We have characterized the human cell line arised from the Epstein–Barr virus (EBV) positive multiple myeloma aspirate subjected to the long-term cultivation. This cell line has acquired the ability to form free-floating spheres and to produce a xenograft upon transplantation into NOD/SCID mice. Cells from both in vitro culture and developed xenografts were investigated with a number of analytical approaches, including pathomorphological analysis, FISH analysis, and analysis of the surface antigens and of the VDJ locus rearrangement. The obtained results, as well as the confirmed presence of EBV, testify that both biological systems are derived from B-cells, which, in turn, is a progeny of the EBV-transformed B-cellular clone that supplanted the primordial multiple myeloma cells. Next we assessed whether cells that (i) were constantly present in vitro in the investigated cell line, (ii) were among the sphere-forming cells, and (iii) were capable of internalizing a fluorescent TAMRA-labeled DNA probe (TAMRA+ cells) belonged to one of the three types of undifferentiated bone marrow cells of a multiple myeloma patient: CD34+ hematopoietic stem cells, CD90+ mesenchymal stem cells, and clonotypic multiple myeloma cell. TAMRA+ cells were shown to constitute the fourth independent subpopulation of undifferentiated bone marrow cells of the multiple myeloma patient. We have demonstrated the formation of ectopic contacts between TAMRA+ cells and cells of other types in culture, in particular with CD90+ mesenchymal stem cells, followed by the transfer of some TAMRA+ cell material into the contacted cell.

An ability of poorly differentiated cells of different genesis, including tumor stem-like cells (TSCs), to internalize extracellular double-stranded DNA (dsDNA) fragments was revealed in our studies. Using the models of Krebs-2 murine ascites carcinoma and EBV-induced human B-cell lymphoma culture, we demonstrated that dsDNA internalization into the cell consists of several mechanistically distinct phases. The primary contact with cell membrane factors is determined by electrostatic interactions. Firm contacts with cell envelope proteins are then formed, followed by internalization into the cell of the complex formed between the factor and the dsDNA probe bound to it. The key binding sites were found to be the heparin-binding domains, which are constituents of various cell surface proteins of TSCs—either the C1q domain, the collagen-binding domain, or domains of positively charged amino acids. These results imply that the interaction between extracellular dsDNA fragments and the cell, as well as their internalization, took place with the involvement of glycocalyx components (proteoglycans/glycoproteins (PGs/GPs) and glycosylphosphatidylinositol-anchored proteins (GPI-APs)) and the system of scavenger receptors (SRs), which are characteristic of TSCs and form functional clusters of cell surface proteins in TSCs. The key provisions of the concept characterizing the principle of organization of the “group-specific” cell surface factors of TSCs of various geneses were formulated. These factors belong to three protein clusters: GPs/PGs, GIP-APs, and SRs. For TSCs of different tumors, these clusters were found to be represented by different members with homotypic functions corresponding to the general function of the cluster to which they belong.

The purpose of the article: Protection from ionizing radiation is the most important component in the curing malignant neoplasms, servicing atomic reactors, and resolving the situations associated with uncontrolled radioactive pollutions. In this regard, discovering new effective radioprotectors as well as novel principles of protecting living organisms from high-dose radiation is the most important factor, determining the new approaches in medical and technical usage of radiation. Materials and methods: Experimental animals were irradiated on the γ-emitter (Cs¹³⁷) with a dose of 9.4 Gy. Radioprotective properties of several agents (total RNA, single-stranded RNA, double-stranded RNA, B-190) were estimated by the survival/death rates of experimental animals within 30-90 days. Pathomorphological examination of internal organs end electron microscope assay were done on days 9-12 after irradiation. Cloning and other molecular procedures were performed accordingly to commonly accepted protocols. For assessment of the internalization of labeled nucleic acid, bone marrow cells were incubated with double-stranded RNA labeled with 6-FAM fluorescent dye. Cells with internalized double-stranded RNA were assayed using Axio Imager M1 microscope. In the other experiment, bone marrow cells after incubation with double-stranded RNA were stained with Cy5-labeled anti-CD34 antibodies and assayed using Axioskop 2 microscope. Results: In the present study, several biological features of the radioprotective action of double-stranded RNA are characterized. It was shown that 160 µg of the double-stranded RNA per mouse protect experimental animals from the absolutely lethal dose of γ-radiation of 9.4 Gy. In different experiments, 80-100% of irradiated animals survive and live until their natural death. Radioprotective properties of double-stranded RNA were found to be independent on its sequence, but strictly dependent on its double-stranded form. Moreover, double-stranded RNA must have “open” ends of the molecule to exert its radioprotective activity. Conclusions: Experiments indicate that radioprotective effect of double-stranded RNA is tightly bound to its internalization into hematopoietic stem cells, which further repopulate the spleen parenchyma of irradiated mice. Actively proliferating progenitors form the splenic colonies, which further serve as the basis for restoration of hematopoiesis and immune function and determine the survival of animals received the lethal dose of radiation.

