Functional properties of individual sub-domains of the fibrin(ogen) αC-domains.

Background: Fibrinogen is a large polyfunctional plasma protein consisting of a number of structural and functional domains. Among them, two αC-domains, each formed by the amino acid residues Аα392-610, are involved in fibrin polymerization, activation of fibrinolysis, platelet aggregation, and interaction with different cell types. Previous study revealed that each fibrinogen αC-domain consists of the N-terminal and C-terminal sub-domains. The major objections of the present study were to test functional role of these sub-domains in the above mentioned processes. Methods: To achieve these objections, we used specific proteases to prepare two truncated forms of fibrinogen, fibrinogen desAα505-610 and fibrinogen desAα414-610, missing their N-terminal and both N- and C-terminal sub-domains, respectively. Results: Our study with these truncated forms using turbidity measurements and electron microscopy revealed that the N- and C-terminal subdomains both contribute to protofibril formation and their lateral aggregation into fibers during fibrin polymerization process. These two sub-domains also contributed to platelet aggregation with the N-terminal sub-domains playing a more significant role in this process. At the same time, the C-terminal sub-domains make the major contribution to the plasminogen activation process. Further, our experiments revealed that the C-terminal sub-domains are involved in endothelial cell viability and migration of cancer cells. Conclusions: Thus, the results obtained establish the functional role of individual sub-domains of the αC-domains in fibrin polymerization, activation of fibrinolytic system, platelet aggregation, and cellular interactions. General significance: The present study expands our understanding of the functional role of individual fibrinogen domains and their specific portions in various fibrin(ogen)-dependent processes.

[Long-term follow-up study of post-COVID-19 patients].

AIM: To evaluate dynamic changes in the lungs, hemostasis system, immune system in different terms after coronavirus pneumonia. MATERIALS AND METHODS: Ventilation-perfusion single-photon emission computed tomography/computed tomography (CT), functional methods of lung investigation, evaluation of hemostasis system, immune status and specific humoral immune response were performed and evaluated in different terms after coronavirus pneumonia. A total of 71 patients were examined according to this protocol. We examined patients with the lesion volume not less than 50% according to chest CT. All patients were divided into 2 groups depending on the distance from the acute stage of coronavirus pneumonia. Group 1 included patients who were examined early (3060 days after hospital discharge), group 2 included patients who were examined later (61180 days after hospital discharge). RESULTS: We obtained gradual regression of pathologically-modified tissue from 67.3% during the inpatient phase to 30.9% during the early period and to 19.7% during the late period of examination, according to CT scan of the chest organs. The same tendency was demonstrated by diffusion capacity of the lungs. Perfusion scintigraphy data showed a decrease in perfusion deficit from 26.012.8% during the early period of examination to 19.46.2% during the late period of examination. On the contrary, ventilatory scintigraphy demonstrates the increase of isotope passage time through the alveolar-capillary membrane over time (from 48.231.3 minutes in the early period to 83.637.2 minutes in the late period). An increase in D-dimer was detected in 24% of patients in the early group. The levels of inflammatory markers, indices of immune status, and specific humoral immune response did not differ in the two described groups. CONCLUSION: The results demonstrate gradual regression of pathological changes caused by coronavirus infection.

High Proliferative Placenta-Derived Multipotent Cells Express Cytokeratin 7 at Low Level.

The purpose of this study was to investigate the immunophenotypes and gene expression profile of high proliferative placenta-derived multipotent cells (PDMCs) population at different stages of culture. We demonstrated that the colonies resulting from single cells were either positive or negative for CK7, whereas only PDMC clones with weak CK7 expression (CK7low-clones) were highly proliferative. Interestingly, vimentin positive (Vim+) placental stromal mesenchymal cells did not express CK7 in situ, but double CK7+Vim+ cells detection in tissue explants and explants outgrowth indicated CK7 inducible expression in vitro. PCNA presence in CK7+Vim+ cells during placental explants culturing confirmed belonging of these cells to proliferative subpopulation. Transcription factors CDX2 and EOMES were expressed in both CK7low-clones and subset of stromal mesenchymal cells of first-trimester placental tissue in situ. Meanwhile, CK7low -clones and stromal mesenchymal cells of full-term placental tissue in situ expressed ERG heterogeneously. SPP1, COL2A1, and PPARG2 mesodermal-related genes expression by CK7low-clones additionally confirms their mesenchymal origin. Inherent stem cell-related gene expression (IFTM3, POU5F1, and VASA) in CK7low-clones might indicate their enrichment for progenitors. Finally, in CK7low-clones we observed expression of such trophoblast-associated genes as CGB types I and II, fusogenic ERVW-1, GCM1, and GATA3. Thus, our results indicate that PDMCs acquired the representative immunophenotype signature under culture conditions.

