We studied the effects of a dual-vector DYSF gene delivery system based on adeno-associated virus serotype 9 capsids on pathological manifestations of dysferlinopathy in skeletal muscles of Bla/J mice lacking DYSF expression. The mice received intravenous injection of 3×1013 genomic copies of the virus containing the dual-vector system. M. gastrocnemius, m. psoas major, m. vastus lateralis, and m. gluteus superficialis were isolated for histological examination in 3, 6, and 12 weeks after treatment. Healthy wild-type (C57BL/6) mice served as positive control and were sacrificed 3 weeks after injection of 150 μl of 0.9% NaCl into the caudal vein. To detect dysferlin in muscle cryosections, immunohistochemical analysis with diagnostic antibodies was performed; paraffin sections were stained with hematoxylin and eosin for morphometric analysis. After administration of gene-therapeutic constructs, muscle fibers with membrane or cytoplasmic dysferlin location were detected in all examined muscles. The proportion of necrotic muscle fibers decreased, the number of muscle fibers with central location of the nucleus increased, and the mean cross-section area of the muscle fibers decreased.

The development of imaging techniques in the experimental oncology has made it possible to model and continuously monitor growth and development of tumors in vivo. For this purpose, fluorescent or bioluminescent proteins are most commonly introduced into the genome of target cells which in turn can be used to create xenograft tumor models. In our study, SH-SY5Y human neuroblastoma cells were genetically modified with a firefly luciferase ffLuc gene or far-red fluorescent protein Katushka2S gene, and bioluminescence and fluorescence of obtained cells were analyzed in vitro. Then, produced cell lines were used to create xenograft tumor models in vivo . The bioluminescence and fluorescence of the obtained tumor models were evaluated in vivo . When evaluated in vitro, ffLuc bioluminescence was firstly detected within fewer number of SH-SY5Y cells compared to Katushka2S fluorescence. However, after in vivo administration, the fluorescence intensity of SH-SY5Y-Katushka2S cells was significantly higher than the bioluminescence intensity of the same number of SH-SY5Y-ffLuc cells. This may be due to the distribution and limited intracellular availability of a luciferase substrate, which is administered intraperitoneally, and requires further analysis.

The brain synaptic circuitry is formed as a result of pre-defined genetic programs and sensory experience during postnatal development. Perineuronal nets ensheath synaptic boutons and control several crucial features of the synapse physiology. Formation of the perineuronal net microstructure during the brain development remains largely unstudied. Here we provide a detailed quantitative description of the 3-dimensional geometry of the synapse and the surrounding perineuronal net in the mouse somatosensory cortex layer IV. We compare the morphology of the synapse+perineuronal net complex in the adult brain formed under normal conditions or in the whisker shaving model of somatosensory deprivation. We demonstrate that the sensory deprivation causes flattening of the 3D PNN mesh geometry and reduction of the VGAT-positive cluster volume in presynaptic boutons. These results reveal a mechanism of the sensory input-dependent synapse morphogenesis during the brain development.

Болезнь Крона (БК) и язвенный колит (ЯК) являются заболеваниями, важным звеном патогенеза которых является повышенная проницаемость слизисто-эпителиального барьера кишечника. Цель исследования – структурный анализ компонентов слизисто-эпителиального барьера при воспалительных заболеваниях кишечника.

Despite the widespread occurrence of ischemic diseases of the lower extremities, including atherosclerosis and diseases with an autoimmune component of their pathogenesis, the pathohistological signs of damage and concomitant chronic ischemia, compensatory tissue responses as intracellular and cellular regeneration remain out of the field of vision in researchers.OBJECTIVE:To assess the signs of damage (the extent of necrosis and apoptosis, capillary density (CD)) and regeneration (the cross-sectional muscle fiber area (CSMFA), the proportion of centrinucleated muscle fibers (CNMFs), and that of connective tissue), by using the gastrocnemius medial head biopsy specimens obtained from patients with heterogeneous forms of chronic lower limb obliterating diseases (CLLODs).SUBJECTS AND METHODS:The investigation included the biopsy specimens obtained from 44 men under 65 years of age (their mean age was 54±9.8 years) with Stage IIB-IV chronic limb ischemia (according to the A.V. Pokrovsky-Fontaine classification) with its history of at least six months. The nosological entities were atherosclerotic lesion in 33 patients (distal atherosclerosis n=13), multistage lesion (n=8), and Leriche's syndrome (n=12); autoimmune-mediated vascular injury in 11 patients (Buerger's disease (n=7) and nonspecific aortoarteritis (n=4)). The similar muscle fragments obtained during autopsy from the deceased without obvious signs of cardiovascular system diseases were examined as a control.RESULTS:It was found that there was a statistically significant difference between the nosological entities, as compared to the control in terms of CD and CSMFA (a decrease), the proportion of CNMFs and that of connective tissue (an increase). No substantial differences were found in the studied parameters between the nosological entities.CONCLUSION:The findings may suggest the universal mechanism for damage to striated muscle tissue because of circulatory hypoxia, regardless of its etiology and the common character of tissue compensatory-adaptive responses (regeneration).

