BACKGROUND: To develop targeted immunotherapy against ovarian adenocarcinoma, direct identification of peptides presented by major histocompatibility complex class I (HLA class I) on the surface of tumor cells can be used. However, standard immunopeptidome analysis protocols may vary substantially in the efficiency of immunopeptidome isolation depending on lysis conditions. Optimizing detergents for immunoaffinity purification of HLA complexes enhances analytical sensitivity, expands the repertoire of identified ligands, and increases the likelihood of detecting clinically relevant peptides suitable for personalized immunotherapeutic approaches. AIM: To evaluate the effect of various detergents on the efficiency of identifying peptide ligands of HLA class I complexes; to identify HLA class I ligands in ovarian adenocarcinoma tumors; and to characterize the surface antigens of ovarian adenocarcinoma tumors presented by HLA class I complexes that are potentially promising for immunotherapy. METHODS: The immunopeptidome of cell lines and postoperative specimens from patients with ovarian adenocarcinoma was isolated using immunoaffinity chromatography followed by liquid chromatography–mass spectrometry analysis. RESULTS: In this study, we tested an immunoaffinity isolation protocol for the immunopeptidome, comparing various detergents (CHAPS, NP-40, SOD, and Triton X-100) for cell lysis, and observed no statistically significant differences in the number of peptides identified. Using NP-40, we identified 5 peptides belonging to cancer/testis antigen proteins in the immunopeptidomes of postoperative specimens from patients with ovarian adenocarcinoma, 3 of which, according to the Human Protein Atlas, are indeed not expressed in normal ovarian tissues. CONCLUSION: Each of the four tested detergents leads to the identification of unique sets of peptides. Immunopeptidome analysis enables the identification of peptides derived from cancer/testis antigen proteins; however, a larger sample of postoperative specimens from patients with ovarian adenocarcinoma and experimental validation of the immunogenicity of the identified peptides are necessary.
Chronic stress elicits a broad spectrum of molecular alterations affecting nearly all levels of metabolic regulation. The primary initiating mechanism involves activation of the hypothalamic-pituitary-adrenal axis by a stressor, resulting in sustained glucocorticoid release. This, in turn, drives a systemic reprogramming of energy metabolism, fosters low-grade chronic inflammation and oxidative stress, and instigates persistent epigenetic modifications. These pathological processes are accompanied by a severe imbalance in pro- and antioxidant systems, significant shifts in adipose and muscle tissue metabolism, including induced lipolysis and gluconeogenesis, and impaired leptin and insulin signaling. Consequently, they elevate the risk for abdominal obesity, metabolic syndrome, type 2 diabetes, and related comorbidities. Of particular significance are stress-induced epigenetic changes, such as DNA methylation, which can serve to entrench a pathological metabolic phenotype over the long term. This review, therefore, aims to systematize and analyze current knowledge on the complex molecular mechanisms underpinning stress-induced changes in metabolism and neuroendocrine regulation in mammals.
BACKGROUND: The number of studies investigating physical methods of cell targeting is steadily growing. Within the field of magnetic cell targeting, two main approaches have recently emerged: the use of microscale structures as magnetic carriers, e.g, porous spheroids, helices, and microrobots, that transport cells, and the use of nano- or microscale magnetic particles that directly label cells. AIM: The study aimed to review experimental studies on the use of magnetic particles for targeted delivery of mammalian cells in order to identify the main parameters of magnetic labeling and targeting systems. METHODS: Scientific data was searched in the PubMed, Cochrane Library, and eLIBRARY.RU databases for the period from January 2019 to September 2024 using the keywords magnetic cell targeting, magnetic cell delivery, magnetic cell localization, and magnetic cell guidance. Original experimental in vitro and in vivo studies were included if they involved labeling mammalian or human cells with magnetic nano- or microparticles for targeted delivery using magnetic fields. Data on study design, cell lines used, characteristics of magnetic particles, magnetic labeling conditions and efficiency, characteristics of magnetic trapping systems, efficiency of magnetic delivery, and clinical effects in in vivo disease models were extracted from the selected articles. RESULTS: A total of 62 articles were included in the analysis, of which 63% (39 studies) involved animal disease models, mainly affecting the nervous system, heart, eyes, urinary system, musculoskeletal system, and cancer. The most common labeled cells were multipotent mesenchymal stromal cells (27 studies), immune system cells (16 studies), and endothelial cells and their progenitors (7 studies). In most studies, superparamagnetic iron oxide nanoparticles were used for cell labeling (82% of studies) with more than 30 types of coatings, whereas neodymium magnets of various configurations at 0.005–1.450 T magnetic induction served as targeting systems. In 84% of studies, the optimal labeling concentration of magnetic particles ranged from 10–100 µg Fe/mL, with a labeling time of 4–24 h. A high degree of labeled cell magnetic controllability was demonstrated in vitro. In 19 animal studies, magnetic targeting resulted in a 1.16- to 20-fold increase in local cell concentration within the target area. In 85% of in vivo studies, magnetic targeting produced a more pronounced therapeutic effect compared with controls without targeting systems. Overall, the analysis confirms the high clinical potential of magnetic targeting in cell therapy. CONCLUSION: Magnetic targeting of cells using magnetic particles is a rapidly developing and promising technology in the field of regenerative medicine and cell therapy.
