The effect of Escherichia coli ZP strain with a conjugation-based colicin E7 delivery on growth performance, hematological, biochemical, and histological parameters, gut microbiota, and nonspecific immunity of broilers.

The Escherichia coli ZP strain (ZP) was constructed based on the known probiotic E. coli strain Nissle 1917. It was genetically modified to carry the colicin E7 synthesis gene encoding DNase on a conjugative plasmid and the colicin E7 immunity gene in the chromosome. The aim of this study was to evaluate the effects of the daily ZP per oral administration (5 × 108 or 5 × 1010 CFU per bird) on the growth performance, hematological, biochemical, histological parameters, gut microbiota, and nonspecific immunity of the 4-24 days old broilers. The ZP administration increased the abundance of genera Bacillus, Butyrivibrio, and Clostridium and did not influence the weight gain of 4-16 days old broilers. The biochemical parameters were within normal ranges for poultry in experimental and control groups. The ZP administration had no effect on the erythrocyte numbers, hemoglobin and immunoglobulin Y concentrations, but significantly increased the serum lysozyme concentration, leukocyte numbers, and reactive oxygen species production by phagocytes compared with the control group. It did not cause inflammatory changes in intestinal mucosa, Peyer's patches, and spleen. Thus, the ZP had no detrimental effects on broiler health and could be an efficient probiotic for the broiler colibacillosis prophylaxis.

Droplet state and mechanism of contact line movement on laser-textured aluminum alloy surfaces.

HYPOTHESIS: Contact line movement over laser-textured metal alloy surfaces depends on the texture, droplet state, inversion of wettability, and liquid flow rate. EXPERIMENTS: Ordered and anisotropic textures were created by a nanosecond pulse laser on aluminum-magnesium alloy surfaces. Microrelief was studied on a profilometer and scanning electron microscope. Changes in wetting properties and the characteristics of spreading were obtained. To implement wetting cycles, liquid was dispensed with a syringe pump using the bottom-up method. FINDINGS: The results provide new, in-depth knowledge to understand the mechanisms of the contact line movement on laser-textured metal surfaces. We have shown that by creating a texture with given parameters (ordered or anisotropic), it is possible to predict a change in wettability from superhydrophilic to hydrophobic, as well as a droplet state. In this paper, we have demonstrated that the contact angle hysteresis determines not only the deviation of the contact angle from equilibrium, but also the adhesion force between the droplet and the surface, a droplet state (Wenzel or Cassie-Baxter), and the wetting anisotropy of local areas on laser-textured alloy. We have recorded two states of the droplet on formed textures: the Wenzel with air cushions on anisotropic and the Wenzel with complete filling of cavities with water on ordered textures.

Effect of Estriol, Chorionic Gonadotropin, and Oncostatin M on the Expression of Recombinase RAG-1 in Regulatory T Lymphocyte Subpopulations.

We studied the effect of estriol, chorionic gonadotropin, oncostatin M, and hormone-cytokine combinations on the expression of recombinase RAG-1 in T-regulatory (Treg) and T helper 17 (Th17) lymphocytes. It was found that estriol in a concentration corresponding to the first trimester of pregnancy increased the level of Treg (CD4+FoxP3+) cells and suppressed the formation of Th17 (CD4+RORC+) lymphocytes. This effect was nor observed after individual administration of chorionic gonadotropin and oncostatin M, but some combinations of the studied hormones with oncostatin M increased the percentage of CD4+FOXP3+ cells. In the presence of oncostatin M, the studied hormones enhanced the expression of RAG-1 in CD4+FoxP3+ cells, but not in CD4+RORC+ cells, thereby initiating of Treg T-cell receptor (TCR) revision. The mechanisms of hormone cytokine control of induction of the immune tolerance during pregnancy are discussed.

Regulation of Recombinase Rag-1 Expression by Female Sex Steroids in Treg and Th17 Lymphocytes: Role of Oncostatin M.

The effect of estradiol (E2), progesterone (P4), and oncostatin M (OSM) on the differentiation of CD4+ T cells to T regulatory (Treg) lymphocytes and T helpers 17 (Th17) was investigated. The possibility of revision of the T cell receptor in these subpopulations by evaluating the expression of RAG-1 recombinase was also studied. E2 at concentrations characteristic of pregnancy trimester I, but no P4 or OSM, increased the Treg level. Combination of sex steroids with OSM increased the percent of CD4+FOXP3+ cells and enhanced RAG-1 expression in these cells, thus promoting the development of immune tolerance during pregnancy. In the study of Th17, such effect of the hormones and OSM was not detected.