A number of sarcolemma proteins are responsible for muscle fiber repair. Dysferlin encoded by the DYSF gene is one of these proteins. Dysferlin promotes membrane repair in striated muscle fibers (MFs). Mutations in DYSF lead to loss of or decreased dysferlin expression, impaired membrane repair in MF, and its destruction, clinically manifesting as dysferlinopathy. Preclinical studies of cell and gene therapies aimed at restoring impaired muscle regeneration require well-characterized small animal models. Our investigation aimed to distinguish the histopathological features of a mouse strain lacking dysferlin expression (Bla/J strain). Ultrastructural changes in the sarcolemma, mitochondria and contractile apparatus were observed. It was shown that postnatal histogenesis of skeletal muscles in genetically determined dysferlin deficiency is characterized by a higher proportion of necrotic muscle fibers, compensatory hypertrophy of muscle fibers with their subsequent atrophy, and decreases in proliferative activity and the level of myogenic differentiation of myogenic progenitor cells compared to wild-type mice (C57Bl/6).

Development of effective methods for cartilage tissue repair is highly relevant for surgical practice. One of the promising approaches in this area is the use of gene-activated materials that capable to change the production of certain factors involved in reparative chondrogenesis regulation in the damaged area within a certain time. In this study, we developed two gene-activated hydrogels based on hyaluronic acid delivering plasmid DNA encoding vascular endothelial growth factor (VEGF-A) gene. One of the hydrogels also contained octacalcium phosphate microgranules. The materials were implanted into the defects of cranial bones, articular and auricle cartilages in rabbits. Control groups consisted of the same materials without plasmid DNA. We found that gene-activated materials formed a larger volume of cartilaginous tissue with complete restoration of the auricle by 90 days. Healing of the articular cartilage and subchondral bone was also more efficient at the 30 and 60 days follow-up, but by 90 days, remodeling of the newly formed cartilage into fibroelastic and fibrous connective tissues was observed in all groups. Cranial bone defects were filled with newly formed bone tissue in the test group by day 90, while in the control a residual defect filled by fibrous connective tissue was retained. Thus, developed variants of the gene-activated hydrogels are promising candidates to be medical devices for treatment of patients with bone and cartilage pathology.

Two variants of gene-activated hydrogels based on sodium alginate containing plasmid DNA with the gene of vascular endothelial growth factor (VEGF-A) are developed. The former represented alginate hydrogel without additional components; the latter contained up to 25 wt % of octacalcium phosphate (OCP) micrograins. Alginate-based hydrogels without OCP are characterized by a honeycomb structure with the pore size of 50–200 μm and mechanical compression strength of 0.2 MPa. Addition of OCP micrograins results in filling of the polymer framework by them and an increase in the compression strength to 0.57 MPa at 16.7 wt % of OCP with a decrease to 0.49 MPa with an increase in the OCP content to 20 wt %. Both variants of gene-activated hydrogels induce reparative myogenesis in the central zone of muscle defect; a larger number of MyoG+ cells and newly formed MyH7B+ muscle fibers are identified as compared to analogous hydrogels without plasmid DNA after two weeks after surgery (p < 0.05).

There is a strong unmet need for bone grafts and substitutes combining osteoinductive capacities and biophysical properties for using them in minimally invasive surgical interventions. We have developed three variants of injectable gene-activated bone substitutes containing three components: octacalcium phosphate microgranules (OCP), plasmid DNA delivering the gene of vascular endothelial growth factor, and one of the hydrogels based on sodium alginate, type I collagen, and hyaluronic acid. The molecules of the gene constructs were contained both in the hydrogel and on the surface of the OCP microgranules. In the model of a critical-sized bone defect in rabbit parietal bone, we found that all the gene-activated hydrogels contributed to bone tissue regeneration, however, the largest amount of newly formed bone, including those in the central part of the defect, was detected in the groups with gene-activated materials based on hyaluronic acid and collagen. Thus, the developed materials can be considered as candidates for medical devices, but additional studies are required to assess the dose-depended effect and optimize the materials composition.

