The Contribution of Mast Cells to the Regulation of Elastic Fiber Tensometry in the Skin Dermis of Children with Marfan Syndrome.

Marfan syndrome (MFS) is a hereditary condition accompanied by disorders in the structural and regulatory properties of connective tissue, including elastic fibers, due to a mutation in the gene encodes for fibrillin-1 protein (FBN1 gene) and the synthesis of abnormal fibrillin-1 glycoprotein. Despite the high potential of mast cells (MCs) to remodel the extracellular matrix (ECM), their pathogenetic significance in MFS has not been considered yet. The group of patients with Marfan syndrome included two mothers and five children (three girls aged 4, 11, and 11 and two boys aged 12 and 13). Normal skin was examined in two children aged 11 and 12. Histochemical, monoplex, and multiplex immunohistochemical techniques; combined protocols of simultaneous histochemical and immunohistochemical staining (the results of staining were assessed using light, epifluorescence, and confocal microscopy); and bioinformatics algorithms for the quantitative analysis of detected targets were used to evaluate mast cells and their relationship with other cells from extracellular structures in the skin dermis. Analysis of the skin MC population in children with Marfan syndrome revealed a considerably increased number of intra-organic populations with the preservation of the specific Tryptase+Chymase+CPA3+ protease profile typical of the skin. The features of the MC histotopography phenotype in MFS consisted of closer colocalization with elastic fibers, smooth muscle cells, and fibroblasts. MCs formed many intradermal clusters that synchronized the activity of cell functions in the stromal landscape of the tissue microenvironment with the help of spatial architectonics, including the formation of cell chains and the creation of fibrous niches. In MCs, the expression of specific proteases, TGF-ß, and heparin increased, with targeted secretion of biologically active substances relative to the dermal elastic fibers, which had specific structural features in MFS, including abnormal variability in thickness along their entire length, alternating thickened and thinned areas, and uneven surface topography. This paper discusses the potential role of MCs in strain analysis (tensometry) of the tissue microenvironment in MFS. Thus, the quantitative and qualitative rearrangements of the skin MC population in MFS are aimed at altering the stromal landscape of the connective tissue. The results obtained should be taken into account when managing clinical signs of MFS manifested in other pathogenetically critical structures of internal organs, including the aorta, tendons, cartilage, and parenchymal organs.

Renal Mast Cell-Specific Proteases in the Pathogenesis of Tubulointerstitial Fibrosis.

Chronic kidney disease is detected in 8-15% of the world's population. Along with fibrotic changes, it can lead to a complete loss of organ function. Therefore, a better understanding of the onset of the pathological process is required. To address this issue, we examined the interaction between mast cells (MCs) and cells in fibrous and intact regions, focusing on the role of MC proteases such as tryptase, chymase, and carboxypeptidase A3 (CPA3). MCs appear to be involved in the development of inflammatory and fibrotic changes through the targeted secretion of tryptase, chymase, and CPA3 to the vascular endothelium, nephron epithelium, interstitial cells, and components of intercellular substances. Protease-based phenotyping of renal MCs showed that tryptase-positive MCs were the most common phenotype at all anatomic sites. The infiltration of MC in different anatomic sites of the kidney with an associated release of protease content was accompanied by a loss of contact between the epithelium and the basement membrane, indicating the active participation of MCs in the formation and development of fibrogenic niches in the kidney. These findings may contribute to the development of novel strategies for the treatment of tubulointerstitial fibrosis.

Burn Wound Healing Activity of Hydroxyethylcellulose Gels with Different Water Extracts Obtained from Various Medicinal Plants in Pseudomonas aeruginosa-Infected Rabbits.

