Nuclear magnetic resonance-based assays in immunodiagnostics

During the research, two different approaches for NMR-based assays were developed. Analytical performance of designed (table) method is acceptable for immunodiagnostics test-systems.This work was supported by Russian Science Foundation (project № 17-15-01116)

Immunoregulatory potential of pregnancy-specific β1-glycoprotein

The embryo, being half an antigenically “foreign” organism, should elicit a maternal immune response. During evolution, however, the mechanisms ensuring successful development of pregnancy have been formed. In particular, among factors providing immune tolerance during pregnancy are some proteins associated with pregnancy. The pregnancy-specific β 1-glycoprotein (PSG, PSG1; SP1; PSβG1) is a dominant fetoplacental protein produced by cyto- and syncytiotrophoblast cells, and it exhibits immunosuppressive properties. Our team of authors possesses a patented method for obtaining native human PSG preparation from blood serum of pregnant women, a mixture of PSG1, PSG3, PSG7, PSG9, and their isoforms and precursors. This review presents an analysis of our results for the period from 2015 to 2020. We studied the immunoregu-latory effects of the obtained PSG preparation at concentrations comparable to those observed in pregnancy (1, 10, 100 |ag/mL). The study was performed with peripheral blood cells obtained from non-pregnant women. It was found that PSG significantly increased the percentage of adaptive Tregs in vitro, as well as expression of CTLA-4, GITR, and production of IL-10 by these cells. It has been shown that PSG has a stimulating effect upon indoleamine-2,3-dioxygenase (IDO) activity of peripheral blood monocytes. For Th17 cells, we have demonstrated that PSG can suppress differentiation and proliferation of these cells, along with reduced production of critical proinflammatory cytokines (IL-8, IL-10, IL-17, IFNγ, MCP-1, TNF α). As for the memory T cells, PSG suppressed CD25 expression and IL-2 production by them, along with simultaneous decreased expression of Gfi1, hnRNPLL genes, thus preventing the formation of the “mature” CD45R0 isoform. PSG has been shown to inhibit naive T cells’ conversion to the terminally differentiated effector subpopulation of helper T cells. When analyzing PSG effects upon cytokine profile of immunocompetent cells, it was found that the protein predominantly suppresses the Th1 cytokine production by the studied cell types, and regulates the Th2 cytokine production in divergent manner. The results obtained are consistent with general concept of immunosuppression during pregnancy. Thus, PSG could be one of the factors preventing formation and implementation of immune response to placental antigens

