High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-ß7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-ß7, LAG3, TGFß/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-ß7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-ß7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Small extracellular vesicles from surviving cancer cells as multiparametric monitoring tools of measurable residual disease and therapeutic efficiency.

Although conventional anti-cancer therapies remove most cells of the tumor mass, small surviving populations may evolve adaptive resistance strategies, which lead to treatment failure. The size of the resistant population initially may not reach the threshold of clinical detection (designated as measurable residual disease/MRD) thus, its investigation requires highly sensitive and specific methods. Here, we discuss that the specific molecular fingerprint of tumor-derived small extracellular vesicles (sEVs) is suitable for longitudinal monitoring of MRD. Furthermore, we present a concept that exploiting the multiparametric nature of sEVs may help early detection of recurrence and the design of dynamic, evolution-adjusted treatments.

Recipient Pericardial Apolipoprotein Levels Might Be an Indicator of Worse Outcomes after Orthotopic Heart Transplantation.

BACKGROUND: End-stage heart failure (ESHF) leads to hypoperfusion and edema formation throughout the body and is accompanied by neurohormonal and immunological alterations. Orthotopic heart transplantation (HTX) has been used as a beneficial option for ESHF. Due to the shortage of donor hearts, the ideal matching and timing of donors and recipients has become more important. PURPOSE: In this study, our aim was to explore the relationship between the clinical outcomes of HTX and the cytokine and apolipoprotein profiles of the recipient pericardial fluid obtained at heart transplantation after opening the pericardial sac. MATERIALS AND METHODS: The clinical data and the interleukin, adipokine, and lipoprotein levels in the pericardial fluid of twenty HTX recipients were investigated. Outcome variables included primer graft dysfunction (PGD), the need for post-transplantation mechanical cardiac support (MCS), International Society for Heart and Lung Transplantation grade ≥2R rejection, and mortality. Recipient risk scores were also investigated. RESULTS: Leptin levels were significantly lower in patients with PGD than in those without PGD (median: 6.36 (IQR: 5.55-6.62) versus 7.54 (IQR = 6.71-10.44); p = 0.029). Higher ApoCII levels (median: 14.91 (IQR: 11.55-21.30) versus 10.31 (IQR = 10.02-13.07); p = 0.042) and ApoCIII levels (median: 60.32 (IQR: 43.00-81.66) versus 22.84 (IQR = 15.84-33.39); p = 0.005) were found in patients (n = 5) who died in the first 5 years after HTX. In patients who exhibited rejection (n = 4) in the first month after transplantation, the levels of adiponectin (median: 74.48 (IQR: 35.51-131.70) versus 29.96 (IQR: 19.86-42.28); p = 0.039), ApoCII (median: 20.11 (IQR: 13.06-23.54) versus 10.32 (IQR: 10.02-12.84); p = 0.007), and ApoCIII (median: 70.97 (IQR: 34.72-82.22) versus 26.33 (IQR: 17.18-40.17); p = 0.029) were higher than in the nonrejection group. Moreover, the pericardial thyroxine (T4) levels (median: 3.96 (IQR: 3.49-4.46) versus 4.69 (IQR: 4.23-5.77); p = 0.022) were lower in patients with rejection than in patients who did not develop rejection. CONCLUSION: Our results indicate that apolipoproteins can facilitate the monitoring of rejection and could be a useful tool in the forecasting of early and late complications.

Nanoinjection of extracellular vesicles to single live cells by robotic fluidic force microscopy.

In the past decade, extracellular vesicles (EVs) have attracted substantial interest in biomedicine. With progress in the field, we have an increasing understanding of cellular responses to EVs. In this Technical Report, we describe the direct nanoinjection of EVs into the cytoplasm of single cells of different cell lines. By using robotic fluidic force microscopy (robotic FluidFM), nanoinjection of GFP positive EVs and EV-like particles into single live HeLa, H9c2, MDA-MB-231 and LCLC-103H cells proved to be feasible. This injection platform offered the advantage of high cell selectivity and efficiency. The nanoinjected EVs were initially localized in concentrated spot-like regions within the cytoplasm. Later, they were transported towards the periphery of the cells. Based on our proof-of-principle data, robotic FluidFM is suitable for targeting single living cells by EVs and may lead to information about intracellular EV cargo delivery at a single-cell level.

Donor Pericardial Interleukin and Apolipoprotein Levels May Predict the Outcome after Human Orthotopic Heart Transplantation.