Macrophages are the immune cells of high‐immunological plasticity, which can exert both pro‐ and anti‐inflammatory activity, as well as repolarize their phenotype to the opposite or neutral one. In this regard, M2 macrophages of the tumor‐associated stroma (TAS) are a promising therapeutic target in treating malignant neoplasms. Using FACS assay, we have estimated the CD11b+/Ly‐6G+/Ly‐6C+ fraction of macrophages from the peritoneum and TAS in intact healthy mice and those with developed Lewis carcinoma, both untreated and treated according to Karanahan technology in combination with group‐specific macrophage activator (GcMAF‐RF). As well, the pattern of pro‐ and anti‐inflammatory cytokines mRNA expression in different groups of experimental and tumor‐bearing animals was assessed. It was found that: (i) exposure of intact mice to GcMAF‐RF results in the increased number of CD11b+/Ly‐6C+ peritoneal macrophages and, at the same time, the expression pattern of cytokines in peritoneal macrophages switches from that characteristic of the mixed M1/M2 phenotype to that characteristic of the neutral M0 one; (ii) combination of Karanahan technology and GcMAF‐RF treatment results in M0/M1 repolarization of TAS macrophages; (iii) in tumor‐bearing mice, the response of peritoneal macrophages to such a treatment is associated with the induction of anti‐inflammatory reaction, which is opposite to that in TAS macrophages.

In this part of the study, the first component of the concept of “natural genome reconstruction” is being proven. It was shown with mouse and human model organisms that CD34+ hematopoietic bone marrow progenitors take up fragments of extracellular double-stranded DNA through a natural mechanism. It is known that the process of internalization of extracellular DNA fragments involves glycocalyx structures, which include glycoproteins/protein glycans, glycosylphosphatidylinositol-anchored proteins and scavenger receptors. The bioinformatic analysis conducted indicates that the main surface marker proteins of hematopoietic stem cells belong to the indicated groups of factors and contain specific DNA binding sites, including a heparin-binding domain and clusters of positively charged amino acid residues. A direct interaction of CD34 and CD84 (SLAMF5) glycoproteins, markers of hematopoietic stem cells, with double-stranded DNA fragments was demonstrated using an electrophoretic mobility shift assay system. In cells negative for CD34, which also internalize fragments, concatemerization of the fragments delivered into the cell occurs. In this case, up to five oligonucleotide monomers containing 9 telomeric TTAGGG repeats are stitched together into one structure. Extracellular fragments delivered to hematopoietic stem cells initiate division of the original hematopoietic stem cell in such a way that one of the daughter cells becomes committed to terminal differentiation, and the second retains its low-differentiated status. After treatment of bone marrow cells with hDNA^gr, the number of CD34+ cells in the colonies increases to 3 % (humans as the model organism). At the same time, treatment with hDNA^gr induces proliferation of blood stem cells and their immediate descendants and stimulates colony formation (mouse, rat and humans as the model organisms). Most often, the granulocyte-macrophage lineage of hematopoiesis is activated as a result of processing extracellular double-stranded DNA. The commitment process is manifested by the appearance and repair of pangenomic single-strand breaks. The transition time in the direction of differentiation (the time it takes for pangenomic single-strand breaks to appear and to be repaired) is about 7 days. It is assumed that at the moment of initiation of pangenomic single-strand breaks, a “recombinogenic situation” ensues in the cell and molecular repair and recombination mechanisms are activated. In all experiments with individual molecules, recombinant human angiogenin was used as a comparison factor. In all other experiments, one of the experimental groups consisted of hematopoietic stem cells treated with angiogenin.

The article reports an original method for producing vitamin D-3-binding protein (DBP) and its conversion into macrophage-activating factor GcMAF-RF. According to an original protocol, DBPs were obtained from human blood plasma using affinity chromatography, purified and modified to GcMAF-RF using cytoimmobilized glycosidases (beta-galactosidase and neuraminidase). The presence of the polypeptide obtained in the Gc group of blood plasma globulins was confirmed by Western blot using specific antibodies. The molecular properties of this polypeptide put it in correspondence with the GcMAF protein described in the literature, which is undergoing clinical trials in the USA, Britain, Israel and Japan (at Saisei Mirai; Reno Integrative Medical Center; Immuno Biotech Ltd; Efranat; and Catalytic Longevity). The biological activity of the GcMAF-RF preparation was detected by the induction of phagocytic activity of macrophages and their ability to produce nitrogen monoxide (NO) in vitro. The phagocytic activity of macrophages was evaluated by their ability to uptake magnetic beads. The degree of activation of macrophages was calculated by the ratio of trapped beads to the total number of macrophages. The level of NO production was estimated by the accumulation of nitrogen monoxide in the culture supernatants of peritoneal macrophages by the colorimetric method using the Griess reagent. It was shown that GcMAF-RF multiplies the phagocytic activity of macrophages and significantly increases their production of nitrogen monoxide. The macrophage activator GcMAF-RF, according to its characteristics, corresponds to similar preparations which are made available to the market by foreign companies, and can be considered as a new biologically active preparation with a wide spectrum of action. Of greatest interest is its ability - through the activation of macrophages - to enhance the adaptive immunity. In this regard, two areas of therapeutic use of the GcMAF-RF are proposed. The preparation will be in demand in the field of cancer treatment, and, in addition, it can be used in the treatment of a number of neurodegenerative pathologies.