Burn injuries represent a significant problem in clinical practice due to the high risk of infection and the prolonged healing process. Recently, more attention has been given to natural remedies such as water extracts of various medicinal plants, which possess anti-inflammatory and wound healing properties. The aim of this study is to evaluate the efficacy and safety of Satureja montana L. and other water extracts in a burn wound model. The study involved male Californian rabbits (n = 52) divided into eight groups. Burn wounds were modeled on the animals and subsequently treated with gels based on Satureja montana L. and other water extracts. The reparative potential of the epidermis (assessed by Ki-67 expression), the state of local immunity (measured by the number of CD-45 cells), and the anti-inflammatory role of mast cells (measured by tryptase levels) were evaluated. Bacteriological and morphological studies were conducted. The most pronounced bactericidal, reparative, and immunostimulatory effects were observed after the treatment using a gel mixture of water extracts from Satureja montana L., Salvia sclarea, Coriandrum sativum L., and Lavandula angustifolia in equal proportions (1:1:1:1). The other gels also demonstrated high efficacy in treating burn wounds, especially when using a strain of Pseudomonas aeruginosa resistant to several antibiotics. Immunohistochemical studies showed a significant increase in the number of Ki-67-positive cells in the basal layer of the epidermis and a decrease in the number of CD-45-positive cells, indicating improved proliferative activity and reduced inflammation. This study confirms the hypothesis that the use of water extract mixtures significantly enhances the reparative potential, improves the immune response in the treatment of burns, and promotes wound healing. These findings pave the way for further research and the application of complex phytotherapeutic agents, specifically water extracts of medicinal plants containing phenols and antioxidants in burn wound therapy.

Morphological aspects of small intestinal mucosal injury and repair after electron irradiation.

Morphological evaluation of the small intestine mucosa and apoptosis activity (caspase-3) is necessary to assess the severity of damage to the small intestine. At the same time, proliferative index based on Ki-67 can be used to assess the regenerative potential of the small intestine. Fragments of small intestine of Wistar rats (n=60) of three groups: I) control (n=20); II) experimental group (n=20; local single electron irradiation at a dose of 2 Gy), III) experimental group (n=20; local single electron irradiation at a dose of 8 Gy) were studied by light microscopy using hematoxylin and eosin staining and immunohistochemical reactions with antibodies to Ki-67 and caspase-3. In all samples of the experimental groups, a decrease in all morphometric indices was observed on day 1 with a tendency to recover on day 3. Small intestinal electron irradiation led to disturbances in the histoarchitecture of varying severity, and an increase in cell apoptosis was observed (increased expression of caspase-3 and decrease in Ki-67). In addition, modulation of the PI3K/AKT and MAPK/ERK signaling pathways was detected. The most pronounced destructive changes were observed in the group of 8 Gy single electron irradiation. Local irradiation of the small intestine with electrons at a dose of 2 and 8 Gy results in a decrease in the number of enterocytes, mainly stem cells of the intestinal crypts.

Genetic changes in the FH gene cause vagal paraganglioma.

Vagal paraganglioma (VPGL) is a rare neuroendocrine tumor that originates from the paraganglion associated with the vagus nerve. VPGLs present challenges in terms of diagnostics and treatment. VPGL can occur as a hereditary tumor and, like other head and neck paragangliomas, is most frequently associated with mutations in the SDHx genes. However, data regarding the genetics of VPGL are limited. Herein, we report a rare case of a 41-year-old woman with VPGL carrying a germline variant in the FH gene. Using whole-exome sequencing, a variant, FH p.S249R, was identified; no variants were found in other PPGL susceptibility and candidate genes. Loss of heterozygosity analysis revealed the loss of the wild-type allele of the FH gene in the tumor. The pathogenic effect of the p.S249R variant on FH activity was confirmed by immunohistochemistry for S-(2-succino)cysteine (2SC). Potentially deleterious somatic variants were found in three genes, SLC7A7, ZNF225, and MED23. The latter two encode transcriptional regulators that can impact gene expression deregulation and are involved in tumor development and progression. Moreover, FH-mutated VPGL was characterized by a molecular phenotype different from SDHx-mutated PPGLs. In conclusion, the association of genetic changes in the FH gene with the development of VPGL was demonstrated. The germline variant FH: p.S249R and somatic deletion of the second allele can lead to biallelic gene damage that promotes tumor initiation. These results expand the clinical and mutation spectra of FH-related disorders and improve our understanding of the molecular genetic mechanisms underlying the pathogenesis of VPGL.

Cyto- and Histopographic Assessment of CPA3-Positive Testicular Mast Cells in Obstructive and Non-Obstructive Azoospermia.