Mechanisms of Leptin and Ghrelin Action on Maturation and Functions of Dendritic Cells.

Molecular mechanisms of the immunomodulatory effects of leptin and ghrelin in concentrations typical for pregnancy on the maturation and functional activity of dendritic cells (DCs) generated from the peripheral blood monocytes of women are investigated. The presence of leptin during DC maturation did not affect the levels of CD83+CD1c+, CD86+CD1c+, and HLA-DR+CD1c+ DCs, but increased the amount and the activity of the enzyme indoleamine 2,3-dioxygenase (IDO). Cell culturing in the presence of ghrelin or combination of leptin and ghrelin reduced the percentage of CD86+CD1c+ DCs but did not affect the levels of CD83+CD1c+ and HLA-DR+CD1c+ DCs. In addition, ghrelin reduced the number of IDO molecules without affecting its activity. Simultaneous presence of leptin and ghrelin increased induced IDO activity without affecting the amount of the enzyme in DCs. The effects of leptin and ghrelin on the investigated functions of DCs in some cases correlated with high levels of cAMP. New mechanisms for leptin and ghrelin regulation of tolerogenic functions of DCs in pregnancy are proposed.

Hormonal Regulation of Dendritic Cell Differentiation in the Thymus.

We studied the effect of hormones estriol, ghrelin, kisspeptin, and chorionic gonadotropin in concentrations corresponding to their content in the peripheral blood in each trimester of pregnancy on the expression of membrane molecules on myeloid and plasmacytoid dendritic cells of the thymus. It was found that thymic myeloid dendritic cells are sensitive to the action of estriol and kisspeptin. Estriol in a concentration of the first trimester of pregnancy reduces the number of myeloid dendritic cells expressing receptor for thymic stromal lymphopoietin (CD11c+TSLP-R+) and inhibitory molecule B7-H3 (CD11c+CD276+). In contrast to estriol, kisspeptin regulates the processes of differentiation of thymic myeloid dendritic cells in concentrations typical of the second-third trimesters and reduced their total number (CD11c+) and the number of cells expressing TSLP-R (CD11c+TSLP-R+). Estriol and kisspeptin do not affect the total number of plasmacytoid dendritic cells (CD303+) and expression of TSLP-R and CD276 by these cells. Ghrelin and chorionic gonadotropin in the studied concentrations had no significant effect on the total number of thymic myeloid and plasmacytoid dendritic cells and on the expression of membrane molecules of TSLP-R and CD276.

Role of PKA and PI3K in Leptin and Ghrelin Regulation of Adaptive Subpopulations of Regulatory CD4+ T-Lymphocyte Formation.

The role of phosphatidylinositol-3 kinase (PI3K) and protein kinase A (PKA) in leptin and ghrelin regulation of formation of adaptive (a) subpopulations of CD4+ T-lymphocytes (helper (h) cells producing interleukin-17A) (aTÒ»17) and of T-regulatory lymphocytes (aTreg) in the context of physiological pregnancy is established. It is shown that leptin at a concentration typical for the second half of pregnancy (trimesters II-III) enhances the differentiation of aTÒ»17 with a high level of interleukin-17A (IL-17A) production and the expression of the chemokine receptor CCR6 with the participation of PI3K. Simultaneously, leptin reduces formation of aTreg expressing the suppressor molecule CTLA-4, which determines the function of these cells. Ghrelin at a concentration characteristic of the first half of pregnancy (trimesters I-II), in contrast, enhances aTreg formation and, in parallel, reduces the level aTÒ»17 (that express CCR6) and the IL-17A production by aTh17. PKA, likewise PI3K, participates in regulatory effects of ghrelin on the formation of aTÒ»17 and aTreg. The combined action of leptin and ghrelin (via PKA participation) enhances formation of only aTreg, which determines the priority of this molecular mechanism and explains why the investigated hormones with reciprocal differentiating potential do not come into antagonism at the level of immune system cells during pregnancy.

MicroRNA in hormonal mechanisms of regulation of NK cell function.

We investigated the effect of human chorionic gonadotropin, estriol, leptin, ghrelin, and kisspeptin on the microRNA expression in separated NK cells. All of these hormones are able to effectively modulate the expression of microRNAs both stimulating and suppressing the cytotoxic potential of NK cells and thereby indirectly regulate the functions of these lymphocytes.

