Irina Alexandrovna Chekmareva

Dysferlinopathy is a rare muscular dystrophy characterized by chronic muscle damage and ineffective regeneration. While late-stage morphological changes, such as fibroadipose replacement, are well described, the early molecular mechanisms driving muscle fiber loss and regenerative failure at the onset of the disease remain largely uncharacterized. To address this gap, we investigated the skeletal muscles of dysferlin-deficient Bla/J mice during the early manifestation stage (3 months of age). This exploratory study aimed to identify primary pathomorphogenetic events by correlating the transcriptomic profile of the tissue with its specific histopathological and ultrastructural alterations. We performed a comparative analysis of the m. gastrocnemius in 3-month-old Bla/J mice versus wild-type controls using RNA sequencing, RT-qPCR, histomorphometry and transmission electron microscopy. The results revealed atrophy and muscle fiber necrosis without the expected induction of Fbxo32 and Trim63 ubiquitin ligases, suggesting ubiquitin-proteasome system-independent muscle mass loss. Furthermore, the absence of Casp3, Bak1, and Bad induction, confirmed by the lack of active caspase-3, excluded apoptosis as the primary death mechanism. A differentiation block in satellite cells was confirmed by the lack of Myf5, Myod1, and Myog induction and a trend toward Tead4 suppression, pointing to an early failure of the reparative program. Exploratory RNA sequencing also identified a suppression of Prkn expression accompanied by LC3B-II-positive autophagosome accumulation. Immunohistochemical and immunofluorescent evaluation of the mitochondrial network (TOMM20) revealed abnormal accumulations and dense clumping, indicating impaired organelle clearance. Furthermore, ultrastructural analysis demonstrated internal organelle damage and the presence of myelin-like structures, consistent with a state of stalled mitophagy. Collectively, this exploratory study demonstrates that early muscle atrophy and myofiber necrosis in dysferlinopathy occur independently of canonical ubiquitin-proteasome and apoptotic pathways. Instead, the disease manifestation stage is structurally characterized by stalled mitochondrial clearance and a delayed regenerative response.

Background. In conditions of increasing bacterial resistance to antibiotics, bacteriophages are becoming particularly important as a promising tool for combating surgical infection of the skin and soft tissues. Scientific studies in recent years have demonstrated the effectiveness of phage therapy both as a treatment method and in order to prevent the development of nosocomial infectious complications, including infections caused by multidrug-resistant strains of microorganisms. Objective. The aim of the study was a comparative clinical, cytomorphological and microbiological analysis of the efficacy and safety of phage therapy in the treatment of purulent wounds of various etiologies and localization in patients over 18 years of age. Materials and methods. The article presents the results of a prospective randomized comparative clinical trial of 80 adult patients with purulent wounds of various etiologies and localization treated at the Department of Wounds and Wound Infections of the A.V. Vishnevsky National Medical Research Center of Surgery of the Ministry of Health of the Russian Federation in 2022—2024 (LEK conclusion No. 7/2NIR-22 dated 05/31/2022). During the study, clinical (edema, pain, the nature of wound discharge, the appearance of granulation tissue), laboratory (the presence and regression of signs of a systemic inflammatory response to the presence of a surgical infection), microbiological (qualitative and quantitative), cytological and morphological parameters were analyzed in all patients. Parametric data were evaluated based on the mean and standard deviation (SD) at 4 study points: point 1 (1±1 day), point 2 (3±1 day), point 3 (7±1 day) and point 4 (9±1 day) after surgical treatment of the purulent focus. The key endpoint was the transition of the wound healing process into the regeneration phase. Results. According to the study protocol, all the patients included in the analysis had purulent-necrotic wounds of soft tissues and bones of different etiology and localization, requiring surgical treatment. To distribute patients into groups, upon admission or intraoperatively, material was collected for qualitative and quantitative microbiological, cytological, morphological and electron microscopic studies. Randomization was carried out using the pseudorandomization method, patients were selected in a 1:1 ratio based on an assessment of the sensitivity of isolated cultures to the drug “Piobacteriophage complex” liquid (Piophage, NPO Microgen JSC, Russia). In the postoperative period, patients of group I (n=20) received systemic and local phage therapy (20 ml 3 times per day, according to the instructions); in subgroup Ia (n=20), bacteriophages were delivered to the focus using negative pressure devices with instillation (–120 mm Hg, permanent mode, VitMedical, Russia); in group II (n=20), systemic and local antibacterial therapy was performed; in group III (n=20)— systemic treatment with local negative pressure (–120 mmHg, permanent mode, VitMedical, Russia). An analysis of the treatment results indicates the high effectiveness of the proposed protocol in all study groups. In group 1a (local negative pressure with bacteriophage instillation in combination with systemic phage therapy) The best microbiological, cytological, morphological and clinical efficacy was noted, indicating the potentiation of both methods of treating purulent wounds of various etiology and localization (the first signs of relief of the inflammatory phase were noted by 3±1 days after surgery). In general, key endpoints (the transition of the wound healing process to the regeneration phase) were achieved in all the studied groups with good tolerability of therapy and a high safety profile. No additional surgical interventions were required in any group, and no deaths were reported. No adverse events requiring discontinuation or change of treatment option were identified. Conclusion. The principles of purulent wounds treatment in our country are based on the method of active surgical treatment developed in the Department of Wounds and Wound Infections of the Vishnevsky Institute of Surgery (now the Vishnevsky National Research Medical Center of Surgery of the Ministry of Health of the Russian Federation). The results of treatment largely depend on the radicality and thoroughness of the primary surgical debridement of the purulent focus, general and local treatment in the perioperative period. The inclusion of bacteriophages in treatment regimens, especially in combination with local negative pressure, allows for a more pronounced antimicrobial and regenerative effect, which makes this approach promising in the treatment of purulent-inflammatory diseases. The combination of active surgical treatment with local negative pressure therapy and bacteriophage instillation demonstrates pronounced synergy: surgical treatment of purulent wounds reduces microbial contamination and destroys biofilms, which increases the availability of phages. In turn, their bacteriolytic and bacteriostatic activity prevents re-colonization and promotes the transition of the wound process to the regeneration phase, which is especially important in the presence of polyresistance.

