Analysis of Extracellular Vesicles Using Magnetic Nanoparticles in Blood of Patients with Acute Coronary Syndrome.

Extracellular vesicles (EVs) are released from various cell types and play an important role in intercellular interactions. In our study, we investigated abundance of individual EVs in patients with acute forms of ischemic heart disease. Previously, we developed an approach for individual analysis of EVs conjugated with magnetic nanoparticles (MNPs), which was applied in the current study for analyzing phenotypic composition of EVs (by staining for markers CD31, CD41a, and CD63). EVs were isolated using fluorescently labeled MNPs containing anti-CD31, CD41a, or CD63 antibodies and analyzed by combining fluorescently labeled anti-CD41a and CD63, CD31 and CD63, or CD41a and CD31 antibodies, respectively. EVs were analyzed in 30 individuals: 17 healthy volunteers and 13 patients with acute coronary syndrome (ACS). Six and seven ACS patients were with acute myocardial infarction and unstable angina, respectively. It was found that patients with ACS and healthy volunteers contained a dominant subset of EVs expressing surface CD41a antigen, suggesting that they originated from platelets. In addition, the total number of EVs isolated using either of the surface markers examined in our study was higher in patients with ACS compared to healthy volunteers. The subgroup of patients with acute myocardial infarction was found to contain significantly higher number of blood EVs compared to the control group. Moreover, increased number of EVs in patients with ACS is mainly due to the increased number of EVs in the subset of EVs bearing CD41a. By analyzing individual EVs, we found that plasma of patients with ACS, particularly upon developing of myocardial infarction, contained dominant platelet-derived EVs fraction, which may reflect activation of platelets in such patients.

[Immunological Analysis of Human Atherosclerotic Plaques in ex vivo Culture System].

THE AIM OF THE STUDY: to analyze the dynamics of lymphocytic composition of human atherosclerotic plaques in ex vivo culture system. MATERIALS AND METHODS: The study included 15 atherosclerotic plaques obtained from patients who underwent carotid endarterectomy. Plaques were cultured as ring-shaped explants on collagen rafts in culture medium of special composition in CO2 incubator according to the previously developed technique. On day 0, and also on the 4th and 19th days of culture we extracted cells from plaque explants and analyzed B- and T-lymphocytic content of the tissue, as well as the percentage of CD16+ natural killer (NK) cells, using multichromatic flow cytometry. For this purpose we digested the explants with an original enzymatic cocktail, which allows preservation of cell surface markers, and we stained extracted cells with fluorescence-labelled monoclonal antibodies against CD45, CD3, CD19, CD4, CD8, CD16. In addition, we estimated the amount of interleukin 2 (IL-2) and interferon-gamma (IFN-)-producing T-cells by means of flow cytometry. RESULTS: After 4 days of culture the amount of lymphocytes in plaques explants decreased, however live lymphocytes were still preserved (2619.3 [1680.4, 3478.2] cells/100mg tissue). Viable lymphocytes population included T cells (2123.4 [484.9; 3181.2] cells/100 mg tissue), B cells (5.6 [3.4, 27.9] cell/100 mg tissue) and CD16+ NK cells (10.6 [1.8, 23.7] cell/100mg tissue). On the 4th day of culture T cells were presented by CD4+CD8- (797 [475.5, 1000.7] cells/100mg tissue, 37.5 [32.1; 46.3]%) and CD4-CD8+ (686.2 [423.6; 1158.4] cells/100 mg tissue, 45.6 [38.1; 47.9]%) populations. The percentage of CD4+CD8- T cell population decreased compared to the 1st day of culture, and this decrease correlated with the increase in CD4-CD8- T cells content (p<0.05). Additionally, after 4 days of culture we found in tissue explants both CD8+ (17.5[13.3;19.9]%) and CD8- (9.9 [6.4; 14]%) IFN--producing T-cells, however, their percentage, as well as the percentage of IL-2-producing T cells tended to decrease. After 19 days of culture explants of atherosclerotic plaques also contained lymphocytes (2830.1 [2350.3, 5900.2] cells/100mg tissue). Lymphocytes population included T cells (2594.5 [2035.7, 5306.7] cells/100mg tissue), presented by CD4+CD8- (1016.8 [671.2, 2201.7] cells/100mg tissue, 42.3 [34.3; 47.8]%) and CD4-CD8+ (1534.3 [813.8; 2207.2] cells/100mg tissue, 50.8 [45.6; 56.5]%) subsets, B cells (31 [18.3; 64.4] cell/100 mg tissue) and CD16+ NK cells (44.9 [33.4; 138.9] cells/100 mg of tissue). DISCUSSION: An ex vivo model of human atherosclerotic plaque culture that we previously developed enables to preserve viability of various lymphocyte subsets for up to 19 days. We also found that cultured tissue explants retain T cells that can maintain T-helper-1-dependent immune response, which demonstrates inflammation in atherosclerotic plaques. Our results allow to perform experiments on immunological mechanisms of atherogenesis and to develop new approaches for treatment of atherosclerosis, devoted to the suppression of local inflammatory processes in atherosclerotic plaques. CONCLUSIONS: An ex vivo model of human atherosclerotic plaque preserves CD4+CD8- and CD4-CD8+ T cells, B cells, and CD16+ NK cells for a long time. Moreover, after 4 days of culture tissue explants also retain IFN-++ T cells.

