Maternal insulin-like growth factors and insulin-like growth factor-binding proteins for macrosomia prediction in diabetic and nondiabetic pregnancy: A prospective study.

OBJECTIVE: Attributable to the insulin-like growth factor (IGF) axis involvement in fetal growth regulation, possible contribution of the maternal IGF axis to antenatal fetal macrosomia diagnosis is a subject of particular interest in diabetic pregnancy. METHODS: A total of 130 women were prospectively enrolled in a longitudinal single-center cohort study. The four study groups were: type 1 diabetes (n = 40), type 2 diabetes (n = 35), gestational diabetes (n = 40), and control (n = 15). IGF-1 and IGF-2 and insulin-like growth factor-binding protein (IGFBP) 1, 3, 6, and 7 serum levels were analyzed in 11- to 14-week and 30- to 34-week samples with a specific immunoassay. RESULTS: In mothers of large-for-gestational-age neonates (90th percentile), higher (median test) first-trimester IGF-1 (P = 0.007) and lower IGFBP-1 (P = 0.035) were observed. The IGF-1/IGFBP-1 ratio was positively associated with neonatal weight (r = 0.434, P < 0.001). Receiver operating characteristic analysis revealed an association between large for gestational age and the first-trimester IGF-1 (area under the curve [AUC] = 0.747, P < 0.001), IGFBP-1 (AUC = 0.334, P = 0.011), and IGF-1/IGFBP-1 ratio (AUC = 0.750, P < 0.001). IGF-1/IGFBP-1 ratio had better performance for prediction of birth weight over 4000 g (AUC = 0.822, P < 0.001). CONCLUSION: The authors detected different first-trimester IGF-1 and IGF-1/IGFBP-1 thresholds applicable for either supposition or rejection of macrosomia diagnosis. Further investigation is needed to determine how the maternal IGF axis can contribute to fetal macrosomia prediction.

Microvesicles Produced by Natural Killer Cells Regulate the Formation of Blood Vessels.

We studied the effect of microvesicles derived from cells of the NK-92 cell line on the formation of tube-like structures by endothelial cells of the ЕА.Hy926 cell line. Microvesicles were isolated by differential centrifugation and their size was controlled by granulometric analysis using dynamic light scattering method. The effect of microvesicles produced by NK cells on angiogenesis was evaluated by cultural methods. In the course of the research, a model of co-culturing of microvesicles and endothelial cells on extracellular matrix Matrigel was developed. It was found that microvesicles derived from NK-92 cells promoted elongation of tube-like structures formed by endothelial ЕА.Hy926 cells. Microvesicles produced by NK cells can modulate functional activity of endothelial cells by affecting their ability to form blood vessels.

[Cys-Flanked Cationic Peptides For Cell Delivery of the Herpes Simplex Virus Thymidine Kinase Gene for Suicide Gene Therapy of Uterine Leiomyoma].

Uterine leiomyoma (UL) is the most common benign tumor in women of reproductive age. Gene therapy using suicidal genes appears to be a promising approach for UL treatment. One of key factors for success of gene therapy is the right choice of genetic construct carrier. A promising group of non-viral carriers for cell delivery of expression vectors is cationic Cys-flanked peptides which form tight complexes with DNA due to electrostatic interactions and the presence of interpeptide disulfide bonds. The paper reports a comparative study of the physico-chemical, toxic, and transfectional properties of the DNA-peptide complexes obtained by matrix polymerization or oxidative polycondensation of Cys-flanked peptides using the chain growth terminator 2-amino ethanethiol. We have demonstrated the therapeutic effect of the delivery of the pPTK-1 plasmid carrying the herpes simplex virus type 1 (HSV-1) thymidine kinase gene into PANC-1, and HEK-293T cell culture as well as into primary UL cells. It has been shown that the carriers obtained by oxidative polycondensation transform primary UL cells more efficiently than those produced by matrix polymerization. Treatment with ganciclovir resulted in the death of up to 40% of UL cells transfected with the pPTK-1 plasmid. The perspectives of use of the polyR6 carrier produced by oxidative polycondensation as a tool for the development of modular peptide carriers for the purposes of UL gene therapy were discussed.