ROLE OF GLYCODELIN IN REGULATION OF MYELOIDDERIVED SUPPRESSOR CELL DIFFERENTIATION

Glycodelin (PP14, PAEP, alpha-2-microglobulin, dimeric glycoprotein with molecular weight of 42 to 56 kDa) is considered as a reproductive tissue receptivity marker. Despite that glycodelin immunosuppressive effects are well-known there still remains uncovered its role in myeloid suppressor cell (MDSC) regulation. MDSC represent the heterogeneous population of immature myeloid cells that acquire suppressor phenotype while inhibiting the immune response under the pathological states. MDSC are known to play an essential role in supporting the immune tolerance in pregnancy and at transplantation. Our hypothesis suggests that glycodelin is capable of inducing the MDSC formation as the level of these cells is elevated during the successful pregnancy, whereas the spontaneous abortion and progression of eclampsia are associated with low circulating glycodelin. Therefore, the aim of the work was to analyze the role of recombinant glycodelin in physiological concentrations in regulation of MDSC differentiation. Peripheral blood mononuclear cells of donor volunteers were separated via centrifugation on density gradient of 1,077 g/cm3 (Ficoll-Hypaque, Sigma-Aldrich) to obtain MDSC generation in vitro. Then cells obtained were cultured in 24-well plate at a concentration of 1 × 106 cell/ml in complete medium with cytokines IL-6 (20 ng/ml), GM-CSF (40 ng/ml) therein for 14 days at 37 °C and 5% CO2. Medium replacement was made by 7th day in culture followed by cytokine re-introduction, and on the 11th day recombinant glycodelin in physiological concentrations (0,2; 2 mkg/ml) was applied while the pharmacological concentration was 50 mkg/ml. The M-MDSC (LinHLA-DRCD33+CD11b+CD14+CD66b- ) and PMN-MDSC (LinHLA-DRCD33+CD11b+CD14- CD66b+) level was evaluated in cultures using flow cytometry (СytoFlexS (Beckman Coulter)) and “R&D Systems” antibodies according to standard protocol. Statistical data processing was realized with GraphPad Prizm software using Friedman test. It was found that glycodelin did not significantly affect cell viability being assessed with flow cytometry (PI). It was revealed that high GdA concentration (50 mkg/ml) being pharmacological did not render significant effect on MDSC differentiation. Meanwhile, glycodelin in concentrations correspanding the healthy pregnancy (0,2; 2 mkg/ml) was stated to increase the MDSC percentage in induced cultures of human mononuclear cells. When analyzing the subsets it was disclosed that this effect was conditioned by the increase in PMN-MDSC level while the M-MDSC level remained significantly unchanged. This result could be interpreted as glycodelin fetoprotective effect as the increase of the PMN-MDSC level is associated with the suppression of the immune response to paternal antigens. The PMN-MDSC level is known to be elevated in peripheral blood of healthy pregnant women at all the stages of pregnancy as compared to nonpregnant subjects whereas the M-MDSC amount remains unaltered. Meanwhile, patients with miscarriage demonstrated more that by 30% lowering in the MDSC amount in blood and endometrium and in I trimester, in particular. During the physiological pregnancy PMN-MDSC accumulate in placenta, but at spontaneous abortion their number is found to be declined. Placental PMN-MDSC efficiently suppress the T-cell response while concurrently polarizing the CD4+ lymphocytes in Th2 phenotype. PMN-MDSC are suggested to play an essential role in inducing and supporting the tolerance to fetal antigens that allows considering these as promising target of therapeutical manipulation in pregnancy complications. As a whole, we have originally demonstrated the GdA effect on MDSC differentiation

GRAPHENE OXIDE NANOPARTICLES IN THE REGULATION OF THE OXIDATIVE ACTIVITY OF HUMAN MONOCYTES

Graphene-based materials have an opportunity for use in biomedicine, thanks to their properties. Nevertheless, due to its cytotoxic effects, the use of graphene-based drugs is problematic. However, the surface modification of graphene oxide (GO) nanoparticles with a polyethyleneglycol (PEG) is one way to reduce the harmful effects of graphene on cells. Applying nanoparticles implies their interaction with the immune system, which protects the body. Monocytes are innate immunity cells and the first line of defenсe of the human organism from microorganisms and other alien objects. One of the monocytes’ reactions to a stimulus of any nature is to produce reactive oxygen species (ROS). Published data shows an incomplete picture of modified graphene oxide nanoparticles’ effects on ROS formation by human monocytes. Thus, it was essential to evaluate the pegylated graphene oxide (GO-PEG and GO-8armedPEG) effect on ROS production by human monocytes, assessed by the luminol-dependent chemiluminescence (LCL). The objects of the study were CD14+-cells isolated from mononuclear cells of healthy donors. ROS production was stimulated by opsonized zymosan (OZ), spontaneous LCL was used as a control. PEG-modified (GO-PEG and GO-8armedPEG) GO nanoparticles with sizes of 100-200 nm (“small”) and 1-5 μm (“big”) with PEG covering ~ 20% were used at concentrations of 5 and 25 μg/ml. The study showed that small size nanoparticles at a low concentration of 5 μg/ml and big nanoparticles coated with 8-armed PEG at both concentrations have a significant suppressive effect on spontaneous ROS production. In the stimulated LCL reaction variant, it was found that small nanoparticles (25 μg/ml) also have a suppressive effect on ROS production, such as big-sized particles coated with linear PEG at the same concentration. Thus, we have established for the first time that graphene oxide nanoparticles functionalized with PEG are capable of inhibiting the ROS production by human monocytes, and therefore, we can speak of the antioxidant activity of GO-PEG

Effect of short PSG peptide fragments on the cytokine profile in Wistar rats during allogeneic transplantation <i>in vivo</i>