The proinflammatory cascade that is activated at the time of brain death plays a crucial role in organ procurement. Our aim of this study was to explore the relationship between the clinical outcome of orthotopic heart transplantation, as well as cytokine and apolipoprotein profiles of the pericardial fluid obtained at donation. Interleukin, adipokine and lipoprotein levels in the pericardial fluid, as well as clinical data of twenty donors after brain death, were investigated. Outcome variables included primary graft dysfunction, the need for posttransplantation mechanical cardiac support and International Society for Heart and Lung Transplantation grade ≥ 2R rejection. Hormone management and donor risk scores were also investigated. Lower levels of IL-6 were observed in primary graft dysfunction (median: 36.72 [IQR: 19.47-62.90] versus 183.67 [41.21-452.56]; p = 0.029) and in the need for mechanical cardiac support (44.12 [20.12-85.70] versus 247.13 [38.51-510.38]; p = 0.043). Rejection was associated with lower ApoAII (p = 0.021), ApoB100 (p = 0.032) and ApoM levels (p = 0.025). Lower adipsin levels were detected in those patients receiving desmopressin (p = 0.037); moreover, lower leptin levels were found in those patients receiving glucocorticoid therapy (p = 0.045), and higher T3 levels were found in those patients treated with L-thyroxine (p = 0.047) compared to those patients not receiving these hormone replacement therapies. IL-5 levels were significantly associated with UNOS-D score (p = 0.004), Heart Donor Score (HDS) and Adapted HDS (p < 0.001). The monitoring of immunological and metabolic changes in donors after brain death may help in the prediction of potential complications after heart transplantation, thus potentially optimizing donor heart allocation.

Differential impact of exportin-1-mediated nuclear export of RNAs on the RNA content of extracellular vesicle subpopulations.

Extracellular vesicles (EVs) are membrane-enclosed subcellular structures released by all cell types. EVs have important roles in both cellular homeostasis and intercellular communication. Recent progress in the field revealed substantial heterogeneity of EVs even within the size-based EV categories. Here we addressed the question whether the exportin-1 (XPO1)-mediated nuclear export of RNAs contributed to the EV heterogeneity. Size-based populations were separated from the conditioned media of three cell lines (U937, THP-1 and 5/4E8) in steady-state condition. The effects of activation and leptomycin B treatment (to inhibit the XPO1-mediated nuclear export of RNAs) were also tested in the case of the two monocytic cell lines. Agilent Pico and Small chips were used to characterize RNAs, fragment analysis was performed, and EV-associated miRNAs were tested by Taqman assays. As expected, we found the highest small RNA/total RNA ratio and the lowest rRNA/total RNA proportion in small EVs (~ 50-150 nm). Profiles of the small RNAs within different size-based EV categories significantly differed based on the activation status of the EV releasing cells. Leptomycin B had a differential inhibition on the tested small RNAs in EVs, even within the same EV size category. A similar heterogeneity of the EV miRNA content was observed upon cellular activation and nuclear export inhibition. Here we complement the already existing knowledge on EV heterogeneity by providing evidence that the RNA cargo varies depending on the EV size-based category, the releasing cell type, the functional status of the releasing cells and the exportin-1-mediated nuclear export of RNAs.

Endoplasmin Is a Hypoxia-Inducible Endoplasmic Reticulum-Derived Cargo of Extracellular Vesicles Released by Cardiac Cell Lines.

Cardiomyopathies are leading causes of human mortality. Recent data indicate that the cardiomyocyte-derived extracellular vesicles (EVs) released upon cardiac injury are present in circulation. This paper aimed to analyze EVs released under normal and hypoxic conditions by H9c2 (rat), AC16 (human) and HL1 (mouse) cardiac cell lines. Small (sEVs), medium (mEVs) and large EVs (lEVs) were separated from a conditioned medium by a combination of gravity filtration, differential centrifugation and tangential flow filtration. The EVs were characterized by microBCA, SPV lipid assay, nanoparticle tracking analysis, transmission and immunogold electron microscopy, flow cytometry and Western blotting. Proteomic profiles of the EVs were determined. Surprisingly, an endoplasmic reticulum chaperone, endoplasmin (ENPL, grp94 or gp96), was identified in the EV samples, and its association with EVs was validated. The secretion and uptake of ENPL was followed by confocal microscopy using GFP-ENPL fusion protein expressing HL1 cells. We identified ENPL as an internal cargo of cardiomyocyte-derived mEVs and sEVs. Based on our proteomic analysis, its presence in EVs was linked to hypoxia in HL1 and H9c2 cells, and we hypothesize that EV-associated ENPL may have a cardioprotective role by reducing cardiomyocyte ER stress.