A family of five male siblings (three survivors at 48, 53 and 58 years old; two deceased at 8 months old and 2.5 years old) demonstrating significant phenotypic variability ranging from intermediate to the myosclerotic like Bethlem myopathy is presented. Whole-exome sequencing (WES) identified a new homozygous missense mutation chr21:47402679 T > C in the canonical splice donor site of the second intron (c.227 + 2T>C) in the COL6A1 gene. mRNA analysis confirmed skipping of exon 2 encoding 925 amino-acids in 94–95% of resulting transcripts. Three sibs presented with intermediate phenotype of collagen VI-related dystrophies (48, 53 and 2.5 years old) while the fourth sibling (58 years old) was classified as Bethlem myopathy with spine rigidity. The two older siblings with the moderate progressive phenotype (48 and 53 years old) lost their ability to maintain a vertical posture caused by pronounced contractures of large joints, but continued to ambulate throughout life on fully bent legs without auxiliary means of support. Immunofluorescence analysis of dermal fibroblasts demonstrated that no type VI collagen was secreted in any of the siblings’ cells, regardless of clinical manifestations severity while fibroblast proliferation and colony formation ability was decreased. The detailed genetic and long term clinical data contribute to broadening the genotypic and phenotypic spectrum of COL6A1 related disease.

The number of studies related with gene-activated matrices is increasing annually; the first-in-class product has been already implemented into clinical practice for bone grafting indications. Considering specificity of the gene-activated matrices mechanism of action determined by gene constructs, there is a demand to standardize the methods allowing to characterize all the stages of biological action in vivo. Here, using on the example of a gene-activated hydrogel consisting of type I collagen and plasmid DNA with the vascular endothelial growth factor gene (VEGF165), the main steps of the plasmid DNA mechanism of action were confirmed by various methods. For this, a fluorescent Cy3, reporter plasmid DNA with the firefly luciferase gene (Luc), RT-PCR and ELISA, immunohistochemical study with antibodies to CD31 were used. The results were compared with the other scientific papers, some recommendations were formulated to determine a minimally required list of studies for the development of gene-activated materials.

Цель. Оценка показателей адаптивного иммунитета слизистой оболочки двенадцатиперстной кишки у детей с неонатальным сепсисом. Методы. Проведено исследование биоптатов слизистой оболочки двенадцатиперстной кишки, полученных при проведении дуоденоскопии новорождённых, имевших признаки эрозивно-язвенного поражения пищеварительного тракта. 5 из них были получены от детей с клинико-лабораторными признаками неонатального сепсиса, 2 — от детей группы сравнения. Проведено иммуногистохимическое окрашивание биоптатов с использованием системы визуализации Novolink Polymer Detection System с коммерческими антителами к CD4, CD8, CD20 и Bcl-2 и системы визуализации CSAII Biotin-free Tyramide Signal Amplification System с коммерческими антителами к каспазе-3 и каспазе-9 в соответствии с инструкциями производителя. Результаты. Было установлено, что количество Т-лимфоцитов CD4 + слизистой оболочки двенадцатиперстной кишки в группе новорождённых с сепсисом в 4 из 5 случаев не отличалось от показателей контрольной группы. Количество лимфоцитов CD8 + у детей с сепсисом в 3 из 5 случаев было даже бόльшим, чем в группе контроля. Количество B-лимфоцитов CD20 + у 4 детей с сепсисом было значительно меньше по сравнению с контролем. Установлена активация апоптоза в клетках слизистой оболочки у детей с сепсисом, что проявлялось бόльшим количеством каспаза-3-позитивных клеток (в 4 из 5 случаев) по сравнению с показателями контрольной группы. Количество каспаза-9-позитивных клеток в исследуемых группах было практически одинаковым. Отмечено значительное уменьшение количества Bcl-2-позитивных клеток слизистой оболочки у всех 5 детей с сепсисом по сравнению с показателями контрольной группы. Вывод. Проведённое исследование выявило умеренно выраженную иммуносупрессию в слизистой оболочке двенадцатиперстной кишки у детей с неонатальным сепсисом, проявившуюся низкими значениями В-лимфоцитов CD20 + на фоне отсутствия значимого снижения количества Т-лимфоцитов CD4 + и CD8 + ; установлена умеренно выраженная активация процессов апоптоза на фоне сниженного антиапоптотического потенциала, что создаёт условия возможной транслокации кишечной микробиоты в кровоток.

