Background: Due to the difficulty of identifying the window of receptivity, a molecular understanding of endometrial changes after fertilization is essential for improving the effectiveness of in vitro fertilization (IVF). This multi-level study examines the compo-sition of cytokines and extracellular vesicles (EVs) in uterine lavage samples obtainable via a noninvasive procedure in mice. The goal is to offer a new perspective on the tim-ing of embryo transfer. Methods: We performed flow cytometric analyses of inflamma-tory cytokines and EVs in uterine lavage samples collected from CD1 mice at the pre-receptive stage (2.5 days post coitum [dpc]) and the receptive stage (4.5 dpc). We also assessed endometrial progesterone receptor expression via immunohistochemistry and evaluated the expression of adhesion molecules related to implantation by RT-PCR. Re-sults: Significant differences were found in the concentrations of inflammatory cyto-kines (IL-6, IFN-γ, and MCP-1), the EV pattern in uterine lavage fluid, and the expres-sion of some genes related to implantation (CD44, CD81, and CD29). These results re-veal uterine lavage fluid markers associated with endometrial receptivity. Using these noninvasive markers can improve the effectiveness of IVF by enabling more precise timing of embryo transfer.

In this study, we investigated the effect of the food preservative tert-butylhydroquinone (tBHQ) on the activation of murine CD4+ T cells. We assessed the role of the nuclear factor erythroid 2-related factor 2 (NRF2) and the aryl hydrocarbon receptor (AHR) and conducted protein interaction studies to identify potential molecular targets of tBHQ. BALB/c mice received tBHQ orally (1.5% [w/w]), after which splenic T cells (Th1, Th2, Th17, Treg) were analyzed for number and activation sensitivity. While the number of effector T cells remained unchanged, their response to stimulation was accelerated in tBHQ treated animals. In contrast, Treg cells increased in number after treatment with this food additive but displayed reduction in count upon activation. In vitro experiments showed that tBHQ modulates the differentiation of Th17 and Treg cells. Pretreating these cells with NRF2 inhibitor trigonelline and/or AHR inhibitor TMF (6, 2, 4'-trimetoxi-flavone) altered the effect of tBHQ on these cells. We found 14 candidate targets of this chemical (AHR, KEAP1, NQO1, RORC, TBX21, IL6, GATA3, BCL2, FOXP3, IFNG, JAK2, ALB, TGFBR1, and HSP90AA1). Network analysis revealed coordinated regulation of pathways associated with inflammation, T cell differentiation, and xenobiotic response. In conclusion, our studies highlight the importance of the T cell response after tBHQ exposure and suggest multiple proteins and pathways that are relevant to the immunotoxicity of this compound.

IntroductionSpirulina maxima (Sm), a blue-green microalgae, is well known for its rich nutritional composition, antioxidant, and anti-inflammatory properties. In this study, we found that small extracellular vesicles (sEVs) isolated from Sm exhibit antifibrotic activity.MethodsSm derived sEVs (Sm_sEV) were purified from the Sm culture medium using tangential flow filtration (TFF), followed by size-exclusion chromatography (SEC). Characterization of the sEVs was performed using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), immunogold TEM, surface-enhanced IR spectroscopy (SEIRS), and flow cytometry (FC). Internalization of DiI-labeled sEVs by human primary peritoneal mesothelial cells (P-MCs) and peritoneal fibroblasts (p-FBs) was examined using fluorescence microscopy. The effect of sEVs on mesenchymal transition of P-MCs and activation of P-FBs was investigated by immunofluorescent staining, MTT cell-proliferation, and Sirius Red collagen accumulation assays, respectively. The antifibrotic relevance of Sm_sEV was further investigated in a chlorhexidine digluconate (CG)-induced mouse model of peritoneal fibrosis.ResultsThe isolated Sm_sEV exhibited a spherical morphology, with a size range of 150.0 ± 67.6 nm, and the protein-to-lipid ratio (P/L_spectr) was 2.27 ± 0.07. The sEVs cargo contained Parkinson’s disease protein 7 (PARK7), and heat shock protein 70 (HSP70). DiI-labeled sEVs were successfully internalized by both P-MCs and P-FBs and inhibited TGF-β-induced mesenchymal transition in P-MCs and the collagen production and PDGF-B-induced proliferation in P-FBs. In vivo, intraperitoneal administration of Sm_sEVs reduced CG-induced submesothelial thickening, fibronectin, and collagen type I alpha 1 immunopositivity, and increased cytokeratin 18, immunopositivity in the mesothelial layer.DiscussionThese findings highlight the antifibrotic effect of Sm_sEV and support their further investigation in the context of fibrosis.

