Institute of Experimental Medicine

In the original publication [...].

Abstract Purpose The aim of this study was to develop and validate StepAn, a novel low-cost software tool for automated gait analysis in rodents. We sought to determine whether this accessible system, which uses standard video from devices like smartphones, could sensitively detect motor deficits in a pharmacological model of Parkinsonian bradykinesia. Methods Wistar rats received a single intraperitoneal injection of saline (control), 0.15 mg/kg haloperidol, or 0.30 mg/kg haloperidol. One hour post-injection, gait was analyzed using both the traditional manual "Footprints" test and the StepAn software, which automates paw detection and stride length calculation from video recordings. Results Both analysis methods detected a significant reduction in average stride length in haloperidol-treated rats compared to controls (p < 0.001), confirming the expected bradykinetic phenotype. No significant difference was found between the two haloperidol doses. StepAn provided equivalent mean values but demonstrated superior precision, evidenced by significantly lower measurement variability compared to manual scoring. Conclusion We successfully validated StepAn as a reliable, precise, and hardware-flexible tool for quantitative gait assessment. Its ability to detect drug-induced Parkinsonian gait deficits comparable to established methods, combined with its low cost and ease of use, makes it a promising resource for broadening access to sophisticated locomotor analysis in preclinical research.

Background/Objectives: The therapeutic potential of selective trace amine-associated receptor 1 (TAAR1) agonists has been established in multiple animal models of depression and anxiety. PTSD is a debilitating psychiatric disorder frequently characterized by anxiety and often comorbid with major depressive disorder. Complex PTSD represents an even more severe clinical presentation, emerging from prolonged or repeated exposure to traumatic events. Recent studies indicate that TAAR1 agonists can attenuate anxiety-like behaviors in experimental models of PTSD; however, the molecular mechanisms underlying this effect remain poorly understood. In this study, we evaluated whether TAAR1 agonism modulates PTSD-related neurochemical and molecular changes within the hippocampus and striatum. Methods: Post-traumatic stress was modeled using predator stress, a validated experimental paradigm relevant to complex PTSD. Treatment consisted of intraperitoneal administration of the TAAR1 agonist LK00764. Monoamine neurotransmitters and their metabolites were quantified, and the expression of genes implicated in noradrenergic, dopaminergic, and serotonergic signaling pathways was assessed. In addition, gene network reconstruction was performed using artificial intelligence to identify TAAR1-dependent regulatory interactions. Results: Treatment with a TAAR1 agonist fully prevented behavioral abnormalities in the experimental model of complex PTSD. Neurochemical analyses revealed decreased 5-HT levels in the hippocampus and reduced dopamine and metabolite concentrations in the striatum following TAAR1 agonism. Moreover, TAAR1 activation was associated with increased expression of the neurotrophic factor BDNF in the striatum. Gene network reconstruction identified a distinct molecular hub within the PTSD network, comprising TAAR1-coexpressed genes, their encoded proteins, and interconnected signaling pathways, suggesting a tightly regulated feedback loop. Conclusions: These findings provide novel evidence that TAAR1 agonists exert protective effects against complex PTSD-related behavioral and neurochemical abnormalities. The reconstructed TAAR1-centered gene network offers mechanistic insight into receptor-dependent regulation of monoaminergic signaling and neuroplasticity, supporting further exploration of TAAR1 agonists as promising therapeutic candidates for PTSD.

Aim: Develop A/H1N1pdm09-based live attenuated influenza vaccines (LAIVs) presenting chimeric hemagglutinin (HA) fused to fragments of Streptococcus pneumoniae (PspA, Spr1875) or S. agalactiae (ScaAB) to elicit combined anti-influenza and anti-bacterial immunity. Methods: Recombinant LAIVs were generated by reverse genetics. Replicative fitness was measured in embryonated chicken eggs (CE; log10 EID50/0.1 mL, n = 5 per delution) and MDCK cells (log10 TCID50/mL, n = 5). BALB/c mice (n = 20 per group; serology n = 6 per group; lung titers n = 5 per group) received intranasal 106 EID50. Systemic IgG and mucosal IgA to influenza and to the recombinant pneumococcal peptide were quantified by ELISA (GMT ± SD). Early cytokine responses were profiled in THP-1 cells. Results: All recombinant strains replicated in CE at 33°C but were temperature-sensitive at 39°C. H1-ScaAB retained relatively high replication and exhibited a cold-adapted phenotype despite a large N-terminal insert. In MDCK cells, H1-PspA showed significantly reduced replication compared with the parental LAIV. In mouse lungs, replication on day 3 post-immunization was significantly lower for H1-ScaAB and H1-PspA compared with the parental LAIV strain (p < 0.05). The parental LAIV induced robust systemic anti-influenza IgG and, uniquely for H1-ScaAB, significant mucosal anti-influenza IgA (p < 0.05). H1-Spr generated stronger antibody responses to the inserted pneumococcal peptide (p < 0.05). THP-1 assays revealed construct-specific cytokine patterns (unmodified H1N1: highest IFN-α; H1-Spr: elevated IL-6; H1-ScaAB: greatest MCP-1). Conclusions: Multiple A/H1N1pdm09-based recombinant LAIVs with chimeric HA can replicate in eggs and murine respiratory tract and induce dual influenza/pneumococcal antigen responses. Expanded biophysical validation, functional antibody assays and challenge studies are needed to optimize insert design without compromising viral fitness.