Infertility is an important personal and society disease, of which the male factor represents half of all causes. One of the aspects less studied in male infertility is the immunological testicular microenvironment. Mast cells (MCs), having high potential for regulating spermatogenesis due to fine-tuning the state of the integrative buffer metabolic environment, are one of the most crucial cellular subpopulations of the testicular interstitium. One important component of the MC secretome is proteases that can act as proinflammatory agents and in extracellular matrix (ECM) remodeling. In the testis, MCs are an important cell component of the testicular interstitial tissue (TIT). However, there are still no studies addressing the analysis of a specific MC protease-carboxypeptidase A3 (CPA3)-in cases with altered spermatogenesis. The cytological and histotopographic features of testicular CPA3+ MCs were examined in a study involving 34 men with azoospermia. As revealed, in cases with non-obstructive azoospermia, a higher content of CPA3+ MCs in the TIT and migration to the microvasculature and peritubular tissue of seminiferous tubules were observed when compared with cases with obstructive azoospermia. Additionally, a high frequency of CPA3+ MCs colocalization with fibroblasts, Leydig cells, and elastic fibers was detected in cases with NOA. Thus, CPA3 seems to be of crucial pathogenetic significance in the formation of a profibrogenic background of the tissue microenvironment, which may have direct and indirect effects on spermatogenesis.

Molecular Mechanisms of Neuroprotection after the Intermittent Exposures of Hypercapnic Hypoxia.

The review introduces the stages of formation and experimental confirmation of the hypothesis regarding the mutual potentiation of neuroprotective effects of hypoxia and hypercapnia during their combined influence (hypercapnic hypoxia). The main focus is on the mechanisms and signaling pathways involved in the formation of ischemic tolerance in the brain during intermittent hypercapnic hypoxia. Importantly, the combined effect of hypoxia and hypercapnia exerts a more pronounced neuroprotective effect compared to their separate application. Some signaling systems are associated with the predominance of the hypoxic stimulus (HIF-1α, A1 receptors), while others (NF-κB, antioxidant activity, inhibition of apoptosis, maintenance of selective blood-brain barrier permeability) are mainly modulated by hypercapnia. Most of the molecular and cellular mechanisms involved in the formation of brain tolerance to ischemia are due to the contribution of both excess carbon dioxide and oxygen deficiency (ATP-dependent potassium channels, chaperones, endoplasmic reticulum stress, mitochondrial metabolism reprogramming). Overall, experimental studies indicate the dominance of hypercapnia in the neuroprotective effect of its combined action with hypoxia. Recent clinical studies have demonstrated the effectiveness of hypercapnic-hypoxic training in the treatment of childhood cerebral palsy and diabetic polyneuropathy in children. Combining hypercapnic hypoxia with pharmacological modulators of neuro/cardio/cytoprotection signaling pathways is likely to be promising for translating experimental research into clinical medicine.

Ultrastructural features of tumor-associated mast cells in parasympathetic paragangliomas (chemodectomas) of the neck.

The mechanisms of the pathogenesis of neck paraganglioma (PGL) and the possible role of mast cells (MCs) in its development and metastasis are still poorly understood. We analyzed MCs' morphologic characterization, activation, and the properties of their cytoplasmic/released granules in PGLs, using light and transmission electron microscopy. Paragangliomas showed a large tumor-associated MC population both in the connective tissue layers of the tumor and between the tumor cells. Notably, MCs were presented by a high expression of specific proteases, size variation, polymorphism, and variable ultrastructural phenotype of granules. A massive number of granules were released surrounding the degranulated MCs while the integrity of MC membrane was maintained. Granules were electron-dense with or without a membrane, ranging from 0.2 to 0.8 µm in diameter. MC plasmalemma was not found at the site of MC-collagen fibrils contact, whereas the secretome and fibrils were directly contacted. We observed direct and mediator-based interactions between MCs and paraganglioma cells. The latter preserved their membrane integrity when MC granules were not in proximity. The effects of the MC secretome on the paraganglioma microenvironment demonstrated its pathogenetic role in tumor progression and allow its application to new diagnostic criteria and the development of protocols for personalized therapy. RESEARCH HIGHLIGHTS: Ultrastructural analysis reveals novel regulatory effects of mast cells via diverse secretory pathways on the pathogenesis of parasympathetic paraganglioma, including fibrous extracellular matrix remodeling and mediator-based interactions between MCs and cells of the tumor microenvironment.

Features of Appendix and the Characteristics of Appendicitis Development in Children with COVID-19.