Hormonal regulation of NK cell cytotoxic activity.

The effects of chorionic gonadotropin, estriol (E3), leptin, ghrelin, and kisspeptin on the intracellular expression of perforin, granzyme A, and granzyme B was studied in separated NK cells. All studied hormones except E3 are could modulate the expression of cytotoxic enzymes in NK cells by suppression of the expression of the most active proapoptotic agents, resulting in increased expression of granzyme A, which is typical of the decidual subpopulation of these lymphocytes.

Roles of leptin and ghrelin in the regulation of the phenotype and cytokine production by NK cells from peripheral blood.

Both leptin and ghrelin used separately at the concentrations corresponding to trimesters II-III of pregnancy increase the number of CD56bright NK cells in mononuclear cell suspension; their combination also enhances the L-selectin expression on the surface of these cells in the culture. These hormones do not affect the production of TGF-ß1, IL-17А, or IFN-γ by NK cells, and they inhibit the production of IL-10. Leptin decreses the IL-4 production by NKp46+ cells, but the presence of ghrelin abrogates this effect.

[The Influence of Chorionic Gonadotropin and Estriol on NK Cells Phenotype and Functional Activity.]

The influence of chorionic gonadotropin (CG) and estriol (E3) at concentrations typical of pregnancy on the expression of phenotypic markers and cytokine production by separated NK cells were studied. It is found that these hormones increase the percentage of CD56brightL-selectin+ NK-cells, but also stimulate the expression of the inhibitory molecule NKG2A in the lymphocytes. In addition, E3 and CG stimulate the production of TGF-B, inhibiting the secretion of all other cytokines by separated NK cells. In general, these hor- mones contribute to the formation of the phenotype and cytokine spectrum characteristic of the regulatory NK3 subpopulation of NK cells during pregnancy.

The effect of kisspeptin on the functional characteristics of isolated NK cells.

The effect of kisspeptin at concentrations typical of pregnancy on the functional activity of isolated cytokine-primed NK cells has been investigated. The hormone has been shown to promote an increase in the proportion of CD56(bright) NK cells, as well as an increase in the L-selectin expression on the cell surface. Assessment of cytokine levels has shown that kisspeptin suppresses the production of IL-4, IL-10, and IFN-γ while stimulating the production of TGF-ß by isolated NK cells. The overall effect of the hormone investigated consisted in the formation of a phenotype and a cytokine spectrum characteristic of the regulatory NK3 subpopulation of NK cells in pregnancy.

Role of leptin and ghrelin in induction of differentiation of IL-17-producing and T-regulatory cells.

We studied isolated and combined effects of leptin and ghrelin on the formation of IL-producing and T-regulatory cells. Leptin in concentrations comparable with its normal blood concentration during pregnancy (trimester II-III) promotes differentiation of peripheral blood CD4(+) cells to IL-17-producing cells and enhances IL-17A production, but suppresses the formation of T-regulatory cells in vitro. In contrast, ghrelin in a concentration typical of trimester I-II of pregnancy reduces the number of IL-17-producing cells, but stimulated the formation of T-regulatory cells. The effects of leptin and ghrelin in combination typical of trimester I-II of pregnancy stimulated the formation of T-regulatory cells, while in combination typical of trimester II-III did not shift the balance of T-regulatory cells and IL-17-producing cells, but stimulated the formation of IL-17A. We conclude that leptin and ghrelin play an important role in the maintenance of the balance of IL-17-producing and T-regulatory cells during pregnancy.

Leptin as an immunocorrecting agent during normal pregnancy.

Experiments were performed with leptin in doses observed during pregnancy. We studied the effect of leptin on expression of membrane molecules and cytokine production by peripheral blood mononuclear cells from women. Leptin increased the expression of HLA-DR on T lymphocytes, stimulated the production of TNF-alpha and IL-6, and did not affect secretion of IL-2, IL-4, and IL-10 by mononuclear leukocytes. Leptin in a dose comparable to that during the 1st trimester of pregnancy increased the percentage of NK cells with membrane CD16 and CD56, stimulated the production of IFN-alpha by mononuclear leukocytes, and did not modulate the number of CD16(+)56(+)NKT cells. Treatment with leptin in a dose for the second-third trimesters of pregnancy was followed by a decrease in the percentage of CD16(+)56(+)NKT cells and increase in the number of NKT cells expressing CD16 and CD56. Our results indicate that leptin play an important role in the regulation of membrane molecule expression and cytokine production by mononuclear leukocytes.