Trophoblast cell influence on peripheral blood natural killer cell proliferation and phenotype in non-pregnant women and women in early pregnancy.

Natural killer (NK) cells are the main population of leukocytes in decidua during the first trimester of pregnancy. NK cells can have contact with trophoblast cells during pregnancy, which raises the possibility of mutual influence. This research aimed to evaluate the proliferation and phenotype of peripheral blood NK cells in the presence of trophoblast cells of the JEG-3 cell line. We showed that trophoblast cells of the JEG-3 cell line (American Type Culture Collection (ATCC), USA) produced TGFß. However, co-culturing of NK and trophoblast cells did not change the SMAD2/3 to pSMAD2/3 ratio within NK cells. These data indicate that the canonical signaling pathway from TGFß is not activated, but do not preclude activation of SMAD-independent signaling pathways through the effect of TGFß and/or other cytokines. We established that trophoblast cells inhibited both constitutive and IL-2-induced expression of Ki-67 proliferation marker by NK cells in vitro in both pregnant and non-pregnant women. Constitutive and induced Ki-67 expression by peripheral blood NK cells was increased in pregnant women compared with non-pregnant women. The influence of trophoblast cells on Ki-67 expression by NK cells was more pronounced in the presence of other mononuclear cells than in their absence. In the presence of trophoblast cells and IL-2, the number of NK cells with the CD16+CD57- phenotype in peripheral blood mononuclear cells (PBMCs) was increased in pregnant and non-pregnant women, compared with culturing with IL-2 only. This might reflect a decrease in the number of NK cells at the terminal stage of differentiation. We also revealed the increased content of NK cells with the CD16-CD56bright phenotype in PBMCs of pregnant women when incubated with trophoblast cells and IL-2, compared with culturing with trophoblast cells only. Our results suggest that NK cells need contact interactions with trophoblast cells and additional cytokine stimulation (IL-2, cytokines of other mononuclear cells) to acquire the CD56bright phenotype.

Two patterns of cytokine production by placental macrophages.

INTRODUCTION: Macrophages participate in the regulation immune and morphogenetic events in the placenta. However, these roles remain unclear for placental macrophages (Hofbauer cells). The aims of this study were to characterize the consecutive steps of cytokine production (intracellular synthesis and secretion) in placental macrophages in early and late gestation and to compare the secretory profiles of placental macrophages and villous tissue. METHODS: Macrophages and villous tissue were isolated from placentas obtained from normal pregnancies at either 9-12 or 38-40 weeks of gestation. Intracellular cytokines were determined by flow cytometry after staining with monoclonal antibodies. Secreted cytokines were quantified by cytometric bead array and ELISA. RESULTS: Two patterns of cytokine production were revealed in placental macrophages. Cytokines in the first group (IL-1, IL-6, IL-8, IL-10, TNFα) demonstrated low basal production and were stimulated by bacterial endotoxin. Cytokines in the second group (IL-11, IL-17A, IL-17F, TGF-ß, VEGF) were characterized by constitutive production and did not respond to stimulation. Gestational age-dependent changes were observed: basal secretion of TNFα and IL-8 increased whereas IL-11 and IL-17 secretion decreased in third-trimester macrophages compared with the first-trimester cells. Comparison of cytokine production at the cellular and tissue levels suggested the contribution of the placental macrophages both in intraplacental and extraplacental cytokine production. DISCUSSION: It would be safe to assume that the two patterns of cytokine production, revealed in our study, correspond to two regulatory roles of placental macrophages: "immune" and "morphogenetic". The inflammatory phenotype of macrophages is attenuated in early gestation and increases with the progression of pregnancy. The cytokines of the first group supposedly contribute to both local and extraplacental levels, whereas the cytokine effects of the second group are more likely confined to the placental tissue.