In idiopathic azoospermia caused by non-obstructive infertility with AZFc deletion, the testicle usually contains an increased number of mast cells (MCs)—which are responsible for collagen synthesis in the testes—as well as Leydig cell hyperplasia. However, the relationship between MCs and telocytes in this pathology remains unexplored. The aim of this study was to examine ultrastructural changes in the interstitial tissue microenvironment of the convoluted seminiferous tubules in the testis, using clinical specimens from men with genetically determined non-obstructive infertility with AZFc deletion. Histological, immunohistochemical, and electron microscopic (EM) studies were performed on surgical materials from 14 patients with AZFc deletion. The IHC study was performed using a panel of antibodies: tryptase, chymase, carboxypeptidase A3, and αSMA. The EM study was performed on ultrathin sections with a thickness of 100–120 nm. MCs were found to be in a functionally active state and characterized by a variety of secretory activities. For the first time, telocytes and their colocalization with MCs and Leydig cells were visualized. It is possibly the telocytes—interacting with MCs—that synchronize the functional activity of the entire MC population of the testis. The interaction of MCs with telocytes, as well as individual secretory granules associated with loci of tropocollagen and collagen microfibril accumulation, leads to the accumulation of collagen fibrils in the interstitium, as observed in idiopathic infertility with AZFc deletion. Even with a small number of MCs in the interstitium of the convoluted seminiferous tubules in the testis, the telocytes are able to synchronize MCs’ activation and secretory activity, supporting the development of a profibrotic phenotype of the tissue microenvironment. The obtained results advance our understanding of idiopathic infertility with AZFc deletion by delineating the ultrastructural landscape of the testicular interstitium and establishing telocytes as key regulators of cellular crosstalk. Telocytes use complex mechanisms for the spatial integration of MCs and fibroblasts in the profibrotic phenotype formation of the convoluted seminiferous tubule tissue microenvironment. Potentially, telocytes can directly be involved in synchronizing such processes by activating the biogenesis and secretion of collagen monomers by fibroblasts; the MC secretome directly affects the polymerization of collagen monomers and dimers into microfibrils in the extracellular matrix, stimulating excessive collagen fiber formation and the development of fibrotic changes.

Objective. To analyze different methods for autologous transplantation of stromal-vascular fraction of adipose tissue combined with skin grafting with split-thickness skin graft 1:6 in patients with extensive deep burns. Material and methods. Open comparative controlled prospective study was devoted to results of autologous skin grafting with split-thickness skin graft 1:6 and autologous cells of stromal-vascular fraction of adipose tissue. Patients were divided into 4 groups depending on transplantation method: injection, aerosol, combined (aerosol+injection) and comparison group (standard skin grafting with split-thickness perforated transplants). There were 43 cases. Inclusion criteria: age 18—65 years, total area of skin burn 10—60% of body surface area, deep burn 10—45% of body surface area. Autologous skin grafting was performed on 10—15% of body surface area. Clinical, laboratory, cytological and histological methods were used to evaluate postoperative outcomes. Results. Combined (injection+aerosol) transplantation method was followed by significantly earlier engraftment (by 5 days, 9.9 ±0.7 days in the main group and 14.5±0.88 days in the control group, Student’s t test 3.518) and reduction of postoperative complications to 6% (in the control group 27%). Conclusion. Analysis of biotechnological methods for surgical treatment of patients with extensive deep burns showed the effectiveness of combined (aerosol+injection) transplantation and skin grafting with split-thickness skin graft. This approach reduced skin recovery time and incidence of postoperative complications.

