[Indices of cell-mediated immunity in rheumatoid arthritis: the role of cytomegalovirus infection.]

The pathogenesis of rheumatoid arthritis (RA) is driven by a combined action of genetic and environmental factors, which can upset the balance between the effector and regulatory components of the immune system. An important actor in maintaining such balance is T cells, especially regulatory T lymphocytes (Treg), but the mechanisms behind the functioning of T cell subpopulations and the roles of individual etiological factors in RA have not been fully elucidated. This study aimed to investigate the indices of cell-mediated immunity, especially T- and Treg cells, in RA patients depending on the disease activity and presence of cytomegalovirus (CMV) infection. The expression of membrane and intracellular molecular markers of lymphocytes was estimated by multicolor flow cytometry. The content of antibodies to CMV in blood plasma was measured by enzyme immunoassays. Patients with RA had reliably reduced numbers of cells with the phenotypes CD4+FOXP3+, CD4+CD25+FOXP3+ correlating with the stage of RA activity. RA patients with CMV infection showed a reduction in the number of regulatory T cells (Treg), CD3+ T lymphocytes, CD3+CD8+ cells in peripheral blood. At the same time, RA involved a rise in the level of B cells and CD4+CD25+ Т cells. The level of antibodies to CMV was observed to grow in line with RA activity. Thus, the data obtained suggest that the presence of CMV infection can significantly influence the state of individual lymphocyte subpopulations during RA development.

[Subpopulations of Regulatory T-lymphocytes in the Peripheral Blood of Patients with Rheumatoid Arthritis].

BACKGROUND: Rheumatoid arthritis (RA) is an inflammatory rheumatic disease associated with a dysfunction of the T cell-mediated tolerance and leading to the disability of working population. The regulatory CD4' T cells play an important role in the regulation of autoimmunity and can suppress immune responses. With that, there is no consensus on the content of these lymphocytes and their role in the pathogenesis of RA. Objective: The aim of the study was to assess the content ofperipheral blood regulatory Tcells (Treg) according to the expression of membrane markers CD4, CD25, CD127 and intracellular FOXP3 marker, as well as the expression of two functional molecules (CTLA-4 and CCR4) in Treg cells of patients with RA. METHODS: Peripheral blood samples of RA patients (median age 65,5 years [54;68,3) and healthy controls (median age 58 years [44; 66]) were analyzed. Cell count and the expression level of molecules were assessed by flow cytometry. RESULTS: Peripheral blood samples of 36 RA patients and 20 healthy donors were analyzed. The number of the cells with Treg-associated phenotypes CD4⁺CD25hi and CD4⁺CD25hiCD127low/⁻ was higher in RA patients in comparison with healthy donors. Increased levels of RA CD4⁺ T cells expressing FOXP3 were also observed. This may be due to increasing in the number of CD4⁺FOXP3⁺CD25⁻ lymphocytes, whereas the content of RA CD4⁺FOXP3⁺CD25⁺ Treg cells was at the level of the control. The expression of the functional molecule CTLA-4 in Treg cells of patients with RA was not different from the control, while the expression level of the chemokine receptor CCR4 which provides migration of lymphocytes at sites of inflammation and barrier tissues was significantly increased in RA patients. CONCLUSION: Increase in the levels of certain Treg-associated lymphocyte populations were detected in peripheral blood of RA patients. During the natural course of RA, alterations in the level of the chemokine receptor CCR4 might indicate the enhanced lymphocyte migration.

[Circulating regulatory T-cells and changes in the subpopulation composition of lymphocytes in colorectal cancer patients].

Data on the frequency of peripheral Treg cells, their functional activity, as well as their relationship to changes in characteristics of cellular immunity in patients with colorectal cancer (CRC, n=42) and healthy donors (n=34) are presented in the article. Assessment of the expression of molecular markers of lymphocytes was performed by multicolor flow cytometry. It was shown that the number of these cells is significantly increased in CRC patients (p<0,05). Probably CD4+CD25+FOXP3+ Treg cells may play a more substantial role in the periphery during the initial period of tumor formation, whereas CD8+FOXP3+ Treg cells are important in the later stages of the disease. In CRC patients, the increase of the functional activity of Treg cells (CD4+CD25+CD127lo/') on the expression of inhibitory molecules CTLA-4 and the marker of cell proliferation Ki-67 was observed. In patients with CRC, the frequency of peripheral Treg cells and the expression level of CTLA-4 were inversely correlated with the number of CD3+CD4+ T-helper cells.

[Alterations of lymphocyte subsets and indicators of immune suppression in patients with acute pancreatitis].