BACKGROUND: By the end of the first quarter of the 21st century, adeno-associated viruses (AAVs) had become a powerful tool for human gene therapy. A number of advantages have placed these viruses in leading positions among gene delivery methods for therapeutic purposes. One current challenge is the development of novel recombinant AAV variants with tropism for specific cell types, including tumor cells of various origins. This can be achieved by mutating the viral capsid using modern capsid engineering approaches. AIM: To develop a recombinant AAV with enhanced tropism for human melanoma cells. METHODS: Using a rational design approach for the viral capsid, we developed a capsid architecture featuring an insertion of an RGD peptide that preferentially binds to αVβ3 integrins on the surface of malignant melanoma cells. To generate a plasmid encoding the mutant capsid protein, the PIPE (polymerase incomplete primer extension) method was used. The transduction efficiency of two recombinant AAV variants (one with the mutant capsid and one with the wild-type capsid) was assessed in the MeWo and HaCaT cell lines. We used a competitive inhibition assay to analyze virus binding to cell surface receptors. RESULTS: We generated a recombinant AAV carrying an RGD15 peptide insertion in the VR-VIII loop of the capsid protein (following asparagine-587 in the amino acid sequence). This mutant virus exhibits reduced tropism for normal skin keratinocytes while retaining the ability to efficiently infect human melanoma cells. Competitive inhibition assays demonstrated that the altered tropism of the AAV with the RGD peptide in the capsid results from a shift in receptor specificity. CONCLUSION: The recombinant AAV with the proposed capsid design can be used further to deliver an expression cassette encoding an oncolytic protein into human melanoma cells. Such an AAV would potentially exhibit dual specificity toward malignant cells, ensuring selectivity first at the stage of viral receptor binding and then at the transgene expression stage. The development of therapeutic agents with this mechanism of action would help address the problem of cancer drug non-selectivity.
BACKGROUND: Replicative aging of human skin fibroblasts is associated with age-related changes in the mTOR signaling pathway, a key regulator of cellular metabolism, proliferation, and autophagy. Despite the established role of mTOR in tissue aging, data on its component Raptor—a subunit of the mTORC1 complex that ensures substrate phosphorylation specificity—in the context of physiological skin aging remain insufficiently studied. AIM: To study dynamic changes in Raptor expression in dermal fibroblasts during human skin development and aging. METHODS: The study material consisted of 134 human skin samples ranging from 20 weeks of gestation to 85 years of age. Dermal fibroblasts were detected by immunohistochemistry using primary polyclonal rabbit antibodies to Raptor, vimentin, and proliferating cell nuclear antigen (PCNA). Morphometric analysis was performed using a Nikon Eclipse 200 light microscope (Japan) with NIS-Elements Br software, calculating the percentage of Raptor+ and PCNA+ fibroblasts relative to the total number of fibroblasts per 1 mm2 of dermis, as well as the absolute count of vimentin+ fibroblasts. Statistical analysis included Student’s t-test (p 0.05), nonparametric Spearman correlation analysis, and one-way ANOVA to identify age-related trends. RESULTS: A statistically significant decrease in the proportion of Raptor+ fibroblasts was observed, from 92.8% at the embryonic stage to 87.2% by age 85 years (p 0.05). The negative trend in Raptor expression correlated with a decrease in the number of PCNA+ and vimentin+ cells, indicating a functional relationship between mTORC1 activity, proliferative potential of fibroblasts, and their metabolic status. CONCLUSION: Raptor is a regulator of replicative aging of dermal fibroblasts, modulating the balance between anabolism and catabolism. These findings expand our understanding of the mechanisms of age-related changes in the number of human skin fibroblasts.