Role of Caspases in the Cytotoxicity of NK-92 Cells in Various Models of Coculturing with Trophoblasts.

Studies of interactions between natural killer (NK) cells and trophoblasts and identification of conditions for the NK cells to perform their cytotoxic function are of fundamental and practical importance for understanding their role in the development of pathological processes and complications during pregnancy. In this study, we examined changes in the content of caspases and studied activation of these enzymes in Jeg-3 trophoblasts in various models of their coculturing with NK-92 cells and demonstrated the necessity of direct contact between these cell populations for the activation of caspase-8 and caspase-3 in the trophoblasts. Contact coculturing of the two cell lines resulted in the appearance of the cytotoxic protein granzyme B in Jeg-3 cells that was accompanied by a decrease in the content of this enzyme in NK-92 cells. Distant coculturing of NK-92 and Jeg-3 cells did not trigger initiator and effector caspases characteristic for the apoptosis development in Jeg-3 cells. The observed decrease in the content of procaspases in the trophoblasts may be associated with alternative non-apoptotic functions of these enzymes.

Changes in expression of Ki-67, CD16 and CD56 by natural killer cells from peripheral blood mononuclear cells in the setting of recurrent miscarriage after in vitro culturing in the presence of trophoblast cells and IL-2.

The aim of this research was to assess the proliferative activity of Natural Killer Cells (NK cells) from Peripheral Blood Mononuclear Cells (PBMCs) in the presence of trophoblast cells in women with a history of recurrent miscarriages. We examined the peripheral blood of women with recurrent miscarriage in the proliferative (n = 12) or secretory (n = 13) phase of their menstrual cycle, and pregnant women with a history of recurrent miscarriage at 6-7 weeks of their current pregnancy (n = 14). Controls were fertile non-pregnant women in the proliferative (n = 11) or secretory (n = 13) phase of their menstrual cycle, and pregnant women at 6-7 weeks of a physiologically normal pregnancy (n = 20). We used IL-2 as a factor maintaining PBMCs viability during long-term culturing. We established that culturing in the presence of IL-2 contributed to an increase in the number of CD56+CD16- NK cells and to a decrease in the number of CD56+CD16+ NK cells from PBMCs compared with these numbers before culturing in both healthy women and in women with recurrent miscarriage. After culturing of PBMCs in the presence of trophoblast cells and IL-2 (compared with culturing without trophoblast cells), the intensity of Ki-67 expression by NK cells was reduced in the whole NK cell population (CD3-CD56+), and in the CD56+CD16- and CD56+CD16+ populations of NK cells in women with recurrent miscarriage and in healthy controls. The intensity of CD56 expression was reduced in the presence of trophoblast cells and IL-2 in non-pregnant women with recurrent miscarriage in the secretory versus the proliferative phase of the menstrual cycle.

[The comparative analysis of immunologic techniques of detection of anti-phospholipid antibodies].