Aims Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment. However, their use often leads to cardiovascular adverse effects, including cardiac dysfunction. Here, we hypothesized that a prior cardiac ischaemic injury could exacerbate cardiac dysfunction due to anti-programmed death protein 1 (PD-1) treatment. Furthermore, we investigated whether abatacept, a T-cell costimulation blocker, could ameliorate the ICI-induced cardiotoxicity in a pre-clinical model.Methods and results In a pre-clinical study, mice were treated with isoprenaline or control to induce reversible cardiac ischaemia. After 16 weeks of follow-up, recovery of cardiac function was confirmed via echocardiography, and mice from both groups were randomly treated with isotype control, anti-PD-1, or anti-PD-1 combined with abatacept, for 2 further weeks. Mice with prior ischaemic injury and anti-PD-1 treatment showed cardiac dysfunction with increased infiltration of T cells and macrophages and elevated expression of pro-inflammatory cytokines. Conversely, cardiac dysfunction and inflammation were less pronounced after anti-PD-1 treatment in mice without prior ischaemic injury. Mice with concomitant abatacept treatment exhibited normal cardiac function and alleviated pro-inflammatory response. In a parallel single-centre retrospective clinical cohort study, 1671 cancer patients receiving PD-1 inhibitors were analysed. Cases were defined as patients who developed incident heart failure (HF) after ICI initiation with a primary aim to test whether pre-existing ischaemic heart disease was associated with an increased risk for HF development post-ICI therapy. Sensitivity analyses included propensity score matching and comparison with non-ICI-treated cancer patients. Among ICI-treated patients, 109 (6.5%) developed HF over a median follow-up of 332 days. Multivariable logistic regression of the matched population showed increased odds of incident HF in patients with prior ischaemic cardiac events (odds ratio 2.11, 95% confidence interval 1.05-4.2, P = 0.033).Conclusion In mice, induction of cardiac inflammation and dysfunction by anti-PD-1 therapy was potentiated by prior transient ischaemic cardiac injury, which was ameliorated by abatacept cotreatment. Cancer patients with pre-existing ischaemic heart disease may be at greater risk for developing ICI-induced new-onset HF. Based on our findings, cardiac surveillance should be considered in patients starting ICI therapy with a prior history of ischaemic heart disease.

Regulatory T lymphocytes are essential for maternal immunotolerance. Their de novo differentiation in the placenta is regulated by local intercellular interactions involving primed uterine immune cells, fetal syncytiotrophoblasts, and the cytokine environment. Trophoblast-derived, HLA-G-positive extracellular vesicles (EVs) can bind to T lymphocytes, thereby influencing their differentiation and cytokine production. Thus, these EVs play a role in establishing and maintaining a tolerogenic environment. In our study, we used the BeWo choriocarcinoma cell line to model the effects of trophoblast-derived EVs. Large EVs derived from BeWo cells (BeWo-12.5K lEVs) reduce IL-6Rα expression on CD4+ T cells. This modifies the IL-6 pathway by downregulating the transcription factors STAT3 and NFKB1, and PIAS3, while upregulating STAT1. This may be caused by specific microRNAs (miRNAs), such as hsa-mir-92a-3p, hsa-mir-520f-3p, and hsa-mir-25-3p. These microRNAs are present in BeWo-12.5K lEVs and target the IL-6 pathway. BeWo-12.5K lEVs induce phenotypic and functional changes in T cells, enhancing the ratio of CD4+/CD25+ T cells that produce IL-10. Pregnancy-associated IL-6Rα downregulation has been demonstrated in clinical samples. Significantly lower levels of IL-6Rα were detected on circulating CD4+CD25+ T cells in healthy pregnant women than in healthy non-pregnant individuals. This finding reflects the in vivo significance of our in vitro studies. Our studies suggest that communication between maternal and fetal cells significantly influences the development and maintenance of local T cell polarity. The microRNA content of HLA-G+ 12.5K lEVs appears to be key to this process, as it alters the IL-6 pathway. ### Competing Interest Statement The authors have declared no competing interest. * EV : extracellular vesicles 12.5K lEV : intermediate-sized extracellular vesicles FSC : Forward Scatter HLA-G : human leukocyte antigen G (major histocompatibility complex, class I, G) IL-6Rα : interleukin 6 receptor alpha miRNA : micro ribonucleic acid PS : phosphatidylserine PSR : phosphatidylserine receptor sEV : small-sized extracellular vesicles SSC : Side Scatter