BACKGROUND: Research on the subject of the influence of SARS-CoV-2 mechanisms on human homeostasis remains an actual problem. Particular interest is the study of pathomorphological changes in the appendix in children with COVID-19. OBJECTIVES: Aim of this study: morphological and molecular biological evaluation of the appendix in children of different age groups with COVID-19. METHODS: Groups were formed on the basis of anamnestic, clinical, and morphological data: I (n = 42; aged 2 to 18 years, average age-10.8 ± 4.79)-with an established clinical diagnosis: coronavirus infection (COVID-19; PCR+); II (n = 55; aged 2 to 18 years, average age-9.7 ± 4.77)-with a confirmed clinical diagnosis of acute appendicitis; collected before the onset of the COVID-19 pandemic in 2017-2019; and III (n = 38; aged 2 to 18 years, average age-10.3 ± 4.62)-the control group. Histological and immunohistochemical studies were conducted using primary antibodies to CD3, CD4, CD68, CD163, CD20, and CD138 and to pro-inflammatory (IL-1, IL-6) and anti-inflammatory (IL-4, IL-10) cytokines. RESULTS: In most samples of appendixes in children with COVID-19, signs of destructive phlegmonous-ulcerative and gangrenous appendicitis were discovered. An increase in CD3+, CD4+, CD68+, CD163+, and CD20+ CD138+ immunocompetent cells was found in the appendix of children with COVID-19. As well, there was an increase in pro-inflammatory (IL-1, IL-6) and anti-inflammatory (IL-4, IL-10) cytokines. CONCLUSIONS: The aforementioned pathological and immunohistochemical changes were more pronounced in the group of children aged 6-12 years (childhood).

Histoarchitecture of stromal collagen fibers in gastrointestinal hollow organs of mice after a 30-day space flight.

The digestive organs are highly sensitive to the influence of orbital flight factors and can limit the professional activities of crew members aboard the International Space Station. Connective tissue, as a system-forming matrix of the integrative-buffer metabolic environment, is of particular relevance in space biomedicine, ensuring the functioning of internal organs under an altered gravitational stimulus. However, the adaptive mechanisms of the fibrous extracellular matrix of the gastric and intestinal connective tissue have not been fully investigated under prolonged microgravity weightlessness. Using histochemical techniques, we experimentally studied the state of collagen fibers in the specific tissue microenvironment of the gastric and intestinal membranes in C57BL/6 N mice after a 30-day space flight, subsequent 7-day ground readaptation, and in animals of the relevant control groups. The 30-day stay of laboratory animals aboard the Bion-M 1 biosatellite resulted in a reduction in the fibrous extracellular matrix of connective tissue in the studied digestive organs, excepting the gastric lamina propria. Increased fibrillogenesis was revealed in the gastrointestinal mucous membranes of animals 7 days after biosatellite landing compared with the parameters of animals in the space flight group. During the experiment with ground simulated orbital flight conditions, changes in collagen fibers were not significant compared to the vivarium control group. Thus, the results obtained evidence gravisensitivity of the fibrous extracellular matrix of the intraorgan connective tissue. This fact also highlights the necessity to further improve gastrointestinal tract-related preventive measures for astronauts during orbital flight.

Comparative Characterisation of Proliferation and Apoptosis of Colonic Epithelium after Electron Irradiation with 2 GY and 25 GY.

Development of new techniques for multimodal treatment and diagnostics of various neoplasms and the improvement of current techniques can significantly increase the life expectancy of patients with carcinomas of the colon and abdominal-cavity organs, since prevention of various side effects of radiation therapy is one of the main problems of oncological care. Electron irradiation is one of the most promising types of radiation therapy. There are no data on proliferation and apoptosis of the colon epithelium after irradiation with electrons, especially in different modes (single and summary). Morphological evaluation of apoptosis and proliferation of colonic epithelium after local irradiation with electrons were conducted at doses of 2 Gy (Gray) and 25 Gy. Colon fragments from sexually mature Wistar rats (n = 50, body weight 200 ± 10 g) were divided into three groups: I-control (n = 10); II-experimental group (n = 20; local single electron irradiation at a dose of 2 Gy); III-experimental group (n = 30) with local fractional irradiation with electrons at a total dose of 25 Gy. They were studied using light microscopy using hematoxylin and eosin staining and immunohistochemical reactions with antibodies to Ki-67 and caspase-3 (Cas3). Morphological disorders were accompanied by increased expression of pro-apoptotic molecules (caspase-3), and the period of regeneration by proliferative marker (Ki-67). Colon electron irradiation led to disturbances in the histoarchitecture of varying severity, and an increase in cell apoptosis was observed (increased expression of caspase-3 and decrease in Ki-67). In addition, modulation of the PI3K/AKT and MAPK/ERK signalling pathways was detected. The most pronounced destructive changes were observed in the group of 25 Gy fractionated electron irradiation.