Synthesis, spectral characterization of novel Pd(II), Pt(II) π-coordination compounds based on N-allylthioureas. Cytotoxic properties and DNA binding ability.

Four novel Pd2+ and Pt2+ mononuclear π-coordination compounds with general formula [M(HL)1,2Cl2], M=Pd2+, Pt2+ have been synthesized by reaction of [PdCl4]2-, [PtCl4]2- anions with N-allyl-4-morpholinethiocarboxamide (HL1) and N-Allyl-N'-tert-butylthiourea (HL2). All complexes have been characterized by single-crystal X-ray diffraction study and 1H, 13C NMR spectroscopy. Cytotoxic, cytostatic and proapoptotic activities of compounds have been determined in vitro on HeLa cell line and compared with cisplatin as etalon drug. All complex compounds possessed pronounced cytotoxic activity with IC50 indexes in range of 2·10-6-1.5·10-4М (IC50 of cisplatin is 5.7∙10-5М) and showed proapoptotic, cytostatic and antisyntetic influence higher or comparable with cisplatin. The comparative influence of cisplatin and synthesized metal complexes on pTZ19R* plasmid DNA was monitored by agarose gel electrophoresis. All compounds showed high affinity to DNA that correlates with observed cytostatic and proapoptotic levels. In general Pd(II) compounds showed higher activity than Pt(II) ones.

The mechanism of VEGF-mediated endothelial cells survival and proliferation in conditions of unfed-culture.

The mechanisms of VEGF-mediated effects on endothelial cells during cancer development and progression is not clear. In present study the biological effects of VEGF, VEGF-rich culture medium of peritoneal macrophages from mice with Lewis lung carcinoma were studied on MAEC cell line under conditions of unfed culture. We have shown that VEGF increased cell proliferation by the 5th day of culturing vs control and anti-VEGF-treated cells. This effect was associated with increased consumption of glucose and NO production by the 2nd day while decreased ­ on the 5th day of cell culturing. VEGF-mediated NO production was dependent on Ca2+ ions. Block of Ca2+-channels (LaCl3) had more pronounced inhibitory effect vs chelator of Ca2+ ions (EDTA). It was shown that peritoneal macrophages are the main suppliers of VEGF at tumor angiogenesis, as evidenced by the data obtained on model system of endothelial cells synchronized in G0/G1 phase.

Effect of Pd(II) and Ni(II) coordination compounds with 4-amino-3-mercapto-5-methyl-1,2,4-triazole on the mitochondrial dehydrogenases activity.

Pd(I) and Ni(II) complex compounds: [Pd(AMMT)2]Cl2 (1), [Pd(AMMT)4]Cl2 (2) and [Ni(AMMT)2(H2O)](NO3)2 (3) with 4-amino-3-mercapto-5-methyl-1,2,4-triazole (AMMT) have been synthesized. The spectral characteristics of 1, 2 were studied by 1H (13C) NMR and UV-Vis spectroscopy. X-ray diffraction studies established that all complexes contain the AMMT molecule, which are coordinated to the central metal ion in the thione tautomeric form. At the ratio M: L = 1:2 ligand is coordinated in bidentate chelate manner by the nitrogen of amino- and sulfur of mercapto group (compounds 1, 3). But the molar ratio M: L = 1:4 leads to monodentate coordination of AMMT molecules only by sulfur of mercaptogroup (complex 2). Vacant coordination sites of the metal ion are occupied by water molecules (complex 3). The screening of complexes 1-3 and starting compounds [AMMT, K2PdCl4 (4), Ni(NO3)2 · 6H2O (5)] by their mitochondrial dehydrogenase activity have been performed by us for the first time, resulting in established that the Pd(II) complexes (1, 2), Pd(II) salt (4) and AMMT normalize the activity of mitochondrial dehydrogenases of cancer HeLa cells, identified by MTT-test. In contrast, the Ni(II) complex (3) and Ni(II) salt (5) do not stimulate the activity of mitochondrial dehydrogenases. It has been found, that all investigated compounds do not affect on the cell cycle and the level of apoptotic cells as well as do not show a toxic effect. Thus, these results indicate that AMMT and Pd(II) complexes may be used as modifiers of mitochondrial respiration, which dysfunction is particularly evident in the tumor cells.