The laboratory diagnostic of anti-phospholipid syndrome consists in detection of anti-phospholipid antibodies using technique of enzyme-linked immunosorbent assay namely in detection of anti-cardiolipin antibodies and antibodies to ß2-glycoprotein. In spite of the fact that serological diagnostic plays a key role in diagnosing anti-phospholipid syndrome application of laboratory tests s complicated by their insufficient standardization. The new approach to detection of anti-phospholipid antibodies became application of immune blotting on the basis of polyvinylidenfluoride membrane. As compared with enzyme-linked immunosorbent assay, the advantage of the mentioned technique is in using hydrophobic solid phase for sorption of antigens. The porous structure of polyvinylidenfluoride membrane orientates hydrophilic areas of phospholipids and by that ensures their more dense distribution imitating bi-lipid layer of membranes of living organism. To specify and compare value of different techniques the comparison was implemented concerning the results of measurement of anti-phospholipid antibodies in enzyme-linked immunosorbent assay test-systems of various manufacturers and reagents kits for immune blotting. The collection was assembled including bio-materials from 47 patients with non-cardioembolic ischemic strokes, 20 patients with recurrent thrombosis of deep veins of lower extremities and 50 patients with obstetrics pathology and also 30 healthy donors. In the given serums aKlaIgG, aKlaIgM, aß2glycoprotein I were measured using enzyme-linked immunosorbent assay technique assisted by test-systems of Euroimmun and Orgentes Diagnostica and the samples with the highest titre using immune blotting technique with reagents manufactured by Medipan. On the basis of measurement of anti-phospholipid antibodies by various enzyme-linked immunosorbent assay test-systems the rate of aß2glycoprotein I amounted to 31% in case of Euroimmun reagents kits for enzyme-linked immunosorbent assay, 78% in case of Orgentec Diagnistica test-systems for enzyme-linked immunosorbent assay, aKlaIgG - 2% and 30%, aKlaIgM - 31% and 54% correspondingly. The measurement of anti-phospholipid antibodies using immune blotting technique on Medipan test-systems in bio-samples with the highest titres detected aß2glycoprotein I in all patients, aKlaIgG in 70% and aKlaIgM in 30% of patients. The convergence between three commercial reagents kits varies from 20% to 88%. The standardization of commercial test-systems still to be achieved. The new technique of immune blotting can be appliedjointly with classic techniques ofserological diagnostic of anti-phospholipid syndrome. The absence of algorithms of diagnostic and standardization of different test-systems for detection of anti-phospholipid antibodies prejudices reliability of serological diagnosis of anti-phospholipid syndrome and therefore existence of anti-phospholipid syndrome as a nosologic unit.

[The application of Fluo-3 AM in measurement of level of cytoplasmic calcium in thrombocytes by flow cytofluorometry].

The study was carried out to evaluate possibility of applying technique of thrombin-induced increasing of concentration of Ca in cytoplasm of Fluo-3-colored thrombocytes as an experimental model of studying mechanism of action of anti-thrombocytes medications in vitro. The effect of anti-thrombocyte substances on thrombin-induced increasing of the level of cytoplasmic Ca in thrombocytes was analyzed on example of acetylsalicylic acid. The measurement of concentration of cytoplasmic Ca was implemented using flow cytometry technique with fluorescent probe Fluo-3 AM. It is established that in the given test acetylsalicylic acid inhibits thrombin-induced increasing of cytoplasmic Ca at 0.125-5.0 mk/mol concentrations. This occurrence testifies that in the mechanism of effect of acetylsalicylic acid the suppression of thromboxane path ceases to be a leading one. The proposed methodical approach permits evaluating anti-thrombocite effect of substances according their impact to the level of cytoplasmic Ca in thrombocytes in vitro. However, this approach has a number of limitations preventing wide-spread application of the given technique.

Effect of Factors Secreted by the Placenta on Phenotype of THP-1 Cells Cultured on a 3D Scaffold.

We studied the effects of secretory products of the placenta obtained from women with normal pregnancy and preeclampsia on the expression of surface markers by THP-1 cells cultured on a 3D Matrigel scaffold. Secretory products of third trimester placentas obtained from women with normal pregnancy reduced the relative number of THP-1 cells expressing CD54 and CD14 molecules and expression of CD14 and CD95 molecules by THP-1 cells in comparison with the effect of secretory products first trimester placentas. In parallel, the intensity of CD49d expression by THP-1 cells increased in the presence of secretory products of third trimester placentas in comparison with the first trimester. No differences in the expression of the studied molecules by THP-1 cells under the effect of placentas from women with physiological pregnancy and patients with preeclampsia were found.

Coordinated expression of TNFα- and VEGF-mediated signaling components by placental macrophages in early and late pregnancy.