Wound healing is a delicately regulated pathophysiological process based on molecular, cellular, and tissue interactions. Mast cells (MCs) are involved in the reparative process in all phases of wound healing, which indicates their general significance in reparative processes. The structural and functional changes in the MCs during the healing process correspond to the phase of the wound process and determine its course. In the inflammatory phase, rapid whole-granular degranulation of MCs with the secretion of biologically active proinflammatory substances that have a stimulating effect on inflammatory cells prevailed. In the proliferation phase, the maximum number of MCs per unit area of wound tissue and the maximum degranulation index were noted. In the phase of granulated tissue remodeling, the amount and functional activity of MCs sharply decrease, which contributes to the completion of the healing process with the formation of a fully fledged normotrophic scar. The gradual degranulation of MCs was characteristic of the proliferation and remodeling phases. The treatment of purulent wounds with a drug from the polyhexamethylene guanidine group with the antiseptic polyhexanide 0.1% contributed to a temporary shift in the phases of the wound process while maintaining its general patterns, while the activation of the process occurred at an earlier time than in the control group of animals without local treatment. The results obtained showed that the use of a drug from the polyhexamethylene guanidine group with the antiseptic polyhexanide 0.1% for the treatment of purulent wounds quickly stops the inflammatory response and creates conditions for the development of the reparative abilities of granulation tissue cells, and primarily, mast cells.

Pancreatic inflammation and subsequent fibrosis drive serious disease complications. However, the pathogenesis of this process and the mechanisms underlying excessive extracellular matrix (ECM) deposition remain poorly understood. Our aim was to study intercellular interactions and ultrastructural changes in mast cells, pancreatic stellate cells, and telocytes, as well as in the extracellular matrix in various degrees of pancreatic fibrosis. Histological, immunohistochemical, and electron microscopic (EM) studies were performed on surgical materials from 17 patients. Mapping of fibrosis fields was performed on scanned images using the QuPath software v0.6.0. The IHC study was performed using a panel of antibodies: CD34, CD117, and SMA. Fluorescent IHC was performed using a panel of antibodies: CD34 and CD117. The EM study was performed on ultrathin sections with a thickness of 100–120 nm. The functional activity of mast cells (MCs) increased in pancreatic fibrosis. Most of the MCs were in a degranulation state, with the formation of intercellular contacts. The activation of pancreatic stellate cells (PaSCs), which underwent ultrastructural and functional changes in pancreatic fibrosis that developed as a result of chronic pancreatitis (CP), was noted. Multiple plasmolemma discontinuities, telopode shortenings, and nuclear fragmentations were observed among telocytes (TCs). The presence of MCs in the inflammatory infiltrate, as well as the destruction of TCs with the activation of exosomal transport, plays an important role in the pathogenesis of fibrosis in CP and provides a promising therapeutic target for the treatment of this pathology.

Varicose disease and other age-related vascular illnesses are extremely prevalent among the adult population. Despite this, research devoted to involutive changes in the veins of the lower extremities is rare and fragmented. Complex morphological evaluation of the wall of the vein related to age and varicose disease can add valuable data to fundamental geriatric and vascular medicine. Objectives: The study was designed to determine the age-related changes in the muscular component of the great saphenous vein and changes associated with varicose disease. Materials and Methods: A morphological study of a specimen of the great saphenous vein was conducted on 55 deceased individuals and 80 patients with varicose disease. Four age subgroups were identified: young, middle-aged, elderly, and senile. A total of 135 fragments of the great saphenous vein were evaluated. Histological, morphometric, and electron microscopic studies were performed. A quantitative analysis of the volumetric fraction of muscular components was calculated using the Shapiro–Wilk test, Kruskal–Wallis (ANOVA) and Mann–Whitney methods with Bonferroni correction. Results: Our study showed that the amount of connective tissue elements between bundles of smooth muscle cells increased with age. In patients with varicose disease, we observed an appearance of connective tissue fibers among smooth muscle cells, more pronounced with the disease progression. The structure of smooth muscle cell changes. Thus, we observed hypertrophy and phenotypic heterogeneity of cells with subsequent destruction of communicative contacts. The values of subintimal longitudinally arranged smooth muscle cells reached their maximum in middle age in both normal and varicose veins, while significant decrease occurred in elderly and senile patients. Quantitative indicators of circularly arranged smooth muscle cells of the middle layer did not change with age but significantly decreased in varicose disease. Age-related changes are characterized by an increase in the proportion of smooth muscle cells in the outer layer. In varicose veins, in young and middle-aged patients, the content of bundles of longitudinally arranged smooth muscle cells in the outer layer was higher compared to the age norm, with a significant decrease in senile age. Conclusions: The age norm of the muscular component of the great saphenous vein wall is characterized by an increase in the volumetric fraction of subintimal longitudinally arranged smooth muscle cells in middle age, the volumetric fraction of circularly arranged smooth muscle cells of the middle layer remains unchanged, and the volumetric fraction of bundles of longitudinally arranged myocytes of the outer layer increases. With age in varicose disease, sclerotic changes progress in the structure of the great saphenous vein at the tissue, cellular, and intracellular levels, leading to a decrease in the volumetric fraction of all muscular components of the great saphenous vein structure.