Combined influence of teichoic acids from Staphylococcus aureus and heterometallik Cu/Cd ethylenediamine complex on peritoneal macrophages and tumor cells.

We investigated the effects of teichoic acid (TA) from Staphylococcus aureus Wood 46 on tumor growth and metastasis of the experimental Lewis lung carcinoma (LLC) in mice. Intranasal administration of TA alone aggravated both tumor growth and metastasis, whereas combined administration of TA with a synthetic bimetallic (copper : cadmium) ethylene diamine complex PO244 resulted in pronounced antitumor and antimetastatic effects. The group of animals subjected to the combined treatment with TA and PO244 manifested the highest degree of lymphocyte infiltration into the tumor tissue, compared to the control group and those exposed to TA or PO244 alone. Moreover, the combined treatment negatively affected the adhesive properties of peritoneal macrophages in the LLC bearing mice. Co-cultivation of the isolated macrophages with primary LLC cultures revealed significant (p < 0.05) cytotoxic and cytostatic effects, detected as an increased level of apoptosis and a reduced fraction of replicating cells.

[Homeostasis changes during rehabilitation period after acute chemical poisoning].

The article deals with review of 78 patients of rehabilitation toxicological unit. The patients received resuscitation and detoxification. All patients were divided into three groups; 1st group--patients after poisoning with psychopharmaceuticals, 2nd group--patients after poisoning with cauterizing liquids and 3rd group--patients with encephalopathy after poisoning with neurotoxin (psychopharmaceuticals, narcotics and ethanol). Disorders of rheology, haemostasis and endotoxicosis accrued in all groups. These disorders were a signs of the erythrocytes and platelets aggregation developing and viscoelasticity disorder. Homeostasis changes during rehabilitation period need an accurate diagnostics for purposeful treatment of the defined disorders.

[Potential cytostatic effect of the maleimide derivative 1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1-H-pyrrol-2,5-dione].

The cytostatic/cytotoxic effects of the maleimide derivative 1-(4-Cl-Benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrol-2,5-dione (MI-1) have been estimated on epithelial derived human cell cultures (Colo 205, MCF-7, and Hela). The anticancer and toxic effects of MI-1 have been investigated on DMH-induced cancer development and normal colon morphology in rats. The results showed that the compound studied has low cytotoxicity but produces a strong antiproliferative effect on cell cultures and partially suppresses colon cancer development in DMH-induced model. The MI-1 effect on normal colon mucosa is insignificant, and no destructive changes have been detected in the intestine of rats. This maleimide derivate can be considered as a promising anticancer drug.

Immunological markers of anti-tumor dendritic cells vaccine efficiency in patients with non-small cell lung cancer.

AIM: To investigate the quantitative and functional status of peripheral blood lymphocytes in patients with non-small cell lung cancer during DC-vaccine therapy and identify the most informative immunological parameters which are associated with clinical outcome. MATERIALS AND METHODS: The study was conducted within the framework of randomized phase III clinical trial of DC-vaccine efficacy in patients with non-small cell lung cancer. Quantitative composition of peripheral blood lymphocytes was determined by flow cytometry. Cytokines mRNA expression level was estimated using real-time RT-PCR. RESULTS: In our study the most pronounced changes in the immune system have been defined after fourth DC-vaccine injection. Immunologic features such as reduction the MIP-1α mRNA expression level, increasing the RANTES mRNA expression level and NK-cells count, retention CD4/CD8 ratio at physiological level were associated with favorable clinical outcome after DC-immunotherapy. CONCLUSIONS: Immunological markers established in our investigation can be used for estimation of DC-immunotherapy efficiency. The results of our research are very promising, but these data should be confirmed in further studies with a large cohort of patients.