THE AIM OF STUDY: Assessment of changes in lymphocyte number and some indicators of the immune suppression (frequency of regulatory T cells (Treg), the level of expression of CTLA-4 (cytolytic T lymphocyte-associated antigen 4) and interleukin (IL)-10) in patients with acute pancreatitis (AP). MATERIALS AND METHODS: Peripheral blood samples of 21 patients with AP (including 11 patients with pancreatic necrosis (PN)) were studied for analysis of lymphocyte subsets and 11 blood samples of healthy donors were estimated as control. Evaluation of the expression of molecules of lymphocytes was carried out by flow cytometry. RESULTS: It was found that percentage of CD3+ T cells and their subpopulation of CD4+ T-helper cells in patients with AP were decreased as compared to the control group. Substantial changes were observed in patients with PN: together with decrease in the number of T cells, CD19+ B cells and CD4+ CD25+ activated T-helper cells were also reduced, and the percentage of CD8+ T cells was higher. The immunoregulatory index (CD4+/CD8+) was lower in the group of patients, than in control group. The frequency of Treg cells (CD4+ CD25hi and CD4+ CD25+ CD127lo) was higher in patients with AP than in the control. The expression level of the inhibitory molecule CTLA-4 in the subset of CD4+ CD25hi Treg cells in patients with AP was also increased. Moreover, it was found that the expression level of anti-inflammatory cytokine IL-10 was higher in T-helper cells from patients with AP. CONCLUSION: Changes in cellular immunity (especially patients with PN) werefound in patients wish AP. These changes may indicate the possible development of secondary immunodeficiency. This is probably related to the generation of immune suppression, since the values of the investigated parameters (frequency of Treg cells, the expression of CTLA-4 and IL-10) in patients with AP were higher than in controls.

[Regulatory T cells and aging].

Regulatory T cells with potent immunosuppressive properties have a pivotal role in the maintenance of immune homeostasis. This review highlights recent findings regarding the age-related changes in numbers and functional activity of human Treg-cells. Some of the mechanisms that keep the balance of Treg-cells during human aging are discussed. The possible roles of Treg-lymphocytes in pathogenesis of several diseases, associated with advanced age are also considered.

[A bioinformatic pipeline for NGS data analysis and mutation calling in human solid tumors]. / Bioinformaticheskii protokol dlia obrabotki NGS-dannykh i identifikatsii mutatsii v solidnykh opukholiakh cheloveka.

We aimed to develop a pipeline for the bioinformatic analysis and interpretation of NGS data and detection of a wide range of single-nucleotide somatic mutations within tumor DNA. Initially, the NGS reads were submitted to a quality control check by the Cutadapt program. Low-quality 3¢-nucleotides were removed. After that the reads were mapped to the reference genome hg19 (GRCh37.p13) by BWA. The SAMtools program was used for exclusion of duplicates. MuTect was used for SNV calling. The functional effect of SNVs was evaluated using the algorithm, including annotation and evaluation of SNV pathogenicity by SnpEff and analysis of such databases as COSMIC, dbNSFP, Clinvar, and OMIM. The effect of SNV on the protein function was estimated by SIFT and PolyPhen2. Mutation frequencies were obtained from 1000 Genomes and ExAC projects, as well as from our own databases with frequency data. In order to evaluate the pipeline we used 18 breast cancer tumor biopsies. The MYbaits Onconome KL v1.5 Panel ("MYcroarray") was used for targeted enrichment. NGS was performed on the Illumina HiSeq 2500 platform. As a result, we identified alterations in BRCA1, BRCA2, ATM, CDH1, CHEK2, TP53 genes that affected the sequence of encoded proteins. Our pipeline can be used for effective search and annotation of tumor SNVs. In this study, for the first time, we have tested this pipeline for NGS data analysis of samples from patients of the Russian population. However, further confirmation of efficiency and accuracy of the pipeline is required on NGS data from larger datasets as well as data from several types of solid tumors.

[Age related changes in rat blood leucocyte count and morpho-metrical parameters of large granular lymphocytes under different light regimens].

The effect of different light conditions on the age related level of blood leucocytes, leucocytes differential count and the morpho-metrical parameters of large granular lymphocytes in rats was investigated. It has been found that the senescent animals demonstrate disturbance in typical lymphocytes/ neutrophils ratio under condition with light and darkness changes natural and regular illumination. The total leucocytes number, the level of neutrophilic bands, eosynophils and large granular lymphocytes increased under continuous light illumination and natural photoperiod in Karelia. The comparatively lower level of lymphocytes and higher of neutrophils was formed earlier under light conditions without light and dark interchange, i.e. under long time light deprivation (DP) and under constant illumination (LL).