Alexander V. Alekhnovich

The article highlights the relevance of developing innovative technologies for the purposes of forensic pathology, describes the possibilities of the multiplex IHC method and shows some areas of its application. The tasks and problems of determining the vitality and age of injuries have been identified, which can be solved by using a multiplex IHC. The prospects of this method are revealed, which consist in the ability to formulate original hypotheses and ideas about the mechanisms of reparative regeneration, determine informative criteria of the local tissue microenvironment for detecting pathological changes, and identify new criteria for the inflammatory-reparative process in order to narrow time intervals and increase the accuracy of determining the prescription of damage in various types of trauma.

Objective. To evaluate the efficacy, safety, and tolerability of nutritional metabolic support (NMS) in surgical patients with burn disease (BD) and to analyze its impact on the postoperative course. Materials and methods. All patients had severe burns with thermal burns covering up to 59% of the body surface area (trunk, chest, abdomen, back, and extremities in various combinations). These patients received conventional therapy appropriate to the stage of their illness (administration of analgesics, sedatives, antibiotics, infusion and transfusion therapy, correction of fluid-electrolyte, acid-base, and blood rheology balances, and surgical treatment of burn wounds). The study group consisted of 31 (61%) burn patients. Their comprehensive treatment included enteral nutritional support using specialized dietary therapeutic and preventive nutrition products — a Detoxifying Protein Cocktail twice daily (morning and lunchtime) and a Restorative Protein Cocktail once daily (evening), manufactured by LEOVIT nutrio — for 21 days. The control group included 20 burn patients who did not receive nutritional support. Burn severity was assessed using the Frank Burn Index (FBI), taking into account the area and depth of the lesion, as well as the presence of respiratory tract burns. Patients in the groups were comparable in severity. Treatment progress was monitored using general clinical laboratory testing methods. Results. The use of therapeutic metabolic enteral nutrition has a beneficial effect on the general condition of patients; Improves healing and epithelialization, accelerates burn scab rejection, improves clinical and biochemical blood parameters, intoxication and inflammation markers, enhances antioxidant defense, reduces inflammation, and improves patients’ quality of life. Conclusion. This study demonstrates the high clinical efficacy of domestically produced metabolic enteral nutrition products in surgical patients with burn disease. Effective domestic enteral nutrition ensures technological sovereignty and import substitution.

The system of management of the epidemiological safety developed by the authors and Public health, sociology, medical and social expertise based on integration of the epidemiological service in the service of quality control of medical care, development of local regulatory legal base (11 orders) with consideration of the current federal framework, creation of a clear algorithm of work (54 standards of operating procedures) and distribution of functional duties (updating of the job instructions for 152 physicians, 298 mid-level medical workers and 123 junior medical workers) contributed to the growth of the medical institution’s epidemiological safety from 63.6% 100%, in particular owing to the comprehensive approach to the organization of internal control of the quality of medical care and safety of medical activity. Joint work of the two mentioned services also resulted in decrease of regular and extraordinary inspections and double redaction in the amount of accounting documents, in particular for the hospital’s epidemiologist.

BACKGROUND: There are currently no published studies on the role of mast cells in the pathogenesis of sporadic medullary thyroid carcinoma. However, their involvement in the progression of a variety of epithelial malignant neoplasms has been demonstrated. Furthermore, mast cell count may be an independent predictor of long-term progression-free survival in patients with pancreatic neuroendocrine tumors. AIM: The work aimed to evaluate the potential of immunohistochemical detection of mast cells in the tumor microenvironment in sporadic medullary thyroid carcinoma. METHODS: Histological specimens of sporadic medullary thyroid carcinoma were assessed using immunohistochemical detection of tryptase in mast cells. A convolutional neural network (CNN) model was then trained to segment positively stained cells, followed by quantitative analysis of the results. RESULTS: Several potentially clinically significant parameters were identified, including correlations between mast cell count in the thyroid stroma and age; correlations between intratumoral mast cell count and T stage according to the TNM (8th edition) classification; and patterns of mast cell colocalization with other cells of the tumor microenvironment. CONCLUSION: The study confirmed the presence of mast cells in the stroma of medullary thyroid carcinoma and revealed quantitative differences depending on tumor size. The observed active interactions of mast cells with atypical cells of sporadic medullary thyroid carcinoma and other components of the tumor microenvironment are a significant criterion for interpreting the biological effects of mast cells in this tumor type. These findings warrant further analysis to develop diagnostic algorithms and improve prognostic accuracy.