[Mitokorrektine stimulates angiogenesis in vitro].

The effect of mitokorrectine (complex native oligopeptides isolated from neonatal pig brain) on endothelial cells in culture was investigated. It was revealed that the drug concentration-dependently induces angiogenesis in vitro. Mitogen effect of Mitokorrectine was shown by MTT-test and routine cell count in concentration diapason (0.1-1 mg/ml) which means an increased number of cells by 25 +/- 5% and cell subpopulation of proliferative pool (G2/M+S) 1,8 times in concentration diapason mitokorrectine (0.01-0.05 mg/ml) in comparison with control. In 3-D culture and in stationary phase we detected induction of differentiation of endothelial cells, a decrease the level of NO production and enhancement of glucose metabolism and stimulation of formation of capillary-like tubes.

[Effect of dendritic cell autovaccine on the treatment outcome in patients with ovarian cancer].

During I/II phase clinical trial in ovarian cancer (OC) patients two types of autologous anticancer vaccines based on dendritic cells have been tested, and a comparative analysis of their effectiveness have been performed. It was shown that the anticancer vaccines based on DC, "loaded" with autologous tumor cell lysate obtained by treatment of tumor cells by cytotoxic lectins B. subtilis had higher efficiency, compared with the standard DC--autovaccine. The presence of antigen-specific immune response observed after at least four vaccinations. Obtained results open the prospects to improve the basic treatment of OC patients by this method. The results of immunological examinations create preconditions for individual optimization of the DC-vaccine therapy.

[Monoclonal antibodies to rubella virus glycoprotein E1].

AIM: To obtain monoclonal antibodies (MCAs) to glycoprotein E1 of rubella virus, to assess their immunochemical characteristics and ability to use fluorescent MCA for rapid identification of rubella virus. MATERIALS AND METHODS: Rubella virus strain C-74 (Moscow), vaccine strains "Orlov" (Saint-Petersburg), Wistar RA 27/3 (USA) as well as strain Judith (Germany) were used. Viral antigens were obtained using diploid cells L-68 and cell lines VNK-21-F and Vero E6. MCAs were produced by conventional method and their isotype was determined: Immunoblotting, immunoenzume assay (IEA), hemagglutination inhibition assay (HIA) and immunofluorescence assay (IFA) were performed. RESULTS: Five monoclonal antibodies--Kh-252.1, Kh-347.2, Kh-183.3, Kh-214.4, Kh-187.5--to antigens of rubella virus strain C-74 were obtained. Isotypes of these antibodies were determined and their reactivity with native and denaturated antigens of other strains ("Orlov", Wistar RA 27/3, Judith) was characterized. IEA showed that all MCAs interacted with rubella virus glycoprotein E1 at high titers ranging from 1/1600 to 1/200,000. Immunoblotting demonstrated that 4 MCAs (Kh-252.1, Kh-347.2, Kh-183.3, Kh-214.4) had aforementioned feature. MCAs inhibited hemagglutinating activity of Judith strain in titer from 1/16 to 1/1024 in HIA. FITC conjugate of MCA Kh-347.2 (most sensitive variant) allowed to detect rubella virus in infected Vero E6 cells after 24 hours since infection, whereas FITC conjugates of 3 MCAs (Kh-183.3, Kh-214.4, Kh-187.5)--after 72 hours since infection. CONCLUSION: Use of FITC conjugates of MCAs is a perspective tool for identification of rubella virus glycoprotein E1 in infected cell cultures and nasopharyngeal swabs.

[Immunoprophylaxis of postoperative inflammatory complications in thoracic and abdominal wounds]. / Immunoprofilaktika posleoperatsionnykh gnoino-vospalitel'nykh oslozhnenii pri raneniiakh grudi i zhivota.

