Vol 14, No 4 (2019)

Duchenne muscular dystrophy (DMD) is a common genetic disease which develops as a result of a mutation in the gene encoding dystrophin. In this review, the main experimental therapeutic approaches based on gene therapy are described. Independence of the type of mutation in the DMD gene is an advantage of the viral delivery of micro- and minidystrophin in muscle cells, but this method provides only a temporary effect. The specificity of the mutation also does not matter with an increase in the level of utrophin, however, this protein cannot fully replace dystrophin. The drugs which promote reading through the stop codon have low efficiency and are suitable for only 10-15% of patients with DMD. The most promising approach for the treatment of DMD is the exon skipping, which will suit 90% of patients. It can be implemented by antisense oligonucleotides or using the CRISPR/Cas9 genome editing system. CRISPR/Cas9-mediated exon skipping is thought to be the most promising approach, because it allows to make the necessary changes in the genome with great efficiency after single application.

The study of the regeneration of a full-layer skin wound under conditions of stimulated angiogenesis was carried out by paravulnare injection of a solution containing the plasmid pCMV-VEGF165. The inflammatory reaction in the experimental group of rats was more intense and already on day 7, the leukocyte shaft in the control was 1.3 times wider, and on day 14 in the experimental group, this indicator was 2.2 times less than the control figures. The indicators characterizing the development of granulation tissue on day 7, in the second group, 1.3 prevail over the control values, after 14 days, due to the maturation of young connective tissue - 1.1 times. The greatest differences in the comparison groups were found in relation to the degree of epithelialization of the defect. The epithelium thickness at the border is 1.4 times the control, the epithelial layer thickness is 1.7 times, and the length is 1.5 times. Compared with the control values, the number of vessels in the experimental group on day 7 were 2.3 times higher, on day 14 - 1.8 times and on day 21 - 1.7 times. The healing of the full-thickness wound of the skin in the experimental group proceeded more intensively compared with the control, and epithelization was completed 2 days earlier. In addition, stimulation of angiogenesis led to the formation of an organ-specific regenerate practically indistinguishable from intact skin.

Endothelial Colony-forming cells (ECFCs) are valuable material for tissue vascular engineering and cell therapy of ischemic tissues. Difficult isolation is the main problem for using of ECFCs. ECFCs isolation from peripheral blood and adipose tissue has been previously described. In the presented research we compared effectiveness of peripheral blood, subcutaneous and epicardial adipose tissue for the ECFCs isolation without cell sorting. ECFCs was isolated from peripheral blood, subcutaneous and epicardial adipose tissue obtained from coronary heart disease patients (males, n=8) undergoing elective coronary artery bypass surgery. The stromal-vascular fraction of subcutaneous (SVF-ST) and epicardial (SVF-ET) adipose tissue as well as the mononuclear blood fraction (MNF) were cultivated in the complied EGM-2 medium. Cell cultures phenotyping was performed by flow cytometry and confocal microscopy. Their angiogenic (Matrigel) and proliferative activity (xCELLigence analyzer) in vitro were studied. ECFCs were isolated from MNF in 50% of cases, from SVF-ST in 12.5% and SVF-ET in 37.5%. The proliferative activity of ECFCs isolated from adipose tissue was low while cultures from MNF showed high and medium activity in 75% of cases. Pure ECFCs (more 99%) were obtained from MNF to third passage without cell sorting. Cultures from adipose tissue were contaminated by mesenchymal-stromal cells (MSCs) and contained ECFCs and MSCs. Thus, peripheral blood is the most effective source of autologous ECFCs compared with adipose tissue for this isolation method. However, adipose tissue is a suitable source of MSC and mixed cultures of MSC and endothelial cells.

