Effect of Short PSG Peptides on Inflammatory Markers in Allogeneic Bone Marrow Cell Transplantation in Wistar Rats.

Short linear peptide fragments of placental trophoblastic ß1-glycoprotein (PSG) (YECE, YQCE, YVCS, and YACS) were studied in the context of their immunomodulatory effects at the level of inflammatory markers. The original host-versus-graft model was used in male Wistar rats without prior conditioning of recipient bone marrow. A composition of PSG peptide fragments was injected to animals after allogeneic transplantation of bone marrow cells in a dynamic experiment, inflammatory markers α1-acid glycoprotein (AGP, orosomucoid), α2-macroglobulin (α2M) were assayed by ELISA, and biochemical parameters (total protein, glucose, creatinine, and urea) were measured. The levels of α2M and AGP increased in response to allotransplantation, whereas administration of PSG peptides normalized serum α2M levels by the end of the experiment. The decrease in α2M level coincided with the independent effect of PSG peptide administration. The levels of total protein, glucose, creatinine, and urea in rat serum after allotransplantation were reduced throughout the experiment. Administration of PSG peptides contributed to normalization of serum total protein, creatinine, and urea levels by the end of the experiment. Administration of PSG peptides after allogeneic transplantation of bone marrow suspension contributed to normalization of the levels of α2M, total protein, creatinine, and urea, which can be interpreted as an anti-inflammatory effect of these peptides.

Interaction of Graphene Oxide Nanoparticles with Human Mononuclear Cells in the Cell-IQ System.

The interaction of graphene oxide nanoparticles with human peripheral blood mononuclear cells was studied using the Cell-IQ continuous monitoring system for living cells. We used graphene oxide nanoparticles of various sizes coated with linear or branched polyethylene glycol (PEG) in concentrations of 5 and 25 µg/ml. After 24-h incubation with graphene oxide nanoparticles, the increase in the number of peripheral blood mononuclear cells at visualization points decreased; nanoparticles coated with branched PEG more markedly suppressed cell growth in culture. In the presence of graphene oxide nanoparticles, peripheral blood mononuclear cells retained high viability in culture after daily monitoring in the Cell-IQ system. The studied nanoparticles were engulfed by monocytes and the type of PEGylation had no effect on this process. Thus, graphene oxide nanoparticles reduced the increase in peripheral blood mononuclear cell mass during dynamic observation in the Cell-IQ system without reducing their viability.

Interaction of Human Dendritic Cells with Graphene Oxide Nanoparticles In Vitro.

We studied the effect of graphene oxide nanoparticles on the differentiation of human dendritic cells and uptake of nanoparticles by these cells in vitro. The objects of the study were mononuclear cells from healthy donors induced into the phenotype of dendritic cells by cytokines (IL-6 and GM-CSF). We used graphene oxide nanoparticles of different sizes functionalized with linear or branched PEG (P-GO or bP-GO) in concentrations of 5 and 25 µg/ml. It was found that graphene oxide nanoparticles did not affect the viability and percentage of dendritic cells in the culture. However, P-GO nanoparticles (25 µg/ml) suppressed the expression of CD83 on the surface of dendritic cells in cultures, thereby suppressing cell differentiation. Dendritic cells internalized P-GO nanoparticles, particles in high concentration were more actively engulfed, but the size of the particles and the type of PEG did not affect the intensity of this process. In general, P-GO nanoparticles in a concentration of 25 µg/ml can regulate differentiation of dendritic cells by suppressing their maturation.

Effect of Pregnancy Specific ß1-Glycoprotein on the Replicative Potential of Naïve T Cells and Immune Memory T Cells.

We studied the effects of pregnancy-specific ß1-glycoprotein (PSG) on the replicative potential of naïve T cells (CD45RA+) and immune memory T cells (CD45R0+) in vitro by evaluating the expression of the hTERT gene in combination with the proliferative activity of cells. Human PSG was obtained by the author's patented method of immunopurification using a biospecific sorbent with subsequent removal of immunoglobulin contamination on a HiTrap Protein G HP column. We used monocultures of CD45RA+ and CD45R0+ lymphocytes isolated from peripheral blood mononuclear cells of reproductive-age women. It was found that PSG in physiological concentrations inhibited the expression of the hTERT gene mRNA in naïve T cells and immune memory T cells and simultaneously reduced the number of proliferating T cells estimated by the differential gating method. At the same time, PSG reduced CD71 expression only on naïve T cells without affecting this molecule on immune memory T cells. Thus, PSG decreased the replication potential and suppressed the proliferation of T cells and immune memory T cells, which in the context of pregnancy can contribute to the formation of immune tolerance to the semi-allogeneic embryo.

Effect of Alpha-Fetoprotein on Differentiation of Myeloid Supressor Cells.