BACKGROUND: With the sharp increase in mine-explosive injuries, one of the key tasks of modern histology is to assess the morphogenesis of high-energy limb tissue damage. AIM: This study aimed to characterize striated skeletal muscle tissue in the zone of mine-explosive limb segment avulsion during the early post-traumatic period (days 1–4). METHODS: Histological methods (hematoxylin and eosin staining and Martius Scarlet Blue [MSB] staining for fibrin age determination), immunohistochemical and immunofluorescence techniques (antibodies to CD3, CD20, CD31 [Platelet Endothelial Cell Adhesion Molecule-1], CD68, and NETs [Neutrophil Extracellular Traps]), transmission electron microscopy, and morphometric analyses were applied. RESULTS: Post-traumatic pathological changes affected both muscle tissue (necrosis, fiber fragmentation, traumatic edema) and endomysial and perimysial connective tissue (traumatic edema, fibrin infiltration). Disruption of the cytoskeletal structure (sarcomeres), loss of myosatellite cells, and endothelial cell death in capillaries were observed. Local hemodynamic disturbances manifested as mosaic thrombosis, embolism, and hemorrhage. Within the first 24 hours, arterial and arteriolar spasms led to reduced tissue perfusion. Partial resolution of the spasms by day 4 likely corresponded to the second stage of traumatic shock (the torpid phase). Among leukocytes, CD68+ macrophages were the first to respond, with extravasation beginning on day 2 after injury. A similar trend was observed for CD3+ T cells. CD20+ B cells showed no morphological signs of reactivity during this period. NET formation was observed both intravascularly and in the extravascular compartment. CONCLUSION: Structural and ultrastructural changes in skeletal muscle tissue within the first 4 days after mine-explosive injury have been characterized.

Tubulinopathies encompass a wide spectrum of disorders resulting from variants in genes encoding α- and β-tubulins, the key components of microtubules. While previous studies have linked de novo or dominantly inherited TUBA4A missense variants to neurodegenerative phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, hereditary spastic ataxia, and more recently, an isolated report of congenital myopathy, the full phenotypic and genotypic spectrum of TUBA4A -related disorders remains incompletely characterised. In this multi-centre study, we identified 13 novel TUBA4A missense variants in 31 individuals from 19 unrelated families. Remarkably, affected individuals in 17 families presented with a primary axial myopathy without any identified CNS involvement or history of such disease. In the remaining two families, we observed probands with cerebellar ataxia and epilepsy accompanying proximal and axial muscle weakness, establishing the first documented association between TUBA4A variants and multisystem proteinopathy. Our cohort exhibited diverse genotypes and associated inheritance patterns: four families demonstrated autosomal dominant transmission through heterozygous variants in TUBA4A , three probands had homozygous TUBA4A variants, where the biallelic genotype was found to be associated with the disease, and the heterozygous carriers were asymptomatic; five probands carried de novo variants, and nine probands with heterozygous TUBA4A variants were classified as "isolated-sporadic cases" where parental samples were unavailable. Clinical phenotypes ranged from mild to severe myopathy, predominantly affecting the axial and paraspinal muscles. We observed a range of disease onset, from congenital to late adulthood. Creatine kinase levels were also variable, ranging from normal to highly elevated. Cardiac function remained preserved across the cohort. Muscle biopsies revealed a range of pathologies, including myofibre size variation, myofibre atrophy, nemaline bodies, core-like regions, internal nuclei, and endomysial fibrosis. Immunohistochemical staining showed evidence of proteinopathy, with autophagic features and TUBA4A accumulation in patient myofibres. Complementary in silico and in vitro investigations suggested that the identified TUBA4A substitutions cause significant protein abnormalities and may differentially impact microtubule dynamics. Our findings establish myo-tubulinopathies as distinct clinical entities, encompassing both primary myopathies and multisystem proteinopathies with muscle involvement. This study broadens the phenotypic and genotypic spectrum of TUBA4A -related disorders beyond autosomal dominant or de novo mechanisms and neurodegenerative presentations. These results underscore the importance of considering TUBA4A variants in the differential diagnosis of axial myopathies and multisystem proteinopathies, regardless of central nervous system (CNS) involvement.