Effects of Immunoregulatory Cytokines (IL-2, IL-7, and IL-15) on Expression of Gfi1 and U2afll4 Genes in T Cells at Different Stages of Differentiation.

Opposite effects of common γ-chain cytokines (IL-2, IL-7, and IL-15) on the expression of Gfi1 and U2af1l4 genes regulating alternative splicing of Ptprc gene in T cells at different stages of differentiation were demonstrated in vitro. Generally, produced a dose-dependent activating effect on T cells, while to the effects of rIL-7 and rIL-15 on T cells at different stages of differentiation were opposite to that of rIL-2: maximum concentrations of recombinant cytokines IL-7 and IL-15 produced the most pronounced inhibitory effect on U2af1l4 and to a lesser extent on Gfi1 gene expression, thus limiting activation of resting cells. This is consistent with their biological effects on T cells. In general, common γ-chain cytokines IL-2, IL-7, and IL-15 prevent differentiation of naïve T cells in vitro and limit activation of primed T cells in the absence of antigenic stimulus, which can contribute to the formation of cytokine imbalance.

[Dose-dependent effects of dexamethasone on functional activity of T-lymphocytes different grade of differentiation].

Glucocorticoids are anti-inflammatory and immunosuppressive agents which have pleiotropic effects on growth, differentiation and functional activity of T-lymphocytes. Under experimental conditions in vitro carried out a comprehensive assessment of the dexamethasone influence on the functional activity of T-cells with different differentiation degrees. It was established that the influence of dexamethasone on the functional activity of CD45RA+ and CD45RO+ T-lymphocytes, in general, has depressing character. It was revealed that in the population of naive (CD45RA+) T-cells dexamethasone exerts a more pronounced inhibitory effect on early (IL-2-dependent, associated with the CD25 expression and IL-2 production) activation stages, whereas in the culture primed memory cells (CD45RO+)--for later (IL-2-independent, associated with the expression of proliferation molecule CD71). Multidirectional effects of dexamethasone on the expression level of telomerase catalytic unit (hTERT) mRNA are associated with the degree of T cells differentiation. It isproposed, that the role of glucocorticoid hormones in immunogenesis is primarily aimed at suppression of excessive T cells growth and on the maintainance of the clonal balance in lymphoid tissue.

[The influence of testosterone and ß-estradiol on T-lymphocytes activation associated with IL-2 production and expression of CD25 (IL-2Rα) molecules].

We have shown that testosterone and ß-estradiol in vitro have effects on naïve (CD45RA+) and priming (CD45RO+) T-lymphocytes, which associated with the production of IL-2 and CD25/IL-2Rα expression. Testosterone can inhibit naïve and priming T-cell function activity in our study. Moreover it was shown that naïve lymphocytes are more sensitive to testosterone than primed. We have found dose dependent suppressive ß-estradiol effect on IL-2 production by activated CD45RA+ and CD45RO+ lymphocytes which leads to uniform decreasing CD25-positive T-cells number.

Dexamethasone effects on activation and proliferation of immune memory T cells.

Dose-dependent effects of dexamethasone on activation and proliferation of donor immune memory T cells (CD45RO(+)) were studied. Activation of memory T cells associated with IL-2 production and membrane expression of CD25 molecule was resistant to dexamethasone. Proliferative activity of memory T cells associated with membrane expression of CD71 molecule was highly sensitive to dexamethasone. Hence, glucocorticoid hormones can maintain the clonal balance in the lymphoid tissue without preventing realization of the immune memory mechanism.

[Influence of immunoregulatory cytokines (IL-2, IL-7 and IL-15) in vitro upon activation, proliferation and apoptosis of immune memory T-cells].

Under the experimental conditions in vitro the effects of immunoregulatory cytokines (IL-2, IL-7 and IL-15) on the activation, proliferation and apoptosis of different subpopulations of immune memory T-cell (Cd45RO+) were investigated in healthy donors. It was demonstrated that the effects of rlL-2 in vitro equally affect the activation and proliferation of CD4+ and CD8+ subpopulations of memory T cells. It has been shown that high concentrations of rIL-2 lead to an increase in the number of CD8+ memory cells expressing apoptotic molecule CD95. Different effect of rIL-7 and rIL-15 on the activation and proliferation of cytotoxic CD8+ memory ells has been revealed in vitro. CD4+ memory lymphocytes have relative resistance to the activation and proliferative effect of rlL-7 and rlL-15, if compared with the effects of rIL-2, which can provide their relative resistance to the activation apoptosis as well as create the necessary conditions for the accelerated implementation of their functional capacity in the development of a secondary immune response.

[Dose-response effect of steroid hormones on the Gfi1 and U2afil4 gene expression in T lymphocytes at different stages of differentiation].