Background/objectivesThe complex interplay between sarcoidosis and COVID-19 remains an important area of research, since COVID-19 leads to long-term changes in the immune system. However, COVID-19 is often followed by autoimmune diseases, including newly manifesting sarcoidosis. The goal of this study is to characterize CD4+ T cell subsets, playing a pivotal role in the regulation of innate and adaptive immunity, in the peripheral blood of patients with sarcoidosis after COVID-19.MethodsThe peripheral blood samples from patients with sarcoidosis (n = 61) were studied. We divided patients into two distinct groups: sarcoidosis patients with no history of COVID-19 (n= 30) and COVID-19 convalescent patients with sarcoidosis within 12–24 weeks after recovery (n = 31). Healthy controls (n = 40) were similar in terms of age and sex to patients with sarcoidosis. Immunophenotyping of peripheral blood cells was performed using a ten-color flow cytometry.ResultsSarcoidosis patients with COVID-19 history had higher levels of T-helper cells (Th) when compared to COVID-19 naïve patients with sarcoidosis, but lower levels when compared to healthy controls. In COVID-19 convalescent patients with sarcoidosis, we noted higher absolute numbers and percentages of CD45RA–CCR7– and CD45RA+CCR7– cells within Th subset. Among COVID-19 convalescent patients with sarcoidosis we also found higher levels of T helper 1 cells and T helper 2 cells (with CXCR5–CCR6–CXCR3+CCR4– and CXCR5–CCR6–CXCR3–CCR4+ phenotypes, respectively) when compared to other groups. We also noted a statistically significant increase in central memory CXCR5+CCR6–CXCR3– follicular Th cells, as wells as effector memory CXCR5+CCR6–CXCR3– and CXCR5+CCR6+CXCR3– follicular Th cells in both groups of patients with sarcoidosis vs. healthy controls.ConclusionsOur study demonstrated Th cells imbalance in patients with sarcoidosis and COVID-19 history. These findings suggest possible clinical and visual progression of chronic lung sarcoidosis in COVID-19 convalescent patients.

BackgroundPediatric brain tumors (PBT) represent a major public health challenge, accounting for approximately 15–20% of all childhood malignancies. The tumor microenvironment (TME) plays a crucial role in tumor progression and prognosis, yet its specific contributions in PBT remain insufficiently explored.ObjectiveThis study aimed to develop and validate a prognostic model based on TME-specific genes in pediatric brain tumors, providing insights into potential therapeutic targets.MethodsWe analyzed gene expression data from a cohort of 70 pediatric brain tumor samples using the ESTIMATE algorithm to classify tumors into high- and low-risk groups based on stromal and immune scores. To identify TME-specific genes, we performed differential gene expression and weighted gene co-expression network analysis (WGCNA). A prognostic model was constructed using LASSO regression and Cox proportional hazards models, comprising eight key genes: CASP10, EPSTI1, FGL2, ITGAX, etc. The model’s predictive accuracy was validated in independent cohorts through ROC curve analysis.ResultsThe model demonstrated strong prognostic capability, with AUC values exceeding 0.8 for 1-year, 3-year, and 5-year survival predictions. Among the identified genes, CASP10 emerged as significantly associated with tumor progression, suggesting its potential as a therapeutic target. Furthermore, Mendelian randomization analysis provided additional support for a causal relationship between CASP10 expression and brain tumor risk.ConclusionThe TME-specific gene-based prognostic model effectively predicts survival outcomes in pediatric brain tumors, offering promising biomarkers for personalized medicine and potential therapeutic targets. However, further research is required to validate these findings across different tumor subtypes and to explore their clinical applications.

