromosomes. Computer-assisted analysis of microscopic images can provide quantitative information on the number of telomeric repeats in individual telomeres. We developed new software to estimate telomere length. The MeTeLen software contains new options that can be used to solve some Q-FISH and microscopy problems, including correction of irregular light effects and elimination of background fluorescence. The identification and description of chromosomes and chromosome regions are essential to the Q-FISH technique. To improve the quality of cytogenetic analysis after Q-FISH, we optimized the temperature and time of DNA-denaturation to get better DAPI-banding of metaphase chromosomes. MeTeLen was tested by comparing telomere length estimations for sister chromatids, background fluorescence estimations, and correction of nonuniform light effects. The application of the developed software for analysis of telomere length in patients with rheumatoid arthritis was demonstrated.
We analyzed telomere length of individual chromosomes in peripheral blood lymphocytes of healthy individuals and patients with rheumatoid arthritis. Quantitative fluorescent in situ hybridization and subsequent computer analysis of metaphase chromosomes showed that distribution of telomere length on individual chromosomes is different under normal and pathological conditions. Patients with rheumatoid arthritis had significantly shorter chromosome 4p telomeres, which can be essential for pathogenesis of this multifactorial disease. Additionally, disease activity inversely correlated with telomere length on chromosome 10p carrying genes involved in T cell differentiation and proliferation.
BackgroundTelomeres are nucleoprotein structures,that protect the ends of chromosomes during cell divisions [1,2,3]. Previously it was found, that average telomere length in immune cells is reduced in atopic and autoimmunity disorders.This fact indicates an early immune aging in immune-mediated diseases [4,5]. The distribution of telomere repeats on different chromosomes has an individual telomere profile in humans [6] and may be a congenital feature, that accelerates immunosenescence.ObjectivesThe purpose of this study was to evaluate the length of telomeres in the arms of individual chromosomes in patients with RA and healthy donors.MethodsThe study included 6 patients with RA and 6 healthy donors (the median age 51.5 (50–54) and 51.5 (49–53) years respectively). Metaphase spreads obtained from PBMCs were used in this study. Written informed consent was obtained from each person enrolled in the study. At the time of sampling, RA inpatients characterized with acute exacerbation of the disease received treatment at the Clinic of Immunopathology,Novosibirsk. RA was diagnosed by clinicians according to ACR/EULAR 2010. For measurement of the telomere length on individual chromosome arms we used Q-FISH with (C3TA2)3 PNA-probe. Inverted DAPI banding was used for chromosome identification according to ISCN 2013. The new MeTeLen software was developed to estimate the telomere repeats relative quantity (in metaphase images.ResultsWhen comparing the telomere length, it was found, that telomere on chromosome 16 p are shorter in patients with RA than in donors. Since each person has an individual telomere profile, we also analyzed the presence of shortened telomere sequences on individual chromosome arms relative to the average length of telomeres for each subject separately. As a result, patients with RA have a larger number of significantly shortened telomeres than donors (see Table).Comparison groupChromosome arms with shortened telomere repeats Healthy volunteers12p, 19p, 2q, 20qPatients with RA7p, 12p,16p, 17p, 19p, 2q, 20q, 21qConclusionsThe revealed features of telomeric profiles of patients with RA may be an indication of a proliferative stress, that occurs as a result of the mass immune cell proliferation in the immunopathology. It can be assumed, that the presence of a great number of shortened telomeres can promote cell death through apoptosis. The observed shortening of the telomere length on chromosome 16 p in RA may be relevant in its pathogenesis. It is known, that telomere shortening can lead to increased gene expression near the telomere DNA region. Thus, in 16 p 13 a number of genes is localized, that are associated with RA or may be involved in its development.References Olovnikov A.M, J. Theor. Biol. 41(1):181–190, 1973.Blackburn E.H., Gall J.G., J. Mol. Biol. 120:33–53, 1978.Hayflick L, Exp. Cell. Res. 37:614–636, 1965.Andrews N.P. et al., Gerontology 56(4):390–403, 2010.Dehbi A. ZA. Et al., Clin. Immunol. 9(12):1193–1204, 2013.Graakjaer J. et al., Mech. ageing dev., 124(5): 629–640, 2003. Acknowledgeme...
Reactive oxygen species (ROS) are highly reactive chemical molecules containing oxygen. ROS play an important role in signaling and cell homeostasis at low and moderate concentrations. ROS could be a cause of damage to proteins, nucleic acids, lipids, membranes and organelles at high concentrations. There are a lot of cells that can produce ROS to maintain functional activity. It is known that metal nanoparticles can increase production of ROS in cells. However, the effect of cucurbiturils on ROS production is still unknown. In our study, we evaluated production of ROS by the immune (T-, B-lymphocytes, NK-cells) and non-immune cells (red blood cells, platelets), as well as tumor cells line (1301, K562) after treatment with cucurbiturils in vitro. Assessment of reactive oxide species (ROS) were provided by using dihydrorhodamine 123 (DHR 123). Fluorescence intensity and percentage DHR123 were measured by flow cytometry. Platelets, erythrocytes and activated T-helpers were changed the level of ROS production in response to stimulation with cucurbiturils. It was found that the percentage of these ROS-producing cells was reduced by cucurbiturils. Thus, cucurbiturils may affect the production of ROS by cells, but further research is needed in this area.
The available data on the contribution of the PD-1 and its ligands to immunoregulatory processes suggest their involvement into development of tolerance during immunotherapy. Currently, allergen-specific immunotherapy (ASIT) is the single treatment option that can influence the outcome of allergic diseases. Our purpose was to evaluate the PD-1/PD-L1 expression on the immune cells in patients with confirmed sensitization to plant pollen allergens in comparison with healthy controls before and after ASIT. The patients with bronchial asthma (BA) (n = 5, age 33.82.7), allergic rhinitis (AR) (n = 7, age 31.62.8), and healthy donors (n = 12, age 32.81.8) were included. Venous blood samples were obtained from the patients three times: before starting ASIT, upon completion of the ASIT course, and during the period of seasonal exacerbation. In patients with AR, the number of B lymphocytes was decreased, and the expression of PD-L1 by B lymphocytes increased after ASIT in comparison with donor parameters. At the same time, B lymphocyte counts were increased in BA patients before ASIT and returned to normal after ASIT. In AR, the CD8+PD-1+T lymphocyte count was reduced before ASIT, however, returning to normal values after ASIT was completed. Meanwhile, the reduced number of CD4+PD-1+T lymphocytes returned to normal only during the pollination season following ASIT. In BA patients, both before or after ASIT, PD-1 and PD-L1 expression on CD4+ and CD8+T lymphocytes did not differ from the donor parameters. The PD-1 expression in the T regulatory cells (Tregs) was decreased comparing with donors before ASIT in BA, and in the patients with AR, both before and after treatment. It was shown earlier that low PD-1 expression in the circulating CD4+ T cells is associated with high specific IgE concentrations. Thus, low PD-1 levels on CD4+ and CD8+T lymphocytes and regulatory T cells may indicate their functional disorders in allergic pathology. In summary, our results show a regulatory role of PD-1/PD-L1 axis in the immune response during ASIT and reflect differences in pathogenesis of allergic disorders, which are associated with imbalance of the cell activation and suppression. Further studies are required to establish the role of PD-1/PD-L1 interactions in the process of ASIT-induced modification of allergic responses.
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