Optimization of the reparative regeneration of striated skeletal muscle tissue is actual for clinical practice. Volumetric muscle loss usually heals through the fibrous scar formation. Herein, there are numerous of methods under developed focused on reparative myogenesis induction. One of the promising approaches in this area is formed by gene-activated materials, particularly, in the hydrogel form. We developed a gene-activated hydrogel based on hyaluronic acid and plasmid DNA with the gene of vascular endothelial growth factor A (VEGF-A). Firstly, we showed a biocompatibility of the product in the subcutaneous test in mice. Using marker plasmid DNA carrying the luciferase gene, prolonged delivery of gene constructs to cells in vivo with a peak in transgene expression at day 7 was confirmed, while the same plasmid DNA in an aqueous solution provided a maximum level of delivery at day 1. Being implanted into a volumetric defect of the anterior tibial muscle in rats the gene-activated hydrogel activated angiogenesis in 2 weeks after surgery and induced MYH7B+-muscle fibers formation in the central zone of the defect at average number 50,0±16,1 and 21,8±10,5 in 2 and 4 weeks, respectively, whereas a hydrogel without plasmid DNA did not have any myogenic effects. Thus, plasmid DNA with VEGFA in the sodium alginate-based hydrogel induced angiogenesis in the volumetric muscle loss model and stimulated reparative myogenesis that could be used for further development of products effective for treatment of patients with muscle pathology.

Aim. To assess of adaptive immunity of the duodenal mucosa in neonates with sepsis. Methods. A study of duodenal biopsy specimens obtained during duodenoscopy from neonates who had signs of suspected ulcerative lesions of the digestive tract. Five of them were obtained from infants with clinical and laboratory signs of neonatal sepsis (NS), two from infants without sepsis (comparison group). Immunohistochemical staining was performed using the Novolink Polymer Detection System imaging system with commercial antibodies to CD4, CD8, CD20 and Bcl-2, and the CSAII Biotin-free Tyramide Signal Amplification System imaging system with commercial antibodies to caspase-3 and caspase-9 in accordance with the manufacturers instructions. Results. It was found that the number of CD4+ T-lymphocytes of the duodenal mucosa in neonatal sepsis group in 4 of 5 infants did not differ from the control group. The number of CD8+ lymphocytes in neonates with sepsis in 3 of 5 cases was even higher than in the control group. The number of CD20+ B-lymphocytes in 4 infants with sepsis was significantly less compared with the control. Activation of apoptosis in mucosal cells was detected, which was manifested by a large number of caspase-3-positive cells (in 4 of 5 cases) in comparison with the control group. The number of caspase-9-positive cells in the studied groups was almost equal. A substantial decrease in the number of Bcl-2-positive mucosal cells in all 5 infants with sepsis was noted as compared with the control indicators. Conclusion. The study revealed moderate immunosuppression in the duodenal mucosa in neonates with sepsis, manifested by low values of CD20+ B-lymphocytes in the absence of a significant decrease in the number of CD4+ and CD8+ T-lymphocytes. The detected moderate activation of apoptosis processes against the background of reduced antiapoptotic potential creates the conditions for a possible translocation of the intestinal microbiota into the bloodstream.

Cilia (cilia) are organelles that are characteristic exclusively for eukaryotes and are found in protozoa, on somatic and germ cells of multicellular, as well as gametes of many plants. In humans, two main types of cilia are distinguished: motile and sensory; also in embryogenesis, it is customary to isolate special nodular cilia necessary for the normal course of gastrulation and possibly subsequent histo- and organogenesis. Motile cilia provide the movement of the liquid medium relative to the cell in the respiratory tract, the ventricular system of the brain and the fallopian tubes, or the movement of the cell itself in the case of sperm. The main function of sensory cilia is the perception of changes in the external environment and the signal molecules inside it and their conversion into intracellular signals that regulate proliferation, differentiation, and programmed cell death. Ciliopathies, a group of pathological conditions associated with impaired development, structure, and functioning of cilia, are of clinical interest. The most studied ciliopathies include polycystic kidney disease, nephronophysis, Barde-Beadle, Joubert, Mekel, Kartagener, Karoli etc. Clinical nephronophthisis and morphological analysis of the case of Caroli, syndrome is given.

The new coronavirus infection is a highly contagious infection caused by the SARS-CoV-2 virus that has become a global public health problem. The pathogenesis of this virus has not yet been clearly understood, the principles of hyperinflammatory immune response in critically ill patients, which leads to acute respiratory distress syndrome and multiple organ failure, innate and adaptive immune responses in the process of structuring the data under study. The interaction of the virus and a macroorganism includes 4 stages: infection, dissemination, cytokine storm, pulmonary fibrosis. This review analyzes the predictors of infection, its possible pathogenesis, the immune response of the macroorganism, as well as the histological characteristics of damage to immune organs; shows receptors for SARS-CoV-2 (ACE2, TMPRSS2) in some organs.