Magnetic liposomes (MLs) are hybrid nanovesicles that combine the biocompatibility of lipid bilayers with the remote controllability of superparamagnetic nanoparticles. To the best of our knowledge, no prior review has systematically covered the literature on MLs published between 2020 and 2024, with a special focus on continuous-flow microfluidic synthesis. Here, we consolidate and critically assess recent advances in MLs' structural design, highlight remaining challenges, and propose future directions for clinical translation. MLs, as one of the types of biomimetic magnetic nanovesicles, are considered promising nanocarriers for biomedical applications. These applications include active drug targeting to specific tissues, magnetic resonance imaging, magnetic hyperthermia, controlled release, and other applications. This review aims to comprehensively classify current knowledge on the main structural types of MLs and their traditional (batch) and modern (continuous-flow) synthesis methods. The current advantages and potential challenges of microfluidics- based MLs synthesis are described. Detailed information on the variants of microfluidicsbased techniques is provided, along with examples and potential biomedical applications. For the main aspects of MLs synthesis and applications, schematic illustrations are provided. Finally, an outlook on the perspectives of further MLs development and applications is presented.

Extracellular vesicles (EVs) are membrane-bound nanoparticles naturally secreted by cells, playing a vital role in intercellular communication and holding significant promise as therapeutic agents. These natural carriers deliver various molecules into cells, including proteins and nucleic acids. There are numerous methods to load and modify EVs, encompassing physical, chemical, and biological approaches. EVs demonstrate the capacity to target specific cells within organs, even requiring blood–tissue transition. The protein corona significantly influences EV availability and cargo delivery, with biomolecules residing both within and conjugated to the EV membrane. Furthermore, embedding EVs within biomaterials such as hydrogels, scaffolds, and nanofibers can enhance their stability, targeting specificity, and therapeutic potential. By addressing cargo loading and cell/tissue-specific targeting, EVs offer a novel therapeutic strategy for various diseases, including cancer, autoimmune disorders, and neurodegenerative diseases. Furthermore, EVs show promise as vaccination tools, delivering messenger RNA and proteins of various pathogens. Advances in EV biology and engineering would provide improved strategies for vesicle targeting, enhanced cargo loading, and safe and effective delivery. The convergence of technological advancements, interdisciplinary collaboration, and an enhanced understanding of EVs promises to revolutionize therapeutic approaches to a wide range of diseases, establishing EV-based treatments as a cornerstone of future medicine.

BACKGROUNDAccording to the literature, significant disorders of gut microbiota are consistently observed in patients with colorectal cancer (CRC). Disorders of gut microbiota composition are manifesting clinically as abdominal pain, dyspeptic symptoms (such as rumbling, bloating, and altered bowel habits, including both constipation and diarrhea), and overall reduced quality of life. Also, negative changes in the microbiota may be associated with a more frequent development of postoperative complications and complications during chemotherapy.CASE SUMMARYTwo patients with CRC underwent surgery (laparoscopic left hemicolectomy) and were prescribed chemotherapy regimen consisted of cisplatin, leucovorin, and fluorouracil. Along with prescribed chemotherapy patients took autoprobiotic enterococci. A fecal sample was collected for autoprobiotic preparation, ensuring that the patient had not taken antibiotics, probiotic supplements, or probiotic-containing foods for at least 10 days. An autoprobiotic contained an indigenous strain of Enterococcus faecium (E. faecium) was formulated. The patients received the autoprobiotic strain E. faecium (liquid form with a concentration of 8 Lg CFU/mL) orally at a dose of 50 mL twice daily during 10 days, regardless of meal times, from the first day of cytostatic treatment, throughout the first course of chemotherapy. As a result, autoprobiotic intake improved patient well-being and prevent side effects associated with the use of cytostatics.CONCLUSIONThe use of autoprobiotics in the treatment of CRC is a promising area to reduce the risks of postoperative complications, increase the tolerability of the basic chemotherapeutic regimen, as well as improve the quality of life.

BACKGROUND: The choroid plexus of the brain, the primary source of cerebrospinal fluid, consists of an inner stromal compartment and an outer epithelium. Epithelial cells renew slowly and, like other brain cells, can accumulate lipofuscin with age. Such age-related changes in laboratory animals, whose lifespan is considerably shorter than that of humans, have not previously been described. AIM: The work aimed to test the hypothesis that epithelial cells of rat choroid plexus accumulate lipofuscin as they age. METHODS: The study used male Wistar rats of different ages: 4–5 months (n = 3), 18 months (n = 3), and 28 months (n = 3). The presence of lipofuscin in tissues was assessed on paraffin-embedded brain sections using confocal microscopy based on its autofluorescence properties. RESULTS: The study found that lipofuscin accumulates in the choroid plexus epithelium of rats as they age. CONCLUSION: Further research into conditions and experimental factors that slow lipofuscin accumulation could aid in the development of novel medical technologies and drug products aimed at delaying aging of brain structures.