Evangelos Ntrivalas

Plasma Components Affect Accuracy of Circulating Cancer-Related MicroRNA Quantitation

Circulating microRNAs (miRNAs) have emerged as candidate biomarkers of various diseases and conditions including malignancy and pregnancy. This approach requires sensitive and accurate quantitation of miRNA concentrations in body fluids. Herein we report that enzyme-based miRNA quantitation, which is currently the mainstream approach for identifying differences in miRNA abundance among samples, is skewed by endogenous serum factors that co-purify with miRNAs and anticoagulant agents used during collection. Of importance, different miRNAs were affected to varying extent among patient samples. By developing measures to overcome these interfering activities, we increased the accuracy, and improved the sensitivity of miRNA detection up to 30-fold. Overall, the present study outlines key factors that prevent accurate miRNA quantitation in body fluids and provides approaches that enable faithful quantitation of miRNA abundance in body fluids.

Expression of killer immunoglobulin-like receptors on peripheral blood NK cell subsets of women with recurrent spontaneous abortions or implantation failures.

Problem:  Decidual natural killer (NK) cells express inhibitory receptors (killer immunoglobulin‐like receptors, KIRs), which bind to ligands on trophoblast cells (human leucocyte antigen, HLA‐C). This interaction appears to block NK cytotoxicity against trophoblast cells. In this study, we investigated the expression of inhibitory and activating receptors in peripheral blood NK cells of women with recurrent spontaneous abortion (RSA) or implantation failures.

Immune etiology of recurrent pregnancy loss and its diagnosis.

Recurrent Spontaneous Abortion of Immunological Origin (RSAI) is currently diagnosed by the occurrence of 2–3 consecutive miscarriages of unknown origin. The psychological trauma incurred by these events is a serious ailment which may be potentially avoided if a method of analysis is derived which may forecast these events and in turn prevent them from occurring. This review intends to examine studies of recurrent spontaneous abortion (RSA) which use laboratory diagnosis and also studies of RSA that do not use laboratory diagnosis. We believe that when laboratory results are incorporated into the diagnosis of RSA/RSAI that treatment is highly successful whereas the absence of laboratory results severely hinders the effectiveness of treatment. It is worth noting that correlating treatment versus outcome is imprudent because of the multiple variables involved in patient cases. It is not imprudent, however, to say that incorporation of laboratory data is essential when diagnosing RSA/RSAI.

Women with multiple implantation failures and recurrent pregnancy losses have increased peripheral blood T cell activation.

Citation Yang KM, Ntrivalas E, Cho HJ, Kim NY, Beaman K, Gilman‐Sachs A, Kwak‐Kim J. Women with multiple implantation failures and recurrent pregnancy losses have increased peripheral blood T cell activation. Am J Reprod Immunol 2010

ORIGINAL ARTICLE: Correlation Between Natural Cytotoxicity Receptors and Intracellular Cytokine Expression of Peripheral Blood NK Cells in Women with Recurrent Pregnancy Losses and Implantation Failures

Problem  Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. We investigated whether women with recurrent pregnancy losses (RPLs) and implantation failures have aberrant correlation between NCRs and intracellular cytokine expression of NK cells.

The N-terminus Domain of the a2 Isoform of Vacuolar ATPase Can Regulate Interleukin-1β Production from Mononuclear Cells in Co-culture with JEG-3 Choriocarcinoma Cells

a2V‐ATPase is the a2 isoform of vacuolar ATPase and is expressed in human trophoblast cells. a2V‐ATPase resides as a 70‐kDa molecule in intracellular vesicles. Upon cell stimulation, it migrates to the surface as a 50‐kDa molecule, after a 20‐kDa portion [N‐terminus domain of the a2V‐ATPase (a2NTD)] is cleaved and secreted to the extracellular environment. Previous studies showed that a2NTD‐regulated cytokine production from stimulated T cells. The aim of this study was to determine if a2NTD can regulate cytokine production from immune cells that were in contact with JEG‐3 cells.