Pregnancy-specific beta-1-glycoprotein (PSG) is a protein with pleiotropic biological effects, particularly immunoregulatory and immunosuppressive potential. The use of recombinant PSG may exert therapeutic effects in experimental animals with induced autoimmune diseases. Recently, a search for the biological effects of short linear motifs (SLiMs) has become a new strategy for designing the pharmacological compounds. Tetrapeptide regions have been identified in the primary structure of several PSGs: YQCE, YECE and YACS, these SLiMs exhibit immunomodulatory activity. The aim of our study was to evaluate the prospectives for usage of PSG peptide fragments as pharmacological agents to modulate transplant immunity. We used an original model of host-versus-graft response in male Wistar rats transplanted with bone marrow, without prior conditioning treatment of recipients. We used a cocktail of the PSG peptide fragments administered to Wistar rats in the course of allogeneic bone marrow transplantation (BM) in dynamic manner, evaluating the cytokine profile as an integral index of immune response. Cytokine levels were determined by multiplex method using Bio-Plex ProTM Rat 23-Plex kit. Statistical processing of the data was performed by means of two-way analysis of variance and Tukey’s post hoc test for multiple comparisons. We have found that the levels of pro-inflammatory cytokines (IFNγ, IL-1α, IL-1β, IL-18), as well as the contents of G-CSF, GM-CSF and IL-7 were increased in the animals injected with BM only. In the group of animals injected with BM + PSG peptides, an increase in IFNγ, IL-6, TNFα was observed, which decreased by the end of the experiment. Increased levels of antiinflammatory cytokines IL-4 and IL-13 were detected in blood serum of the animals on day +14. Moreover, administration of PSG peptides also led to increase in IL-2, M-CSF, MCP-1, and RANTES levels on day 14 from the beginning of the experiment, and to a gradual decrease in their levels till the end of the experiment. Meanwhile, control group showed a marked tendency for increase of these and other cytokines. Thus, it was shown that the use of PSG peptides upon development of immune response to BM allograft may promote a return to normal levels for the most cytokines studied, thus presuming the immunopharmacological potential of these peptides. The obtained data can be used to develop a pharmacological preparation of the studied peptides to correct the imbalance of immune system

THE ROLE OF PREGNANCY-SPECIFIC GLYCOPROTEIN IN REGULATION OF MOLECULAR GENETIC DIFFERENTIATION MECHANISMS OF IMMUNE MEMORY T CELLS

The role of pregnancy-specific β1-glycoprotein (PSG) in the regulation of molecular genetic factors determining the functional activity of naїve T cells and T cells of immune memory in vitro was studied. Human PSG was isolated with a proprietary immuno-purification method using a biospecific sorbent followed by removing of immunoglobulin contamination with a HiTrapTM Protein G HP column. Physiological concentrations of PSG were used in the experiments. They corresponded to PSG levels in the peripheral blood of pregnant woman: 1, 10 and 100 μg/ml (I, II, III trimester, respectively). The objects of study were monocultures of naїve T cells (CD45RA+) and memory T cells (CD45R0+), obtained by immunomagnetic separation from the peripheral blood of women of reproductive age.It was established that at the level of naїve T cells (CD45RA+) PSG inhibited the expression of CD28 (1, 10, 100 μg/ml) and CD25 (100 μg/ml), without affecting the interleukin-2 (IL-2) production by these cells. At the same time, PSG in all concentrations studied suppressed the expression of CD25 at the immune memory T-cell (CD45R0+) surface but increased the IL-2 production. Expression of U2af1l4, Gfi1, hnRNPLL genes regulating the alternative splicing of the Ptprc gene encoding CD45 was also evaluated. It was found, that PSG reduced the expression of the Gfi1 (1, 10, 100 μg/ml), hnRNPLL (10, 100 μg/ml) genes, but increased the expression of the U2af1l4 gene (1, 10, 100 μg/ml) in the naїve T cells. It was shown that at the immune memory T-cells’ level the effects were similar, with PSG rendering them in all concentrations used. The revealed changes in the mRNA transcription of U2af1l4, Gfi1 and hnRNPLL genes in the studied T cell subsets may lead to the inhibition of CD45 “mature” isoform formation – CD45R0.Thus, PSG reduces the functional activity of naїve T cells and immune memory T cells associated with the expression of costimulation/activation molecules CD25 and CD28 and is involved in the regulation of Ptprc gene alternative splicing, which determines the ratio of CD45 molecule variants. Apparently, using these mechanisms, PSG regulates the functional activity of the memory T cell circulating pool, which is potentially capable of carrying out antigen-specific cytotoxic reactions against fetal antigens in vivo. In general, the data obtained broadens the notion of the PSG role in the regulation of molecular-genetic mechanisms of naїve T cells and immune memory T cells differentiation