Immune system (IS) was studied in 228 wounded with penetrating knife and shotgun wounds of the thorax and abdomen complicated by hemorrhage more than 1000 ml. Six variants of IS reaction to trauma, hemorrhage, surgical stress and intensive therapy 1-2 days after surgery were registered. Comparative analysis of laboratory and clinical efficacy of prophylactic use of leukinferon in 99 patients (test group) was carried out. Control group consisted of 129 patients matched by gender, age, type of wound, hemorrhage volume and fluid therapy. High immunoprophylactic efficacy of leukinferon was demonstrated. Period of immunorehabilitation (normalization of leukogram, correction of immune imbalance) in patients of the test group was shortened twice. Purulent complications rate reduced from 24.9 to 1.0%, hospital stay--from 41.7 +/- 3.6 to 16.3 +/- 1.1 days.

Synergistic effects in antigen capture ELISA using three monoclonal antibodies directed at different epitopes of the same antigen.

Using a panel of monoclonal antibodies (mAb) against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture enzyme-linked immunosorbent assay (ELISA) may be significantly increased by the simultaneous immobilization on a solid phase of two co-operating capture mAbs. This method ("a three-site ELISA") uses three mAbs at different epitopes of the same antigen (two capture/one tracer), unlike the traditional two-site assay, using one capture and one tracer mAbs. We established two-site and three-site ELISA assays for Mb, by varying capture and tracer mAbs. Three-site assays showed 4-6 fold increase in sensitivity, if compared with two-site assays. The model for the effect has been suggested, according to which in three-site ELISA the high-affinity cyclic configurations may be formed by an antigen, two-capture mAbs and the surface of solid phase.

Triple-site antigen capture ELISA for human myoglobin can be more effective than double-site assay.

Using a panel of monoclonal antibodies against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture ELISA can be significantly increased by simultaneous immobilization of two cooperating capture monoclonal antibodies on a solid phase. This method ("triple-site ELISA") uses three monoclonal antibodies to different epitopes of the same antigen (two capture/one tracer) unlike the traditional double-site assay using one capture and one tracer monoclonal antibody. We developed double- and triple-site ELISA for Mb by varying the capture and tracer monoclonal antibodies. Triple-site assays showed 4-6-fold increase in sensitivity compared to the double-site assays. A model for this effect is suggested; according to the model, in triple-site ELISA, high-affinity cyclic configurations can be formed by an antigen, two capture monoclonal antibodies, and the surface of the solid phase.

Single-step sandwich immunoassay of myoglobin with bifunctional monoclonal antibodies.

Two protocols for sandwich antigen-capture ELISA of human myoglobin were compared. In the first (routine) variant, 14D6 monoclonal antibodies conjugated to horseradish peroxidase were used as the secondary antibodies. Bifunctional antibodies specific for myoglobin/peroxidase were used as the secondary antibodies in the second variant. The myoglobin-binding site of the bifunctional antibodies was similar to that of the 14D6 antibodies, and the second antigen-binding site of the bifunctional antibodies was bound to horseradish peroxidase. When comparing standard calibration curves, the effective concentration of the bifunctional antibodies and that of antibodies conjugated to horseradish peroxidase were made equal. It is shown that the use of bispecific antibodies as the secondary antibodies does not improve the quality of the parameters tested, i.e., the sensitivity of the assay does not increase and the slope of the calibration curve remains constant.

[Expression of receptors for human alpha- and gamma-interferons on the surface of peripheral blood mononuclear cells in viral infections]. / Ekspressiia retseptorov dlia chelovecheskikh alpha- i gamma-interferonov na poverkhnosti mononuklearnykh kletok perifericheskoi krovi pri virusnykh infektsiiakh.

Expression of receptors on peripheral blood mononuclears of donors and patients with viral infections during therapy was studied using FITC-labeled monoclonal antiidiotypical antibodies with the "internal image" of human alpha- and gamma-interferons and monoclonal antibodies to these interferons. Activation of the immune system caused by infection modifies the expression of interferon receptors and leads to appearance of membrane-bound interferons, mainly gamma-interferon.