Nowadays titanium and its alloys are the most widely used metallic materials in medicine. In comparison with other metals, titanium has several advantages including biocompatibility, good mechanic properties and corrosion resistance. This research was focused on the studies of proliferative and secretory characteristics of human fibroblasts, cultured on nanotube-layered titanium surfaces as well as the levels of collagen and non-collagenous proteins deposition. Experiments were performed with 2 fibroblast lines isolated from skin samples of 2 donors. Fibroblasts were grown on titanium disks with untreated and anodized surfaces and on the tissue culture treated plastic. Cells were fixed after 3, 5, 7 and 9 days of cultivation. At each time point six samples were analyzed for each surface type. Cell density was estimated by counting cell nuclei, stained with DAPI. IL-6, IL-8/CXCL8 and pro-collagen I concentrations were measured by ELISA, the quantity of collagen and non-collagenic proteins on surfaces was calculated by measuring the level of absorption of Sirius Red and Fast Green dyes, respectively. The results of experiments indicate that the modification of titanium surface with nanotubes does not trigger the formation of fibrous capsule during osseointegration. However, elevated levels of secreted chemokine IL-8/CXCL8, which attracts neutrophils, were observed on anodized samples thus implying possible increased inflammatory response. To get more insights on the role of nanotubes in osseointegration further research is needed.

Adoptive T- and NK-cells transfer with chimeric antigenic receptors (CAR) is considered as a promising anticancer strategy. Chimeric antigenic receptors are artificial molecules that provide activation of the cells carrying them when they contact with a specific antigen to induce cell death. Unlike T cells, NK cells have no T-cell receptors, which prevent "graft-versus-host” disease under allograft transplantation. The aim of this work was to study antimetastatic activity of a double-modified human NK cell line YT - Cyto-CAR-YT-Lact cells carrying CAR to the PSMA protein and expressing antiapoptotic protein lactaptin. The BrCCh4e-134 breast carcinoma line with high PSMA expression was constructed by lentiviral transduction. The YT double modified NK cell line, Cyto-CAR-YT-Lact, expressing functional anti-PSMA CAR and carrying a deletion of the Shp-2 gene encoding the Shp-2 protein, a negative regulator of NK cell activation, was used as effector cells. The cytotoxic activity of Cyto-CAR-YT-Lact cells was tested on PSMA-positive BrCCh4e-134 cells and on parental BrCCh4e cell in vitro in real-time mode. The antimetastasis activity of Cyto-CAR-YT-Lact cells was analyzed in SCID and NOD/SCID mice with spontaneously metastases and intravenously transplanted PSMA-positive BrCCh4e-134 cells. Cyto-CAR-YT-Lact cells efficiently reduced the viability of PSMA-positive tumor cells and moderately PSMA-negative target cells in vitro. As a tumor model, we used human breast cancer cells that overexpress PSMA as a transgene and are characterized by metastasis to the mediastinal lymph nodes being transplanted on mice. It was shown that a single intravenous administration of the Cyto-CAR-YT-Lact cells suppressed the development of metastases in the spontaneous metastasis model and when tumor cells were introduced into the bloodstream. The reaction of the primary tumor node to Cyto-CAR-YT-Lact therapy was not detected. Thus, the use of modified NK cells can be considered as a promising therapeutic approach for suppressing metastasis, but not for suppressing the growth of the primary tumor node.

The giant cell tumors of maxillofacial bones include several barely differentiable diseases with diverse prognoses. A case of a giant cell tumor in an 18-year-old man is described. Clinical, instrumental and laboratory examination did not allow to establish an accurate diagnosis. Molecular genetic test by New generation sequencing (NGS) of tumor tissues and blood identified mutation p. 1424 C> A; p.Pro475His in the SH3BP2 gene, previously described in patients with the so-called cherubism, that determined the tactic of treatment. Carrying out an oncogenetic study allows the differential diagnosis of such morphologically similar conditions with a giant cell component as a multiorgan form of fibrous dysplasia, reparative granulomas, Noonan syndrome, Sturge-Weber-Crabbe syndrome, juvenile ossifying fibroma.