Application of NMR for quantification of magnetic nanoparticles and development of paper-based assay

H1 NMR relaxometry is a method that is extremely sensitive to the presence of magnetic nanoparticles, which significantly affect the transverse relaxation time of the water proton. Accordingly, the use of magnetic nanoparticles as labels allows detection of even extremely small amounts of the test substance. This paper analyzes the prospects for applying the method of solid-phase NMR-relaxometric determination of biologically active molecules. The nitrocellulose membranes are chosen as a solid phase and nanoparticles based on iron core with a carbon shell are used as magnetic labels. The possibility of detecting small concentrations of magnetic particles in porous medium is demonstrated. Finally, the ability to detect extremely low concentrations of an analyte, in this case, streptavidin protein (0.5 ng/ml to 100 ng/ml), which is actively used in various fields of biology and medicine, is demonstrated. © Published under licence by IOP Publishing Ltd.Russian Science Foundation, RSF: 17-15-01116The work was carried out within the Russian Science Foundation project 17-15-01116. equipment of the Ural Center for Shared Use Modern nanotechnology UrFU was used

THE ROLE OF GLYCODELIN IN THE REGULATION OF THE IMMUNE SYSTEM IN THE CONTEXT OF DEVELOPING PREGNANCY

The review presents data on the role of glycodelin A (GdA, PP14, α2-PEG, EP15, PEP, AUP, PAEP) in regulating the functions of the immune system in the context of the formation of feto-maternal immune tolerance during pregnancy.Glycodelin was first isolated and identified in 1976 by D.D. Petrunin. and Tatarinov Yu.S. with colleagues as a new placenta antigen, which was named chorionic α2-microglobulin. Since then, a huge amount of scientific data has been obtained on the structure, properties, and biological effects of this glycoprotein. This protein has four differentially glycosylated isoforms, namely GdA, GdF, GdC, and GdS, which are secreted in different parts of the reproductive system.The most studied isoform, glycodelin A (GdA), is secreted by the decidual glandular epithelium and accumulates in the amniotic fluid and maternal serum during pregnancy. GdA level is a sign of endometrial fertile function. GdA has diverse biological effects, in particular, it modulates endocrine function and differentiation of trophoblast cells.The role of GdA in the regulation of the immune system is to inhibit the proliferation of T and B lymphocytes, suppress the cytotoxicity of NK cells, induce apoptosis of activated CD4+ cells, monocytes and NK cells, inhibit the activity of cytotoxic T lymphocytes and suppress the functional activity of macrophages and dendritic cells. In addition, GdA increases the level of regulatory T cells, regulates the Th1/Th2 balance towards Th2, and induces a tolerant phenotype of dendritic cells.The immunomodulating activity of GdA depends on the degree of its glycosylation, which, in turn, is associated with the preparation obtaining method. Therefore, the review analyzed the features of the immunomodulating effects of the native and recombinant types of glycodelin on the immune system cells.However, the cumulative effects of GdA on the cells of the immune system make it possible to consider it as one of the main factors shaping the feto-maternal immune tolerance during pregnancy. It is also important to note that clinical studies have revealed a correlation between low levels of circulating GdA and repetitive spontaneous abortions that confirms the importance of this protein in fetoprotection.In general, it is obvious that GdA has the potential as a medicinal preparation for autoimmune deseases treatment, post-transplant complications and in vitro “reprogramming” of autoreactive T-cell clones for further cellular immunotherapy