The effect of recombinant alpha-fetoprotein (AFP) on human myeloid suppressor cell (MDSC) differentiation was studied in vitro in the presence of cytokines IL-6 (10 ng/mL) and GM-CSF (10 ng/mL). It was found that AFP at concentrations of 50 and 100 IU/mL increased the number of MDSC (CD33+ HLA-DR-/lowCD11b+) in culture. Analysis of MDSC subpopulations showed that the increase was due to monocytic M-MDSC (HLA-DR-/lowCD33+CD11b+CD14+CD66b-). There was no modulating effect of AFP on granulocytic PMN-MDSC (HLA-DR-/lowCD33+CD11b+CD14-CD66b+). The effects of recombinant AFP on MDSC differentiation were thus demonstrated for the first time.

Effect of Graphene Oxide Nanoparticles on Differentiation of Myeloid Suppressor Cells.

We studied the effect of graphene oxide (GO) nanoparticles on differentiation of human myeloid suppressor cells (MDSC) in an in vitro system. Separated mononuclear cells of healthy donors were induced with cytokines (IL-6 and GM-CSF) into the MDSC phenotype (both polymorphonuclear (PMN-MDSC) and monocyte (M-MDSC) subsets of these cells were taken into account). Pegylated GO nanoparticles (GO-PEG; mean size 569±14 nm, PEG content ~20%) were used. GO-PEG in low concentrations (2.5 and 5 µg/ml) increased the percentage of MDSC in cultures, but reduced their content in high concentration (10 µg/ml). After exposure to GO-PEG (2.5 and 5 µg/ml), the MDSC content increased at the expense of M-MDSC, while the level of PMN-MDSC did not change. The decrease in MDSC levels after exposure to high doses of GO-PEG (10 µg/ml) was due to a decrease in PMN-MDSC. Thus, GO-PEG nanoparticles can oppositely regulate differentiation of MDSC by inhibiting or stimulating differentiation of these cells depending on the concentration.

Role of α-Fetoprotein in Regulation of Proliferation and Functional Activity of Naïve T Cells and Immune Memory T Cells.

We studied the role of native α-fetoprotein preparation in the regulation of proliferation and functional activity of naïve T cells and immune memory T cells in vitro. The study was carried out on separated fractions of naïve T cells (CD45RA+) and immune memory T cells (CD45R0+) incubated with α-fetoprotein under conditions of TCR activation. At the level of naïve T cells, α-fetoprotein in a concentration of 100 U/ml reduced the expression of CD28, but increased the expression of CD25, while at the level of immune memory T cells α-fetoprotein (50 and 100 U/ml) only suppressed the expression of CD25. No effects of α-fetoprotein on the proliferative status of the studied lymphocyte subpopulations and on the expression of CD71 (proliferation marker) by these cells were detected. Addition of α-fetoprotein in a concentration of 100 U/ml increased the level of IL-2 in naïve T cell culture supernatants, while production of IL-2 by memory T cells remained unchanged. These data demonstrated the priority aspects of regulation of the functional activities of naïve T cells and immune memory T cells.

Role of α-fetoprotein in differentiation of regulatory T lymphocytes.

The effect of native α-fetoprotein (AFP) on the expression of T-regulatory lymphocyte (Treg) markers by activated CD4+ lymphocytes with different proliferative status was studied. α-Fetoprotein did not affect the ratio of proliferating and non-proliferating activated CD4+ cells. In the study of Treg differentiation, it was found that AFP at concentrations of 50 and 100 µg/mL significantly inhibited the number of nonproliferating CD4+FOXP3+ and CD4+FOXP3+HELIOS+ lymphocytes without affecting the expression of Treg markers by proliferating CD4+ lymphocytes.

Effect of pregnancy-specific ß1-glycoprotein on indoleamine-2,3-dioxygenase activity in human monocytes.

The role of heterogenic human pregnancy-specific glycoprotein (PSG), obtained by the authors' technology, in the regulation of the indoleamine-2,3-dioxygenase (IDO) activity in female blood monocytes has been studied in vitro. PSG stimulated IDO activity under the conditions of induction of the monocytes by interferon-γ. Upon the induction of cell proliferation by lipopolysaccharides, the stimulating effect was obtained only with 10 µg/mL of PSG. Enhanced IDO activity is probably a factor of peripheral immunological tolerance and antimicrobial protection against intracellular infections in the gestation period.

[The study of human pregnancy specific ß1-glycoprotein immunomodulating effects].