INTRODUCTION: Mononuclear phagocytes are thought to significantly contribute to cytokine regulation at the maternal-foetal interface, but the role of placental macrophages has been poorly investigated. TNFα and VEGF were demonstrated to have regulatory effects on basic structures of the placenta, particularly the trophoblast and blood vessels. The aims of this study were to determine the expression of TNFα, VEGF and related receptors in placental macrophages, and how does the participation of placental macrophages alter with gestational age in TNFα- and VEGF-mediated signaling. METHODS: Macrophages were isolated from placental villous tissue from normal pregnancies at either 9-12 or 38-40 weeks gestation. Cell surface receptors (TNFR1, TNFR2, VEGFR1, and VEGFR2) and intracellular TNFα and VEGF were quantified by flow cytometry after antibody staining. Basal and stimulated secretion of both cytokines and soluble TNF receptors was quantified by cytometric bead arrays. Secreted VEGFR1 was measured by ELISA. RESULTS: The expression of TNFR1 and VEGFR1 was remarkably variable and did not change from first to third trimester. There was minimal basal TNFα production in the placental macrophages, but nearly all cells in the population produced VEGF. TNFα and VEGF secretion increased with gestational age accompanied by decreased secretion of the antagonists sTNFR1 and sVEGFR. Macrophages isolated from early term placentas were less effective in responding to bacterial endotoxin. Lipopolysaccharide induced increases in the secretion of TNFα, TNFR1, TNFR2, and VEGFR1 but did not affect the production of VEGF. In late pregnancy, a significant correlation was observed between TNFR1 and VEGFR1. DISCUSSION: The progression of pregnancy is accompanied by the concerted increase in TNFα and VEGF secretion and decrease in the production of their soluble receptors, but the expression of cell surface receptors does not depend on gestational age. The observed patterns of basal and stimulated expression of TNFα and VEGF may reflect the dual immune and morphogenetic roles of placental macrophages in gestation. Compatible patterns of TNFR1 and VEGFR1 expression suggest common regulatory pathways for these receptors.

Comparative phenotypic characterization of human cord blood monocytes and placental macrophages at term.

The expression of surface molecules in cord blood monocytes and placental macrophages was studied using flow cytometry. When compared with monocytes, macrophages presented a decrease in HLA-DR and LAP/TGF-ß1 levels and increased expression of alternative activation markers, especially CD206. No difference in the production of the apoptotic factors TRAIL and TWEAK was observed, whereas the levels of cytokine receptors in monocytes were significantly higher than in macrophages. Most remarkable was the difference in the expression of IL-17 and TNFα receptors. A strong correlation between VEGF and TNFα receptors was revealed in both cell populations. The results obtained in this study provide antigenic phenotypes for two related cell populations and outline the feasible functional alterations during tissue macrophage differentiation.

Effects of placental tissue secretory products on the formation of vascular tubules by EA.Hy926 endothelial cells.

The formation of vascular tubules by EA.Hy926 endothelial cells was studied in the presence of placental secretory products from women with normal gestation at early and late periods and with gestosis. The factors secreted by placental tissues at the early stages of placental development stimulated the branching angiogenesis, while the products of the end of pregnancy stimulated nonbranching angiogenesis. In gestosis the placental tissue secreted products stimulating even more intense nonbranching angiogenesis, which manifested by a lesser number of branchings of vascular tubes formed by EA.Hy926 endothelial cells.

Phenotypical characteristics of peripheral blood monocytes in normal pregnancy and gestosis.

Monocyte migration from the peripheral blood to the uterine decidual tissue is essential for the regulation of placental development. We evaluated the phenotypical changes in the peripheral blood monocytes in pregnant women. The peripheral blood counts of monocytes expressing CD11b, CD47, and integrin ß7 were elevated in women with normal gestation in comparison with nonpregnant women; the intensity of CD62P, CD11b, CD11c, CD29, CD31, and CD54 expression was higher in pregnancy. The counts of monocytes expressing adhesion molecules were similar in normal pregnancy and gestosis. Gestosis was characterized by higher counts of monocytes expressing IFN-γ receptor (CD119) and more intense expression of this receptor. Changes in the monocyte phenotype can promote their adhesion to the uterine vascular endothelium and further migration of these cells to the decidual tissue.