Extracellular vesicles (EVs) are lipid membrane-enclosed particles released by all cells and can be isolated from various sources, even from solid tissues. This study focuses on isolating and characterizing EVs from mouse lymph nodes (LNs). Male C57BL/6 mice were injected with complete Freund’s adjuvant, with or without ovalbumin. Inguinal and popliteal LNs were incised 9 days after immunization, and EV isolation was carried out using a combination of differential centrifugation and size-exclusion chromatography. The characteristic morphology of small and large EVs was confirmed by transmission electron microscopy. Particle size distribution and concentration were determined by nanoparticle tracking analysis, while protein and lipid contents were measured by bicinchoninic acid assay, and sulfo-phospho-vanillin assays, respectively, to calculate the protein-to-lipid ratio. Immune and EV markers were analyzed by using flow cytometry and Western blot assay, revealing significant changes between immunized mice compared to controls. This study establishes a novel protocol for isolating and characterizing EVs from LNs and highlights the impact of immunization on EV properties, offering insights into their roles in immune processes.

Tamoxifen is a well-established selective estrogen receptor modulator (SERM) widely used in breast cancer treatment, yet its efficacy varies across tumor types. To enhance its antitumor potential, we previously synthesized and investigated novel ferrocene-linked (T5, T15) derivatives. This publication is a close continuation of this work, introducing a new indene-based (T6) derivative. Objectives: The main aim of this study was to further broaden our knowledge of the mechanism behind the increased antitumor effect of the ferrocene-linked drugs (T5 and T15) and compare it with a new, indene-based tamoxifen derivative, T6. The indene moiety was selected as a rigid, hydrophobic aromatic unit to probe pharmacological effects independent of ferrocene’s redox activity. Methods: The compounds were tested on MCF7, MDA-MB231 and PANC1 cells. Cell viability was assessed with the AlamarBlue assay and the xCELLigence SP system. Reactive oxygen species (ROS) production was measured with the ROS Glo assay. Flow cytometry and RT-qPCR experiments were conducted to assess apoptosis and ROS regulation as well. Results: The modified compounds demonstrated an increased cell-viability-decreasing effect in breast (MCF7, MDA-MB-231) and pancreatic (PANC1) cancer cell lines, influencing both estrogen-receptor-dependent and -independent pathways. T6 led to G2/M phase arrest in PANC1 cells. Beyond cell cycle disruption, these derivatives significantly elevated ROS levels, contributing to apoptosis. Conclusions: Our findings suggest that these structural modifications retain tamoxifen’s pharmacophore properties while expanding its mechanism of action, particularly through universal interactions independent of the ER status of tumor cells. The enhanced antitumor effects highlight the potential of these derivatives as promising candidates for improved cancer therapies.

IntroductionLight exposure of embryos during assisted reproduction affects embryo quality and implantation capacity in a wavelength dependent manner. We investigated the molecular mechanism of these light-induced changes through the comparative analysis of gene expression and regulatory miRNA profile of murine embryos cultured in dark environment and those exposed to white- or red filtered light. miRNA sequencing was used to assess the role of embryo-derived extracellular vesicles in the endometrium-embryo dialogue.MethodsIn vitro cultured mouse embryos at 3.5 days post coitum (dpc) were exposed to white or red filtered light. After 24 hours mRNA and miRNA content of the embryos as well as the miRNA content of embryo-derived extracellular vesicles were isolated and RNA-sequencing was performed. Differential expression analysis and functional enrichment analysis were used for evaluating the transcriptome results.ResultsLight exposure caused transcriptomic changes in the embryos. White light upregulated apoptotic pathways, while red filtered light gave rise to the activation of regeneration pathways, including DNA repair mechanisms. Embryo-derived extracellular vesicles enclosed wavelength dependently unique miRNA cargos the target genes of which play a role in embryo implantation.DiscussionWhite light upregulates apoptotic pathways, at both the transcriptome and regulatory miRNAs levels. Red filtration partially counterbalances these negative effects by shifting the cellular processes towards regeneration, including DNA repair mechanisms. Extracellular vesicles of light exposed embryos play a role in blastocyst-decidua communication through the horizontal transfer of regulatory miRNAs. Our data prove that light exposure during in vitro fertilization modifies cell function that might affect the outcome of implantation.