Hydrogen Inhalation Reduces Lung Inflammation and Blood Pressure in the Experimental Model of Pulmonary Hypertension in Rats.

Hydrogen has been shown to exhibit selective antioxidant properties against hydroxyl radicals, and exerts antioxidant and anti-inflammatory effects. The monocrotaline-induced model of pulmonary hypertension is suitable for studying substances with antioxidant activity because oxidative stress is induced by monocrotaline. On day 1, male Wistar rats were subcutaneously injected with a water-alcohol solution of monocrotaline or a control with an only water-alcohol solution. One group of monocrotaline-injected animals was placed in a plastic box that was constantly ventilated with atmospheric air containing 4% of molecular hydrogen, and the two groups of rats, injected with monocrotaline or vehicle, were placed in boxes ventilated with atmospheric air. After 21 days, hemodynamic parameters were measured under urethane narcosis. The results showed that, although hydrogen inhalation had no effect on the main markers of pulmonary hypertension induced by monocrotaline injection, there was a reduction in systemic blood pressure due to its systolic component, and a decrease in TGF-ß expression, as well as a reduction in tryptase-containing mast cells.

Smooth Muscle Actin as a Criterion for Gravisensitivity of Stomach and Jejunum in Laboratory Rodents.

Smooth muscle tissue (SMT) is one of the main structural components of visceral organs, acting as a key factor in the development of adaptive and pathological conditions. Despite the crucial part of SMT in the gastrointestinal tract activity, the mechanisms of its gravisensitivity are still insufficiently studied. The study evaluated the content of smooth muscle actin (α-SMA) in the membranes of the gastric fundus and jejunum in C57BL/6N mice (30-day space flight), in Mongolian gerbils Meriones unguiculatus (12-day orbital flight) and after anti-orthostatic suspension according to E.R. Morey-Holton. A morphometric analysis of α-SMA in the muscularis externa of the stomach and jejunum of mice and Mongolian gerbils from space flight groups revealed a decreased area of the immunopositive regions, a fact indicating a weakening of the SMT functional activity. Gravisensitivity of the contractile structures of the digestive system may be due to changes in the myofilament structural components of the smooth myocytes or myofibroblast actin. A simulated antiorthostatic suspension revealed no significant changes in the content of the α-SMA expression level, a fact supporting an alteration in the functional properties of the muscularis externa of the digestive hollow organs under weightless environment. The data obtained contribute to the novel mechanisms of the SMT contractile apparatus remodeling during orbital flights and can be used to improve preventive measures in space biomedicine.

Space-Flight- and Microgravity-Dependent Alteration of Mast Cell Population and Protease Expression in Digestive Organs of Mongolian Gerbils.

Mast cell (MC)-specific proteases are of particular interest for space biology and medicine due to their biological activity in regulating targets of a specific tissue microenvironment. MC tryptase and chymase obtain the ability to remodel connective tissue through direct and indirect mechanisms. Yet, MC-specific protease expression under space flight conditions has not been adequately investigated. Using immunohistochemical stainings, we analyzed in this study the protease profile of the jejunal, gastric, and hepatic MC populations in three groups of Mongolian gerbils-vivarium control, synchronous experiment, and 12-day orbital flight on the Foton-M3 spacecraft-and in two groups-vivarium control and anti-orthostatic suspension-included in the experiment simulating effects of weightlessness in the ground-based conditions. After a space flight, there was a decreased number of MCs in the studied organs combined with an increased proportion of chymase-positive MCs and MCs with a simultaneous content of tryptase and chymase; the secretion of specific proteases into the extracellular matrix increased. These changes in the expression of proteases were observed both in the mucosal and connective tissue MC subpopulations of the stomach and jejunum. Notably, the relative content of tryptase-positive MCs in the studied organs of the digestive system decreased. Space flight conditions simulated in the synchronous experiment caused no similar significant changes in the protease profile of MC populations. The space flight conditions resulted in an increased chymase expression combined with a decreased total number of protease-positive MCs, apparently due to participating in the processes of extracellular matrix remodeling and regulating the state of the cardiovascular system.