Alternative splicing of Ptprc gene is a key event in memory T cell differentiation. This gene encodes transmembrane tyrosine phosphatase CD45. One of potential mechanisms of alternative splicing regulation is based on antagonistic effects of auxiliary splicing factor U2AF26 and transcription factor Gfi1. These two proteins regulate antigen-dependent T cell activation. We have shown that steroid hormones have different effects on U2af1l4 and Gfi1 transcription regulation in dissimilar differentiation stage cell culture, subjected to antigen-independent stimulation. Low concentrations of glucocorticoid (Dex) and female sex hormone (Est) can activate expression of U2af1l4 in re-stimulated cells that probably induce terminal receptor CD45 isoforms formation mechanism, whereas high doses of hormones inhibit the process. In the same conditions Dex in a wide range of concentrations (10(-5)-10(-7) M) and Est (10(-6) and 10(-7) M) activate U2af1l4 gene expression that probably leads to "surrogate memory T cells" formation. Dose dependent testosterone (Test) effect is opposite to Est and Dex effect on priming (CD45RO+) and naive (CD45RA+) lymphocytes. The role of steroid hormones in memory T cell differentiation in antigen-independent stimulation conditions is of great interest for the understanding of chronic hormonal and immune disbalance mechanisms.

Critical role of Nox4-based NADPH oxidase in glucose-induced oxidative stress in the kidney: implications in type 2 diabetic nephropathy.

Molecular mechanisms underlying renal complications of diabetes remain unclear. We tested whether renal NADPH oxidase (Nox) 4 contributes to increased reactive oxygen species (ROS) generation and hyperactivation of redox-sensitive signaling pathways in diabetic nephropathy. Diabetic mice (db/db) (20 wk) and cultured mouse proximal tubule (MPT) cells exposed to high glucose (25 mmol/l, D-glucose) were studied. Expression (gene and protein) of Nox4, p22(phox), and p47(phox), but not Nox1 or Nox2, was increased in kidney cortex, but not medulla, from db/db vs. control mice (db/m) (P < 0.05). ROS generation, p38 mitogen-activated protein (MAP) kinase phosphorylation, and content of fibronectin and transforming growth factor (TGF)-ß1/2 were increased in db/db vs. db/m (P < 0.01). High glucose increased expression of Nox4, but not other Noxes vs. normal glucose (P < 0.05). This was associated with increased NADPH oxidase activation and enhanced ROS production. Nox4 downregulation by small-interfering RNA and inhibition of Nox4 activity by GK-136901 (Nox1/4 inhibitor) attenuated d-glucose-induced NADPH oxidase-derived ROS generation. High d-glucose, but not l-glucose, stimulated phosphorylation of p38MAP kinase and increased expression of TGF-ß1/2 and fibronectin, effects that were inhibited by SB-203580 (p38MAP kinase inhibitor). GK-136901 inhibited d-glucose-induced actions. Our data indicate that, in diabetic conditions: 1) renal Nox4 is upregulated in a cortex-specific manner, 2) MPT cells possess functionally active Nox4-based NADPH, 3) Nox4 is a major source of renal ROS, and 4) activation of profibrotic processes is mediated via Nox4-sensitive, p38MAP kinase-dependent pathways. These findings implicate Nox4-based NADPH oxidase in molecular mechanisms underlying fibrosis in type 2 diabetic nephropathy.

Hypoxia induces hypersensitivity and hyperreactivity to thromboxane receptor agonist in neonatal pulmonary arterial myocytes.

PPHN, caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane-prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd-6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O2 for 3 days; control myocytes from normoxic piglets were cultured in 21% O2. PPHN was induced in newborn piglets by 3-day hypoxic exposure (Fi(O2) 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca2+ mobilization to thromboxane mimetic U-46619 and ATP was quantified using fura-2 AM. Three-day hypoxic exposure in vitro results in increased basal [Ca2+]i, faster and heightened peak Ca2+ response, and decreased U-46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O2. Blockade of Ca2+ entry and store refilling do not alter peak U-46619 Ca2+ responses in hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca2+ channels inhibits hypoxic augmentation of peak U-46619 response. Ca2+ response to ryanodine alone is undetectable; ATP-induced Ca2+ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca2+ pool mobilization. We conclude hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca2+, thromboxane hypersensitivity, and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression.

[Effects of a cardiac peptide preparation on the myocardium in ischemia]. / Vliianie peptidnogo preparata iz serdtsa na miokard v usloviiakh ishemii.

Based on experimental studies the possibility of cordialin use in acute ischemia is being substantiated. On the first day of the animals' mortality and increased life duration of cells in ischemia zone, delaying the injury region expansion. But later in rats, that were given cordialin, slowing down of injury zone recovery and scar tissue formation was demonstrated. Cordialin use in early stages of myocardial infarction is suggested. In experiments on isolated heart cordialin is reported to decrease the intensity of processes of lipids' peroxide oxidation in intact and ischemic myocardium. But in reperfusion cordialin activates LPO, that is associated with heart contracting activity inhibition. The results of the study may serve as an experimental basis for cordialin use on the first day of MI development. Its further use needs the correction of its ability to slow down the processes of necrotic tissue recovery.

[Current problems in the morphometry of the heart]. / Sovremennnye problemy morfometrii serdtsa.

Cardiac morphometric data reported by different authors are difficult to compare. For linear measurements the processing methods need to be standardized and important considerations are the age-thanatogenetic features, and homogeneity of the autopsy material. An essential requirement is determination of the tissue compression coefficient. The specific volumes of structures should be measured in semithin sections. The largest disagreement among measurements have been noted for volumes of cardiac muscle cells and nuclei and for the nucleocytoplasmic ratio. The rules of stereometry are frequently violated in calculating parameters per unit sectional area. Although more than a hundred parameters have been proposed for cardiac morphometry, only two or three of them are of real diagnostic value. Standardizing morphometry of the heart, and of other organs as well, is a matter of high priority.