Effects of placental secretory factors on cytokine production by endothelial cells.

We studied the effects of soluble products of the placental tissue from women with normal pregnancy and gestosis on the cytokine secretion by endothelial EA.Hy926 cells. The secretory products of the placental tissue induced the production of angiogenin, bFGF, IL-8, MCP-1, and RANTES by endothelial cells. The secretion of bFGF by EA.Hy926 cells increased, while IL-8 secretion decreased under the effects of factors produced by the placental tissue in gestosis but not in normal pregnancy. This could be aimed at reduction of inflammation intensity in the placental tissue and maintenance of endothelial and trophoblast cells viability.

In vitro expression of vascular endothelial growth factor and its receptors by placental macrophages.

The expression of VEGF and membrane-bound and soluble forms of the VEGF-R1 receptor in cultured placental macrophages (trimesters I and III of pregnancy) was studied by flow cytometry, cytometric bead array, and ELISA. Nearly all population of placental macrophages (98%) was capable of producing VEGF during the early and late gestational periods. However, the expression of cellular VEGF-R1 varied from 3.4 to 92%. VEGF secretion was relatively low in the first and third trimesters (0.5 and 1.1 pg/10(5) cells, respectively). Cultured placental macrophages produced soluble receptor sVEGF-R1 in the first and third trimesters (86.4 and 36.4 pg/10(5) cells, respectively). Stimulation with LPS was followed by a 4-fold increase in sVEGF-R1 secretion. Our results indicate that placental macrophages are involved in the autocrine and paracrine regulation in chorionic villi. The data suggest that these cells have a physiological and pathogenetic role in gestation.

Changes in the profiles of chemokines secreted by endothelial cells and monocytes under different coculturing conditions.

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.

Expression of thrombospondin-1 gene mRNA and protein in the placenta in gestosis.

The expression of TSP-1 gene mRNA and TSP-1 protein in the placental tissue was studied during normal pregnancy and in gestosis. The formation of placental tissue in normal gestation was associated with expression of TSP-1 gene mRNA and of TSP-1 protein. Gestosis was associated with inflammatory reaction in the placenta characterized by increased counts of lymphocytes and macrophages in the villous stroma and involution degenerative changes in tissue. Disorders in placental villi maturation and branching in gestosis were paralleled by hyperexpression of TSP-1 gene mRNA by placental cells and hyperexpression of TSP-1 protein predominating in the stromal elements of terminal villi and near villous vessels.

Regulatory mechanisms for apoptosis in placental tissue during normal pregnancy and gestosis-complicated pregnancy.

The localization of apoptosis and expression of proapoptotic and antiapoptotic factors by the placental tissue were compared during normal pregnancy and gestosis-complicated pregnancy. The degree of apoptosis did not differ in the third trimester of normal pregnancy and gestosis-complicated pregnancy. Increased expression of Fas, caspase-8, and caspase-3 in placental tissue during normal pregnancy was shown to contribute to the suppression of angiogenesis and growth of placental tissue. No differences were found in the expression of FasL (CD95L), caspase-2, caspase-9, and Mcl-1 by placental cells during normal pregnancy and gestosis-complicated pregnancy. Increased expression of TRAIL by trophoblast cells is a protective mechanism from apoptotic signals of maternal cytotoxic lymphocytes and NK cells during gestosis.

Expression of VEGF and VEGF-R3 receptor by placental endothelial cells in health and gestosis.

We carried out a comparative analysis of changes in VEGF secretion and expression of VEGF-R3 receptor by placental endothelial cells in health and gestosis and of changes in VEGF-R3 expression by EA.hy926 human endothelial cells during culturing with supernatants conditioned by placental explants from women with normal pregnancy and patients with gestosis. Reduced secretion of VEGF and expression of VEGF-R3 by placental endothelial cells in gestosis can be caused by functional deficiency of the endothelial cells and low viability of endothelial cells.