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AIM: Study the effect of components of destroyed streptococci on human blood monocyte functions related to processes of trans-endothelial migration in vitro. MATERIALS AND METHODS: Mononuclear leukocytes, isolated from blood of healthy donors, endothelial cells of EA.hy 926 line and supernatant of ultrasound disintegrated Streptococcus pyogenes (DSS) were the objects of the study. Evaluation of adhesion and monocyte migration, level of expression of adhesion molecules and phosphokinases on monocytes was carried out by flow cytometry using monoclonal antibodies. Cytokine concentration was determined by using standard commercial test systems in enzyme immunoassay. RESULTS: Under the effect of DSS, expression of adhesion molecules CD162 and CD11b, as well as phospho-p38 MAPK changed, IL-6 and IL-8 secretion induction took place. DSS caused enhancement of migration and adhesive activity of monocytes, however, inhibited intensity of trans-endothelial migration. CONCLUSION: Products of destroyed streptococci have a multi-directional effect on human blood monocytes, that could be explained by the presence of components with varying biological activity in DSS.

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Angiogenesis and vascular remodeling are vital components of inflammation. As an inflammation evolves, vessels expand to supply nutrients and inflammatory mediators, sustaining the accumulation of activated immune cells in the affected tissues. This study demonstrates that ultrasonic supernatant of Streptoccocus pyogenes has anti-angiogenic properties: inhibit EA.hy 926 human endothelial cells metabolism, adhesion, migration, proliferation. At the same time Streptococcal components inhibit signaling pathways that involve FAK and ERK1/2. These effects are not associated with necrosis or apoptosis in cell culture. Taking together, our results suggest that impairing angiogenic function of endothelial cells might contribute to the reduced tissue perfusion, hypoxia, and subsequent regional tissue necrosis caused by Streptococci group A.

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Imiquimod (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine) is an active immunomodulator with antiviral effects. In addition to its stimulatory effect on cell-mediated immunity, in vivo studies have detected its antiviral and antiangiogenic effects. Possible direct effect of imiquimod on endothelial cells remains not studied. We have shown that imiquimod inhibited proliferation and migration of human endothelial cells (EA.hy 926 strain) in vitro and induced apoptosis (but not necrosis) of endothelial cells and production of IL-6 cytokine. These results suggest that imiquimod inhibits angiogenesis via direct modulation of endothelial cell function.

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Secretion of chemokines under different conditions of monocyte and endothelial cell coculturing was compared. Secretion of all the studied chemokines was recorded in cocultures: IL-8/CXCL-8, MCP-1/CCL2, RANTES/CCL5, and IP-10/CXCL10. The presence of TNF-α increased the concentrations of all chemokines, the concentrations of IL-8/CXCL-8, MCP-1/CCL2, and IP-10/CXCL10 decreased significantly in transendothelial migration. Addition of IFN-γ to cocultures significantly increased only IP-10/CXCL10 concentration; in transendothelial migration, the concentration P-10/CXCL10 decreased, while the concentrations of RANTES/CCL5 and MCP-1/CCL2 increased. Cell coculturing with IL-4 reduced the concentrations of all chemokines; the concentration of RANTES/CCL5 significantly increased in transendothelial migration. These results demonstrate the important role of monocyte-endothelial interactions in the regulation of the constitutive and cytokine-induced secretion of chemokines.

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Two subsets of monocytes were identified in humans and other mammals blood based on different levels of CD14 and CD16 expression. These subsets have different patterns of adhesion molecules and chemokine receptors, which suggest different modes of interaction with endothelium and tissue traffic. Here, we investigated the ability of CD14+CD16+ and CD14++CD16- monocytes to adhesion to endothelial cells monolayer in presence and in the absence of pro- and anti-inflammatory cytokines. We demonstrated that CD14+CD16+ monocytes had higher level of adhesion to intact endothelial cells monolayer than CD14++CD16- monocytes. Significant increase in adhesion of CD14++CD16- and CD14+CD16+ monocytes subpopulations was observed in the presence of both TNF alpha and TNF alpha combinations with other cytokines. IFN gamma and IL-4 showed no independent effects on adhesion of monocytes. These results have demonstrated that both CD14++CD16- and CD14+CD16+ monocytes can be recruited to inflamed endothelium, but, in the absence of inflammation, CD14+CD16+ monocytes adhere to endothelial cells two times stronger than CD14++CD16- monocytes.