This study investigates T cell subsets in pericardial fluid samples obtained from heart transplantation donors, heart transplantation recipients, and coronary artery bypass graft patients. Using flow cytometry, we characterized regulatory T cells (Tregs), tissue-resident memory T cells (Trm), and exhausted T cells based on specific markers. Our results showed significant alterations in the CD4+ and CD8+ T cell subsets, migration (CXCR3, CCR5), and exhaustion markers (PD-1, TIM3) across the groups. Notably, Tregs and Trm cells were enriched in recipients, while markers of T cell exhaustion showed a complex regulation. These findings provide novel insights into the local immune regulation in cardiac disease and transplantation.

Objectives: We aimed to assess the relationship among circulating extracellular vesicles (EVs), hypoxia-related proteins, and the conventional risk factors of life-threatening coronary artery disease (CAD) to find more precise novel biomarkers. Methods: Patients were categorized based on coronary CT angiography. Patients with a Segment Involvement Score > 5 were identified as CAD patients. Individuals with a Segment Involvement Score < 5 were considered control subjects. The characterization of EVs and analysis of the plasma concentration of growth differentiation factor-15 were performed using multicolor or bead-based flow cytometry. The plasma protein levels of glycogen phosphorylase, muscle form, clusterin, and carboxypeptidase N subunit 1 were determined using an enzyme-linked immunosorbent assay. Multiple logistic regression was used to determine the association of the biomarkers with the CAD outcome after accounting for established risk factors. The analysis was built in three steps: first, we included the basic clinical and laboratory variables (Model 1), then we integrated the plasma protein values (Model 2), and finally, we complemented it with the circulating EV pattern (Model 3). To assess the discrimination value of the models, an area under (AUC) the receiver operating curve was calculated and compared across the three models. Results: The area under the curve (AUC) values were 0.68, 0.77, and 0.84 in Models 1, 2, and 3, respectively. The variables with the greatest impact on the AUC values were hemoglobin (0.2 (0.16-0.26)) in Model 1, carboxypeptidase N subunit 1 (0.12 (0.09-0.14)) in Model 2, and circulating CD41+/CD61+ EVs (0.31 (0.15-0.5)) in Model 3. A correlation analysis showed a significant impact of circulating CD41+/CD61+ platelet-derived EVs (p = 0.03, r = -0.4176) in Model 3. Conclusions: Based on our results, the circulating EV profile can be used as a supportive biomarker, along with the conventional laboratory markers of CAD, and it enables a more sensitive, non-invasive diagnostic analysis of CAD.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide, and atherosclerosis is the key factor promoting its development. Carotid intima-media thickening and the presence of carotid plaques are important indices of cardiovascular risk. In addition, inflammation is a major and complex factor in the development of atherosclerosis. The relationships between carotid atherosclerosis and certain inflammatory markers have rarely been studied in healthy individuals. Therefore, we aimed to investigate the associations between subclinical carotid atherosclerosis and various inflammatory biomarkers in a large Caucasian population free of evident CVD. In addition to recording study participants’ demographic characteristics, anthropometric characteristics, and atherosclerotic risk factors, laboratory tests were performed to measure levels of hemoglobin A1c (HbA1c), high-sensitivity C-reactive protein, and inflammatory cytokines/chemokines, including interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, IL-33, interferon (IFN)-α2, IFN-γ, tumor necrosis factor-α, and monocyte chemoattractant protein (MCP)-1. This study included 264 asymptomatic individuals with a median age of 61.7 years (interquartile range, 54.5–67.5 years); 45.7% of participants were male. Participants were divided into two groups according to their carotid status: the normal carotid group, comprising 120 participants; and the pathological carotid group, comprising 144 participants. Compared with the normal carotid group, hypertension and diabetes mellitus were significantly more common and serum levels of HbA1c, IL-8, and MCP-1 were significantly higher in the pathological carotid group. Multivariate regression analysis revealed significant positive associations between pathological carotid findings and serum levels of IL-8 (highest tertile, OR: 2.4, p = 0.030) and MCP-1 (highest tertile, OR: 2.4, p = 0.040). Our results suggest that IL-8 and MCP-1 may serve as early indicators of subclinical atherosclerosis, thereby helping to identify individuals at increased risk of CVD before the onset of clinical symptoms.