Expression of Natural Cytotoxicity Receptors and a2V-ATPase on Peripheral Blood NK Cell Subsets in Women with Recurrent Spontaneous Abortions and Implantation Failures

Problem  Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. a2V‐ATPase is expressed on subsets of PBMC and regulates the extracellular environment, which facilitates NK cytotoxicity or cytokine secretion. In this study, we aim to investigate the expression of NCRs and a2V‐ATPase in peripheral blood NK cells of women with recurrent spontaneous abortions (RSA) or implantation failures.

ORIGINAL ARTICLE: Women with Multiple Implantation Failures and Recurrent Pregnancy Losses have Increased Peripheral Blood T Cell Activation

Citation Yang KM, Ntrivalas E, Cho HJ, Kim NY, Beaman K, Gilman‐Sachs A, Kwak‐Kim J. Women with multiple implantation failures and recurrent pregnancy losses have increased peripheral blood T cell activation. Am J Reprod Immunol 2010

An In Vitro Coculture Model to Study Cytokine Profiles of Natural Killer Cells During Maternal Immune Cell-Trophoblast Interactions:

Objectives: The cytokine milieu at the implantation site plays a role in human pregnancy. Th2 cytokines, such as interleukin (IL)-4 and IL-10, stimulate growth and development of placenta, whereas Th1 cytokines, such as tumor necrosis factor-alpha (TNF-α), are associated with pregnancy complications. Natural killer (NK) cells predominate at the implantation site. The aim of the present study is to investigate cytokine expression in NK cells when they are in close contact with JEG-3 trophoblast-like cells using an in vitro coculture model. Methods: Female peripheral blood mononuclear cells (PBMCs) were cocultured with JEG-3 cells for 24 hours. PBMCs were harvested from the cocultures and stimulated with 25 ng/mL phorbol myristate acetate and 1 μmol/mL ionomycin in the presence of 2 μmol/mL monesin. NK cells were analyzed by flow cytometry for intracellular TNF-α, interferon-gamma (IFN-γ), and IL-4 and IL-10 cytokines. Controls were PBMCs cultured without JEG-3 cells. Results: The proportion of CD56+ /TNF-α+ NK cells was significantly decreased when they were in coculture with JEG-3 cells (26.1%) as compared to without JEG-3 cell coculature (40.8%) (P < .05). There was no difference in the proportion of CD56+ NK cells expressing intracellular IFN-γ, IL-4, and IL-10. Down-regulation of CD56+ /TNF-α+ NK cell levels was dependent on direct cell-to-cell contact between NK cells and JEG-3 cells. The expression of human leukocyte antigen (HLA)-G on trophoblast cell lines did not affect CD56+ /TNF-α+ NK cell levels under these experimental conditions. Conclusion: We report that JEG-3 cells induce down-regulation of intracellular CD56+ /TNF-α+ NK cell levels. It is speculated that trophoblasts may secure themselves from NK cell cytotoxicity via this mechanism.

The Placental Immunomodulatory Cytokine Regeneration and Tolerance Factor is also Expressed by Both Human Cycling and Early Pregnant Endometrium

Problem:  Regeneration and tolerance factor (RTF) has been recently suggested to contribute to the control of fetal‐ablating immunity at the maternal–fetal interface through the induction of T helper 2 (Th2)‐dominated response. The protein consists of a membrane‐associated domain and an extracellular portion which is proteolitically cleaved to yield a soluble peptide. In humans, it has been shown to be expressed by invading cytotrophoblasts and decidual lymphoid cells, to be increased on peripheral blood B lymphocytes during a normal gestation and on circulating natural killer cells during unsuccessful pregnancies. However, the expression of RTF in other cell types and, specifically, in non‐hematopoietic maternal cells of the human uterus has not been characterized in detail. Thus, we have specifically studied the expression and modulation of the cytokine in human endometrium obtained in different phases of the cycle and in early pregnancy.