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The flow cytometry method could support physicians' decisions in the diagnosis and treatment monitoring of immunodeficient patients. Most clinical recommendations are focused on the search for alterations in T- and B-lymphocyte subsets, less commonly natural killer (NK) cells and granulocytes. While reference values for clinically meaningful lymphocyte subsets have been published ubiquitously among numerous countries, we have not found significant data for a population of adult Polish habitats; thus we determined reference values for T, B, and NK subsets according to sex and age. The female group showed a higher percentage of lymphocytes (CD45++), T helper lymphocytes with a higher absolute count, as well as CD4/CD8 ratio, marginal zone-like B cells, class-switched B cells, and CD21low B cells than the male group. The male group was found to have elevated percentages of naïve B lymphocytes, transitional B cells, and plasmablasts. A weak positive correlation with age was found among double positive T lymphocytes, natural killer T cells (NKT) lymphocytes, and CD21low B cells. A negative correlation with age for double negative T lymphocytes, marginal zone-like B cells, and plasmablasts was noted. The results indicated the importance of creating distinct reference ranges regarding sex and age concerning immunophenotype. [ABSTRACT FROM AUTHOR]

Problem. Natural killer (NK) cells are the most abundant leukocyte population in the uterus. The interactions of the maternal NK expression of killer cell immunoglobulin-like receptors (KIRs) and human inhibitory receptor Ig-like transcript (ILT) with fetal HLA determine the activation of NK cells and pregnancy outcomes. Moreover, dNK cells release numerous angiogenic factors including VEGF. Our previous study showed that sildenafil citrate (SC) significantly decreased peripheral blood NK (pbNK) cell activity and improved intrauterine blood flow, which correlated with a successful pregnancy outcome. Thus, in this study, we investigated whether SC influenced the expression of KIR or ILT receptors on induced decidual NK (idNK), the apoptosis of cells, and VEGF-A production in the culture supernatants of idNK cells. Method of Study. pbNK cells from 24 healthy women and 23 women with RPL were converted to idNK cells under hypoxia, IL-15, and TGF-β conditions. The cultures were prepared with or without SC. Changes in KIR2DL1 (CD158a), NKG2A (CD159a), ILT-2 (CD85j), and ILT-4 (CD85d) expression on CD56+CD16- cells and their apoptosis were determined via flow cytometry. VEGF-A level was established in culture supernatants with the ELISA method. Results. KIR2DL1 and ILT-2 expression on idNK cells was higher in healthy women than in RPL patients. Sildenafil enhanced NKG2A expression in RPL patients. VEGF concentration was higher in fertile woman idNK cell cultures. idNK cells were more sensitive for necrosis in RPL than in fertile women. SC did not influence VEGF production or idNK cell apoptosis. Conclusions. A combination of hypoxia, IL-15, and AZA promotes the conversion of pbNK into idNK cells CD56+CD16--expressing KIR receptors and produces VEGF. Alterations in KIR2DL1 and ILT-2 expression as well as impaired VEGF production were associated with RPL. SC affects NKG2A expression on RPL idNK cells. SC had no effect on VEGF release or idNK cell apoptosis. [ABSTRACT FROM AUTHOR]

In our previous study, we showed that sildenafil citrate (SC), a selective PDE5A blocker, modulated NK cell activity in patients with recurrent pregnancy loss, which correlated with positive pregnancy outcomes. It was found that NK cells had a pivotal role in decidualization, angiogenesis, spiral artery remodeling, and the regulation of trophoblast invasion. Thus, in the current study, we determined the effects of SC on angiogenic factor expression and production, as well as idNK cell activity in the presence of nitric synthase blocker L-NMMA. Methods: NK cells (CD56+) were isolated from the peripheral blood of 15 patients and 15 fertile women on MACS columns and cultured in transformation media containing IL-15, TGF-β, and AZA—a methylation agent—for 7 days in hypoxia (94% N2, 1% O2, 5% CO2). Cultures were set up in four variants: (1) with SC, (2) without SC, (3) with NO, a synthase blocker, and (4) with SC and NO synthase blocker. NK cell activity was determined after 7 days of culturing as CD107a expression after an additional 4h of stimulation with K562 erythroleukemia cells. The expression of the PDE5A, VEGF-A, PIGF, IL-8, and RENBP genes was determined with quantitative real-time PCR (qRT-PCR) using TaqMan probes and ELISA was used to measure the concentrations of VEGF-A, PLGF, IL-8, Ang-I, Ang-II, IFN–γ proteins in culture supernatants after SC supplementation. Results: SC downregulated PDE5A expression and had no effect on other studied angiogenic factors. VEGF-A expression was increased in RPL patients compared with fertile women. Similarly, VEGF production was enhanced in RPL patients' supernatants and SC increased the concentration of PIGF in culture supernatants. SC did not affect the expression or concentration of other studied factors, nor idNK cell activity, regardless of NO synthase blockade. [ABSTRACT FROM AUTHOR]

Sildenafil citrate (SC), a PDE5 inhibitor, a drug for erectile dysfunction (ED) and pulmonary hypertension (PAH), was found to exert a positive effect on pregnancy outcomes when administered intravaginally before conception. In our previous studies, sildenafil increased endometrial thickness and significantly decreased peripheral blood NK cell activity after the intravaginal administration in women with recurrent pregnancy loss (RPL). No data are available to confirm the effect of sildenafil on maternal T cell populations involved in shaping fetal-maternal tolerance and NK cell activity. Thus, the present study aimed to establish if SC influences NKT cells or the axis of Th17/Treg cells and Th1/Th2 cytokine production. Materials and methods: Twenty-one healthy fertile women and twenty-two nonpregnant women with idiopathic RPL were studied. The ELISA method was used to evaluate the production of cytokines, including IL-2, IL-12p40, IL-4, IL-10, IL-6, IL-17, IL-21, TGF-β, TNF-α, and IFN-γ in PBMC culture supernatants before and after supplementation with the physiological concentration of SC. The percentages of NKT (CD56+CD3+CD44+CD161+), Treg (CD4+CD25+FOXP3+) and Th17 (CD4+CD25+IL-17A+) cells were determined with flow cytometry method. Results: Unexpectedly, we found that the PBMCs of patients with RPL produced a significantly lower level of inflammatory cytokines (TNF-α and IL-6) and a higher level of anti-inflammatory cytokines (TGF-β and IL-10). SC significantly decreased IL-6, IL-12 and increased TGF-β cytokine concentration in fertile women. In the case of RPL patients' PBMCs, SC improved the production of TNF-α and IL-10. Conclusions: Lower concentration of proinflammatory cytokines in idiopathic RPL women compared to fertile women might suggest the exhaustion of the immune system. The emphasized production of IL-10 by SC partially explains the previously observed downregulation of NK cell activity in RPL patients. The immunomodulatory effect of the drug might be utilized in anti-inflammatory therapies and help achieve positive pregnancy outcomes in women with reproductive failure due to a Th1/Th2 imbalance. [ABSTRACT FROM AUTHOR]