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We studied the anti-angiogenic properties of sutent (SU11248) and celecoxib in human endothelial cell line EA. hy 926 in vitro. Sutent 0.05-0.5 microg/ml suppressed their proliferation and migration depending on dose while celecoxib did the same at 5.0 microg/ml. We were the first to demonstrate that endothelial cell incubation was followed by increase in 5'-nucleotidase activity in the presence of sutent while celecoxib did not produce such effect. It may be suggested that elevated 5'-nucleotidase concentration at the membranes of endothelial cells might in turn contribute to the pool of extracellular adenosine to stimulate antiinflammatory effect. Our data also contribute to the knowledge about the anti-angiogenic properties of sutent and celecoxib.

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Developer of the phagocytosis theory I.I Mechnikov forecasted the most fruitful directions of its development. Macrophages express on the plasma membranes broad spectrum of receptors, which mediate their interaction with altered organism's own components as well as with exogenous agents, including various microorganisms. Recognition leads to changes of expression of surface molecules, enhancement of phagocytic activity as well as production and secretion of cytokines, presentation functions, signaling and genes expression. This reflected on maintenance of homeostasis, as well as on host defense effectiveness, including mechanisms of innate and adaptive immunity.

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AIM: To study the influence of lypopolysaccharide (LPS) of Gram-negative bacterium (Escherichia coli O55:B5) and lysate of Gram-positive bacteria (Streptococcus pyogenes - group A, type M1, strain 40/58) on the level of expression of important surface molecules of monocyte-derived cells from continuous cell line THP-1 and endothelial cells from continuous cell line EA.hy 926. MATERIALS AND METHODS: Expression of surface molecules HLA-DR, CD11b, CD14, CD16, CD32, and CD54 was assessed using FITC- or PE-labeled monoclonal antibodies (Beckman Coulter, USA). Intensity of fluorescence was measured by flow cytometer Epics Altra manufactured by Beckman Coulter (USA). RESULTS: Studied components of Gram-positive and Gram-negative bacteria stimulated expression of CD14, CD16, CD32, and CD54 molecules on cells from THP-1 line; incubation of cells from EA.hy 926 line in the presence of the same bacterial components increased expression levels of CD54 and HLA-DR molecules. CONCLUSION: Endothelial cells of EA.hy 926 line was less sensitive to LPS of E. coli and lysate of S. pyogenes compared to monocyte-derived cells of THP-1 line. Usage of THP-1 cells allowed to reveal differences between effects of components of Gram-positive and Gram-negative bacteria. The stimulating effect of LPS was more pronounced compared to effect of S. pyogenes lysate in relation to expression of HLA-DR, CD11b, and CD54 molecules, whereas lysate of S. pyogenes better stimulated expression of CD14, CD16, and CD32 molecules.

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The level of expression of CD11b and HLA-DR surface molecules on monocyte-like THP-1 cells increased significantly as a result of transmigration of these cells through a monolayer of endothelial cells. The expression of all studied markers (CD11b, HLA-DR, and CD-14) increased significantly after transendothelial migration in the presence of TNF-alpha and IFN-gamma. The changes in surface phenotype of THP-1 cells after transendothelial migration in the presence of TNF-alpha were more pronounced than in the presence of IFN-gamma. Transendothelial migration of THP-1 cells in the presence of IL-4 caused less pronounced changes in the surface phenotype, which did not differ from changes in transmigration without cytokines.

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Immunological parameters were studied at randomization in 60 surgical patients during the similar operation--cholecystectomy made under combined endotracheal low-flow general anesthesia using N2O:O2+fentanyl in 32 patients and Xe:O2 in 28 patients. The time course of changes in cellular immunity and cytokines was closely related to the type of an anesthetic. Unlike N2O:O2+fentanyl, Xe did not show such a marked proinflammatory activity, exerted a mild normalizing effect on leuko- and lymphopoiesis, had an immunostimulating activity, and reduced the frequency of postoperative inflammatory complications and the length of stay at hospital. The differences in the action of the anesthetics were due to the fact that Xe had a greater narcotic potential, a protective action on neuroendocrine function, and no toxicity. Xe is indicated to patients with baseline immunodeficiency.