Effect of human Pregnancy specific ß1-glycoprotein (PSG) in physiological concentrations was analyzed against the expression of natural killer (NK)-, T-cells with natural killer functions (N KT-) and T-regulatory lymphocyte (Treg) markers, as well as on the activity of monocyte indolamine-2,3-dioxygenase (IDO) and lymphocyte apoptosis in vitro. It was revealed that PSG in high concentration (100 µg/mL) suppressed the CD16/56 expression by NK-cells, while inhibiting the cytolytic activity of these cells. Meanwhile, PSG in low concentrations (1 and 10 µg/mL) enhanced the CD16/56 expression by NKT-cells that was related to cytokine-producing activity. It was found that PSG increased the number of adaptive Tregs in culture (CD4+FOXP3+ and CD4+CD25(bright)FOXP3+). In addition, PSG promoted the IDO activity in peripheral monocytes, while further potentiating the Treg generation. In general, PSG rendered anti-apoptotic action on lymphocytes. Therefore, indicated effects can determine the PSG contribution to the development of immune tolerance in pregnancy.

Immunochromatographic system for diagnostics of pseudotuberculosis.

We developed a model immunochromatographic test-system for serological express-diagnostics of pseudotuberculosis. Nitrocellulose membrane sensitized with species-specific antigen, Yersinia pseudotuberculosis thermostable toxin, was used as the immunosorbent. Detection of antibodies to the pathogen was performed using functionalized carbon nanoparticles. System performance was verified in testing of the reference pseudotuberculosis serum. Tests with salmonella, escherichia, and enteral yersinia diagnostic sera and blood serum from a healthy man demonstrated high specificity of the system.

[Application of diagnosticum based on functionalized carbon nanoparticles for monitoring of affine immunoglobulin purification].

A diagnostic reagent application based on carbon nanoparticles covalently functionalized by streptococcus G-protein was investigated in order to construct a system for the monitoring and optimization of the affinity purification technology of rabbit polyclonal antibodies to alpha-fetoprotein. The developed system allows semiquantitative assessment ofthe immunoglobulin content (IgG) of most higher animals and human beings in blood serum samples and eluate in a short time (45 min). IgG detection sensitivity using the carbon-G-protein diagnosticum was 80 ng/mL. Approaches to stabilizing the components of the analysis system, which ensures the preservation of their functional properties during long storage, were developed. The storage life of the diagnosticum was more than 20 years, and that of immunosorbents was more than year and a half. A technique of long-term immunosorbent storage, used in the analysis, was developed. Application of the test-system is possible without the presence of registration equipment.

Solution of the problem of nonspecific binding in solid-phase noninstrumental dot immunoassay.

In this work, we evaluated blocking properties of various reagents used in dot immunoassay test systems on nitrocellulose membranes employing carbon-based diagnosticum to determine antibodies to tetanus anatoxin, thermostable toxin of Yersinia pseudotuberculosis, and HIV-I. The results support the usage of a nonionic detergent Tween 20 which efficiently inhibited nonspecific protein binding to the solid-phase reagent surface and did not decrease sensitivity of the analysis.

Technological features of surface functionalization of nanosized particles of various nature.

The technological universality of the developed methods of surface functionalization was exemplified by carbon (160 nm) and ferrocarbon (4-6 nm) nanoparticles. Based on universal technological scheme, the study demonstrated the possibilities of methodical variants with due account for the intrinsic properties of the particles such as carbonic surface and magnetism. The basic analytic properties of the developed diagnostic systems were examined to assess their prospective use in detection of the stereospecific molecules and their interaction. The model systems have been developed, which demonstrated the analytic potencies of the reagents obtained.

[Colloidal carbon particles in the noninstrumental diagnostic systems].

A technology has been developed to design test systems based on the one-stage determination of the conjugate containing the ligand-determined stereospecific compound that is covalently conjugated with colloidal carbon particles. Due to the high optical density of such conjugates in the visible light spectrum region, a ligand may be directly visually inspected as black spots (dots), stripes, or the arbitrary pattern ("+", "-") onto the work surface of a solid phase, which forms after a short (minutes) time of addition of a conjugate regardless of the format of analytical system. These systems may be effectively used in various tests in the ambulatory, hospital, and prehospital settings, including extreme clinical and epidemiological cases, as well as in various household and field diagnostic kits.

[Role of the energy status of Escherichia coli in cell membrane phospholipid composition changes during aerobic-anaerobic transitions]. / Rol' énergeticheskogo statusa Escherichia coli v izmenenii fosfolipidnogo sostava kletochnykh membran pri aérobno-anaérobnykh perekhodakh]

The aerobic to anaerobic transition of E. coli is accompanied with interrelated changes of the adenylate pool, energy charge, respiration rate, as well as the phospholipid content of the cell membranes and the activity of the polyamine synthesizing system. The role of the cellular energy status in the control of the relative content of membrane phospholipids is discussed. The control is based either on energy redistribution in phospholipid metabolism or on the effect on the activity of the polyamine synthesizing system.