[Proteasomes on thyroid tissue allotransplantation under induction of donor specific tolerance in rats].

The proteasomes in the liver of August rats (RT1C) were investigated 30 days after the allotransplantation of Wistar rat (RT1u) thyroid tissue under renal capsule with/without induction of donor specific tolerance by donor splenocyte intraportal administration. The level of the total proteasome pool, immune proteasomes containing the LMP2 and/or LMP7 subunits, proteasome 19S- and 11S-regulators was defined. The intact and sham-operated August rats were used as control groups. The level of all immune proteasome forms and 11S regulator increased while the level of the total proteasome pool and 19S regulator decreased in the liver of experimental animals compared to the control groups that indicated changes of liver functional state after transplantation. The 19S/11S ratio increased in the liver of non-tolerated rats compared to tolerated animals. In the liver of tolerated rats with survived transplants, the quantity of mononuclear cells, expressing the immune subunit LMP2, greatly increased in comparison with control and non-tolerated animals. Study of the survived transplants showed the increase of the ratio of LMP2/LMP7 immune subunits and 19S/11S regulators in them compared to the tissue replacing the rejected transplants. In the control intact thyroid tissue, the immune proteasomes were almost not revealed, while 19S/11S ratio was maximal. Thus, the development of the immune reaction or its suppression is accompanied by change of the balance between different proteasome forms. The immune subunit LMP7 and 11S regulator are connected with the response against donor tissue. On the contrary, the immune subunit LMP2 and 19S regulator are likely to be important for the immune tolerance development and survived tissue functioning. The low content of the immune proteasomes in the follicle cells was found by immunofluorescence assay. The formation of antigens for major histocompatibility complex class I molecules was impaired by low immune proteasome content that led to immunological tolerance to hormone-producing follicle cells.

Decrease in pool of T lymphocytes with surface phenotypes of effector and central memory cells under influence of TCR transgenic ß-chain expression.

Peripheral T lymphocytes can be subdivided into naïve and antigen-experienced T cells. The latter, in turn, are represented by effector and central memory cells that are identified by different profiles of activation markers expression, such as CD44 and CD62L in mice. These markers determine different traffic of T lymphocytes in the organism, but hardly reproduce real antigenic experience of a T lymphocyte. Mechanisms of homeostasis maintenance of T lymphocytes with different activation phenotypes remain largely unknown. To investigate impact of T cell receptor (TCR) transgenic chains on formation of T lymphocytes, their peripheral survival and activation surface phenotypes, we have generated the transgenic mouse strain expressing transgenic ß-chain of TCR 1D1 (belonging to the Vß6 family) on the genetic background B10.D2(R101). Intrathymic development of T cells in these transgenic mice is not impaired. The repertoire of peripheral T lymphocytes in these mice contains 70-80% of T cells expressing transgenic ß-chain and 20-30% of T cells expressing endogenous ß-chains. The ratio of peripheral CD4⁺CD8⁻ and CD4⁻CD8⁺ T lymphocytes remained unchanged in the transgenic animals, but the percent of T lymphocytes with the "naïve" phenotype CD44⁻CD62L⁺ was significantly increased, whereas the levels of effector memory CD44⁺CD62L⁻ and central memory CD44⁺CD62L⁺ T lymphocytes were markedly decreased in both subpopulations. On the contrary, T lymphocytes expressing endogenous ß-chains had surface phenotype of activated T cells CD44⁺. Thus, for the first time we have shown that the pool of T lymphocytes with different activation phenotypes depends on the structure of T cell receptors.

[Generation of transgenic animals, expressing alpha- and beta-chains of autoreactive TCR].

Transgenic animal studies has become a key approach for gene function analysis as well as for modeling of different human diseases, including autoimmune diseases caused by activation of T-lymphocyte clones whose TCRs possesses high affinity for syngeneic MHC molecules. In this study we cloned genes, encoding alpha- and beta- chains of autoreactive TCR of hybridoma 7, specific for syngeneic MHC class II molecules A(b). Amplified DNA fragments, containing rearranged genomic DNA of alpha- and beta-chains of hybridoma 7 were cloned into special cassette vectors, containing natural promoter and enhancer elements for direct expression of genes encoding TCR alpha- and beta-chains in T-lymphocytes of transgenic animals. Using this cassette vectors we generated animals in which most of peripheral T-lymphocytes carry alpha-chain, as well as animals with expression of beta-chain transgene of autoreactive TCR. Obtained animals may serve to explain a number of intrathymic selection processing features and T cell maturation as well as to serve as experimental models for development of new approaches to therapy of autoimmune diseases.