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Angiogenesis is a complicated process, which is regulated by numerous cytokines and growth factors. Besides, the interaction of endothelial cells with extracellular matrix components, with other cell types and with each other is essential for the formation on new blood vessels. The initiation, continuation and completion of angiogenesis depend on the balance of pro- and antiangiogenic factors in the endothelium microenvironment. Factors that influence endothelial cell proliferation are necessary for vascular development and their normal functioning. The influence of new pharmaceutical agents on angiogenesis is commonly evaluated by results of in vivo assays, i.e. chick chorioallantoic membrane and rabbit cornea assays. However, reported results are not always objective. So, the aim of our study was to elaborate methods of estimation of endothelial cell proliferation as one of important stages of angiogenesis.

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We studied long-lasting consequences of the low-doses irradiation on the immune system of 71 clean-up workers who participated in the emergency work after the Chernobyl Plant accident in 1986 and 25 healthy donors from Belarus. In sera of the workers the level of autoantibodies to thyroid gland antigens (thyroglobulin and microsomal fraction of thyroid gland) was increased in 48% of cases, the level of autoantibodies to lens oculis antigen was increased in 44% of cases; the level of circulating immune complexes was elevated in 55%, and the serum level of thyroglobulin in 60% of people. Immunological disorders were found without any definite clinical evidences of diseases and this allows us to consider the examined contingent as a group of risk for the development of autoimmune pathology in the future.

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Depending on the type of autonomous regulation, differences in basic levels of interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) were revealed under conditions of hyperthermia in healthy subjects aged 19-21. A parasympathetic type of autonomous regulation corresponded to higher initial levels of proinflammatory cytokinesis, whereas a dominating sympathetic type corresponded to lower levels of the IL-1 beta and TNF alpha. The subjects with the latter type of regulation revealed an increase in the IL-1 beta TNF alpha combined with a higher heat tolerance. The subjects with the former type of regulation revealed a lower heat tolerance. The increase in the alpha2-macroglobulin appeared to be a most typical acute phase response of the human body to hyperthermia.

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The paper provides a brief review of recent basic studies made by the Department of Immunology, Institute of Experimental Medicine, Saint Petersburg. They show that with a panel of macrophage-like cell lines of varying maturation (P388D, THP-1, U937, HL-60), the ability to produce TNF alpha spontaneously and to synthesize TNF alpha in response to atherogenic low-density lipoprotein stimulation correlates with the degree of cell differentiation that can be in turn induced by agents such as phorbol myristic acetate. The TNF alpha molecular site responsible for the macrophage-activating action of TNF alpha was identified as peptide 123-131. The latter was demonstrated to be uninvolved in the cytotoxic activity of TNF alpha. In addition to its activating and modulating effects against neutrophils, monocytes, and lymphocytes, the acute-phase reactant C-reactive protein was found to be able to bind the antiinflammatory cytokines IL-4 and TGF beta.

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Clinicians of different profiles often direct their patients to immunological tests because of a high incidence of various immunopathological syndromes, such as immunodeficiencies and allergic and autoimmune diseases. The efficacy of an immunological study depends on the task of the laboratory verification of the tentative clinical diagnosis, on the disease stage, and planned treatment. Biological material for investigation is collected depending on the task of the study and localization of the pathological process. It is not only the traditional material-blood, but other biological fluids of the organism as well. The choice of an adequate and informative complex of methods is also determined by the tasks of investigation. Special attention is paid now to methods for assessing the cytokine status of the organism. Interpretation of the results is the most intricate step of immunological tests because the notion of "immunological norm" is ambiguous and it is necessary to take account of the individual shifts of immunological parameters in the course of disease.

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Effects of AML and normal mononuclear phagocyte conditioned media (CM) on the proliferation and differentiation of monoblastic human cell line U-937 have been studied. The normal mononuclear phagocyte CM inhibited proliferation and weakly stimulated differentiation of U-937 cells, whereas AML CM exerted no effect. Activation of both normal and AML macrophages with phorbol ether (TPA) was followed by enhancement of CM effect. TPA treatment corrected defects of secretory activity of AML mononuclear phagocytes. A possible role of different monokines in the regulation of proliferation and differentiation of leukaemic cells is discussed.

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