Endometriosis is a multifactorial disease, hence the difficulty to explain its cause with a single factor. Since the causes of endometriosis are not clear, it is difficult to screen and treat the disease. Currently, the most common treatment methods for endometriosis are hormonal drugs – such as GnRH agonist injections, progestogen implants – and surgical removal of the affected tissue or reduction of its size. Currently, there is no reliable non-invasive method to confirm endometriosis without surgery, and many patients feel that hormone therapy and surgery are ineffective in preventing recurrence. Based partly on our own results and partly on the basis of extensive literature, our research group concluded that the microbiome of the lower and upper female genital tract and the gut play a role in the development of endometriosis. A more detailed mapping of the bacterial community of the mentioned areas can be an important step towards a better understanding of the pathomechanism of endometriosis and provide additional new diagnostic methods for the clinician. Future prospective studies with larger samples and a more rigorous methodology combined with standardization of patients are needed to clarify the role of the microbiome in the pathogenesis and clinical features of endometriosis and to clarify the effect of possible interventions. Orv Hetil. 2024; 165(1): 3–13.

Among patients on peritoneal dialysis (PD), 50–80% will develop peritoneal fibrosis, and 0.5–4.4% will develop life-threatening encapsulating peritoneal sclerosis (EPS). Here, we investigated the role of extracellular vesicles (EVs) on the TGF-β- and PDGF-B-driven processes of peritoneal fibrosis. EVs were isolated from the peritoneal dialysis effluent (PDE) of children receiving continuous ambulatory PD. The impact of PDE-EVs on the epithelial–mesenchymal transition (EMT) and collagen production of the peritoneal mesothelial cells and fibroblasts were investigated in vitro and in vivo in the chlorhexidine digluconate (CG)-induced mice model of peritoneal fibrosis. PDE-EVs showed spherical morphology in the 100 nm size range, and their spectral features, CD63, and annexin positivity were characteristic of EVs. PDE-EVs penetrated into the peritoneal mesothelial cells and fibroblasts and reduced their PDE- or PDGF-B-induced proliferation. Furthermore, PDE-EVs inhibited the PDE- or TGF-β-induced EMT and collagen production of the investigated cell types. PDE-EVs contributed to the mesothelial layer integrity and decreased the submesothelial thickening of CG-treated mice. We demonstrated that PDE-EVs significantly inhibit the PDGF-B- or TGF-β-induced fibrotic processes in vitro and in vivo, suggesting that EVs may contribute to new therapeutic strategies to treat peritoneal fibrosis and other fibroproliferative diseases.

Age-related disorders are closely linked to the accumulation of senescent cells. The senescence-associated secretory phenotype (SASP) sustains and progresses chronic inflammation, which is involved in cellular and tissue dysfunction. SASP-related growth and differentiation factor-15 (GDF-15) is an immunoregulatory cytokine that is coupled to aging and thus may have a regulatory role in the development and maintenance of atherosclerosis, a major cause of cardiovascular disease (CVD). Although the effects of GDF-15 are tissue-specific and dependent on microenvironmental changes such as inflammation, available data suggest that GDF-15 has a significant role in CVD. Thus, GDF-15 is a promising biomarker and potential therapeutic target for atherosclerotic CVD.

Hypercholesterolemia (HC) induces, propagates and exacerbates cardiovascular diseases via various mechanisms that are yet not properly understood. Extracellular vesicles (EVs) are involved in the pathomechanism of these diseases. To understand how circulating or cardiac-derived EVs could affect myocardial functions, we analyzed the metabolomic profile of circulating EVs, and we performed an in-depth analysis of cardiomyocyte (CM)-derived EVs in HC. Circulating EVs were isolated with Vezics technology from male Wistar rats fed with high-cholesterol or control chow. AC16 human CMs were treated with Remembrane HC supplement and EVs were isolated from cell culture supernatant. The biophysical properties and the protein composition of CM EVs were analyzed. THP1-ASC-GFP cells were treated with CM EVs, and monocyte activation was measured. HC diet reduced the amount of certain phosphatidylcholines in circulating EVs, independently of their plasma level. HC treatment significantly increased EV secretion of CMs and greatly modified CM EV proteome, enriching several proteins involved in tissue remodeling. Regardless of the treatment, CM EVs did not induce the activation of THP1 monocytes. In conclusion, HC strongly affects the metabolome of circulating EVs and dysregulates CM EVs, which might contribute to HC-induced cardiac derangements.