Endometritis features an inflammatory milieu in the endometrium, accompanied by the recruitment of immunocompetent cells, including mast cells (MCs). The mechanisms underlying MC involvement in chronic endometritis (CE) and fibrous niche formation remain poorly understood, particularly regarding spatial intercellular interactions in situ. In this study, we used multiplex immunohistochemistry and quantitative immunofluorescence analysis to map the spatial phenotype of MC distribution. Standard histochemical techniques, monoplex and multiplex immunohistochemical staining technologies, light-field microscopy, epifluorescence, and confocal microscopy with multispectral imaging, combined with quantitative immunofluorescence analysis with AI application, were used to identify the spatial phenotyping of quantitative and qualitative features of the endometrial MC population in CE. The increased intensity of endometrial inflammation was accompanied by a rise in the profile of MC content in the endometrium; this accounted for a 0.014% increase in the control and 0.067%, 0.113%, and 0.206% increases in mild, moderate, and severe CE, respectively. We are the first to map the number of MCs that demonstrated loci of accumulations in the endometrium coinciding with foci of fibrous changes. The number of these foci correlated with the severity of chronic endometritis and the development of clinical signs. The frequency of juxtacrine and paracrine MC colocalization with other immunocompetent cells increased with increased CE activity and fibrotic changes: For CD8⁺ lymphocytes, colocalization increased from 4.6% in the control to 11.6%, 18.5%, and 28.0% in mild, moderate, and severe CE, respectively. For monocytes, colocalization increased from 5.6% in the control to 18.7%, 26.8%, and 28.8% in mild, moderate, and severe CE, respectively. For type 1 macrophages, colocalization increased from 5.6% in the control to 13.5%, 17.4%, and 24.6% in mild, moderate, and severe CE, respectively. For type 2 macrophages, colocalization increased from 3.4% in the control to 9.6%, 9.1%, and 21.5% in mild, moderate, and severe CE, respectively. Spatial patterns of juxtacrine and paracrine MC interactions with other immune cells may provide diagnostic algorithms for chronic endometritis, enabling targeted therapy and preventing fibrotic changes.

Objective. To evaluate the efficacy, safety, and tolerability of nutritional metabolic support (NMS) in surgical patients with polytrauma involving the gastrointestinal tract, as well as to analyze its impact on the postoperative period. Material and methods. Eighty patients with gastrointestinal tract trauma were included in the study using an open randomization method. Most patients were in a serious or moderate condition. Twenty patients in the first main group received the «Protein Detoxification Cocktail» (PDC), and those in the second main group received the «Protein Recovery Cocktail» (PRC), one serving twice daily. The third main group included 20 patients with polytrauma who received one serving of PDC in the morning and one serving of PRC in the evening (PDC+PRC) for 21 days. Twenty patients in the control group did not receive additional NMS. Anthropometric parameters, laboratory data, and questionnaire data were assessed on Day 1 of the study and after 21 days. Results. The studied enteral metabolic nutrition reduced gastrointestinal and metabolic disorders, normalized body weight, increased blood levels of total protein and albumin, improved markers of intoxication and inflammation, enhanced wound healing, increased vital energy, accelerated the recovery of patients’ quality of life, and significantly improved overall condition. The medical nutrition did not cause intolerance or allergic reactions. Patients did not refuse to take it and consumed the entire servings. Conclusion. The study demonstrated the high clinical efficacy of the metabolic enteral nutrition products in the perioperative period. The enteral nutrition developed In Russia ensures technological sovereignty and import substitution.

Cutaneous melanoma remains one of the most aggressive tumors, yet the role innate immunity plays in its progression remains poorly understood. Effector elements with high regulatory potential, capable of both promoting and inhibiting tumor growth—mast cells (MCs), are of particular interest. This includes quantitatively characterizing the interactions between tryptase-positive mast cells (MCs) with atypical Melanin—A⁺ cells and describing their spatial phenotype, in relation to the stage of cutaneous melanoma. A retrospective analysis was carried out on samples retrieved from 128 patients with cutaneous melanoma (AJCC 8th edition: IA–IIID). Histological analysis, histochemistry (toluidine blue, Giemsa), and diplex /multiplex IHC for tryptase and Melan-A were performed; as well as Fluorescence imaging, 3D reconstructions and quantitative mapping in QuPath v 0.6.0. Proximity was assessed by the nucleus-to-nucleus distance: 20 μm (out of interaction). The relative amount of MCs in the intratumoral zone was lower than in the intact dermis, with a simultaneous increase in their absolute density per mm² in the melanoma microenvironment, maximum in the peritumoral area and most pronounced at stage II. Three types of interactions were identified: (i) juxtaposition without secretion, (ii) degranulation of MCs directed to tumor cells, (iii) melanosecretion of Melanin—A⁺ cells directed towards MCs, followed by phagocytosis of melanocores. An inverse intratumoral connection between the number of MCs and the number of Melanin—A⁺ cells was noted; MCs with elongated forms, extensive contacts and polarized tryptase secretion, including granule localization near/at the nuclei of adjacent cells, were frequently observed. The obtained data indicate stage-region-dependent bidirectional cross-talk between melanin and MCs, forming tissue spatial signals, potentially useful as biomarkers and targets for personalized therapy.

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.

Background: Phosphaturic mesenchymal tumours secreting fibroblast growth factor 23 (hereinafter referred to as FGF23+ PMT) are rare neoplasms that can cause hypophosphataemic osteomalacia, owing to excessive FGF23 production. Mast cells (MCs) play a key role in tumour biology by modulating proliferative activity of atypical cells, resistance to innate and acquired immunity, angiogenesis, and metastatic behaviour. However, MCs associated with FGF23+ PMT have not previously been investigated. This study, to our knowledge, is the first to characterise features of the tumour microenvironment through spatial phenotyping of the immune and stromal landscape, together with histotopographic mapping of intercellular MC interactions with other subcellular populations in FGF23+ PMT. Methods: Histochemical staining (haematoxylin and eosin, toluidine blue, Giemsa solution, picro-Mallory protocol, silver impregnation), as well as monoplex and multiplex immunohistochemical staining with spatial phenotyping, were performed to detect atypical FGF23-secreting cells, immune cells (CD3, CD4, CD8, CD14, CD20, CD38, CD68, or CD163), stromal components (CD31, α-SMA, or vimentin), and specific MC proteases (tryptase, chymase, or carboxypeptidase A3). Bioinformatics analysis using artificial intelligence technologies was applied for spatial profiling of MC interactions with tumour, immunocompetent, and stromal cells in the tumour microenvironment. Results: Bioinformatic analysis of the entire tumour histological section, comprising over 70,000 cells stained using monoplex and multiplex immunohistochemical protocols, enabled identification of more than half of the cell population. The most abundant were CD14+ (30.7%), CD163+ (23.2%), and CD31+ (17.9%) cells. Tumour-associated MCs accounted for 0.7% of the total pool of immunopositive cells and included both mucosal and connective tissue subpopulations, predominantly of the tryptase + chymase-CPA3-specific protease phenotype. This pattern reflected combined multidirectional morphogenetic processes in the patient’s FGF23+ PMT. More than 50% of MCs were colocalized with neighbouring cells of the tumour microenvironment within 20 μm, most frequently with monocytes (CD14+CD68+), M2 macrophages (CD68+CD163+), and endothelial cells (CD31+). In contrast, colocalization with atypical FGF23-secreting cells was rare, indicating minimal direct effects on tumour cell activity. Interaction with T lymphocytes, including CD8+, was also infrequent, excluding their activation and the development of antitumour effects. Mapping of MC histotopography validated the hypothesis of their inductive role in monocyte differentiation into M2 macrophages and probable polarisation of macrophages from M1 into M2, thereby contributing to slow tumour growth. MCs were further involved in extracellular matrix remodelling and participated in the formation of pro-osteogenic niches within the FGF23+ PMT microenvironment, leading to pathological osteoid development. Conclusions: This study demonstrated active MC participation in the evolution of the FGF23+ PMT microenvironment. The findings may be applied in translational medicine to develop novel algorithms for personalised therapy in patients with FGF23-secreting tumours, offering an alternative when surgical removal of the tumour is not feasible.