High fat diet and PCSK9 knockout modulates lipid profile of the liver and changes the expression of lipid homeostasis related genes.

BACKGROUND: High fat diet (HFD) increases the likelihood of dyslipidemia, which can be a serious risk factor for atherosclerosis, diabetes or hepatosteatosis. Although changes in different blood lipid levels were broadly investigated, such alterations in the liver tissue have not been studied before. The aim of the current study was to investigate the effect of HFD on hepatic triglyceride (TG), diglyceride (DG) and ceramide (CER) levels and on the expression of four key genes involved in lipid homeostasis (Pcsk9, Ldlr, Cd36 and Anxa2) in the liver. In addition, the potential role of PCSK9 in the observed changes was further investigated by using PCSK9 deficient mice. METHODS: We used two in vivo models: mice kept on HFD for 20 weeks and PCSK9-/- mice. The amount of the major TGs, DGs and CERs was measured by using HPLC-MS/MS analysis. The expression profiles of four lipid related genes, namely Pcsk9, Ldlr, Cd36 and Anxa2 were assessed. Co-localization studies were performed by confocal microscopy. RESULTS: In HFD mice, hepatic PCSK9 expression was decreased and ANXA2 expression was increased both on mRNA and protein levels, and the amount of LDLR and CD36 receptor proteins was increased. While LDLR protein level was also elevated in the livers of PCSK9-/- mice, there was no significant change in the expression of ANXA2 and CD36 in these animals. HFD induced a significant elevation in the hepatic levels of all measured TG and DG but not of CER types, and increased the proportion of monounsaturated vs. saturated TGs and DGs. Similar changes were detected in the hepatic lipid profiles of HFD and PCSK9-/- mice. Co-localization of PCSK9 with LDLR, CD36 and ANXA2 was verified in HepG2 cells. CONCLUSIONS: Our results show that obesogenic HFD downregulates PCSK9 expression in the liver and causes alterations in the hepatic lipid accumulation, which resemble those observed in PCSK9 deficiency. These findings suggest that PCSK9-mediated modulation of LDLR and CD36 expression might contribute to the HFD-induced changes in lipid homeostasis.

Initial and ongoing tobacco smoking elicits vascular damage and distinct inflammatory response linked to neurodegeneration.

Tobacco smoking is strongly linked to vascular damage contributing to the development of hypertension, atherosclerosis, as well as increasing the risk for neurodegeneration. Still, the involvement of the innate immune system in the development of vascular damage upon chronic tobacco use before the onset of clinical symptoms is not fully characterized. Our data provide evidence that a single acute exposure to tobacco elicits the secretion of extracellular vesicles expressing CD105 and CD49e from endothelial cells, granting further recognition of early preclinical biomarkers of vascular damage. Furthermore, we investigated the effects of smoking on the immune system of healthy asymptomatic chronic smokers compared to never-smokers, focusing on the innate immune system. Our data reveal a distinct immune landscape representative for early stages of vascular damage in clinically asymptomatic chronic smokers, before tobacco smoking related diseases develop. These results indicate a dysregulated immuno-vascular axis in chronic tobacco smokers that are otherwise considered as healthy individuals. The distinct alterations are characterized by increased CD36 expression by the blood monocyte subsets, neutrophilia and increased plasma IL-18 and reduced levels of IL-33, IL-10 and IL-8. Additionally, reduced levels of circulating BDNF and elevated sTREM2, which are associated with neurodegeneration, suggest a considerable impact of tobacco smoking on CNS function in clinically healthy individuals. These findings provide profound insight into the initial and ongoing effects of tobacco smoking and the potential vascular damage contributing to neurodegenerative disorders, specifically cerebrovascular dysfunction and dementia.

Acidification of blood plasma facilitates the separation and analysis of extracellular vesicles.

BACKGROUND: Blood plasma is available with minimal invasive sampling, it has significant diagnostic utility, and it is a valuable source of extracellular vesicles (EVs). Nevertheless, rich protein content, the presence of lipoproteins (LPs) that share similar biophysical properties, and relatively low abundance of EVs, especially those of rare subpopulations, make any downstream application a very challenging task. The growing evidence of the intricate surface interactome of EVs, and the association of EVs with LPs, impose further challenges during EV purification, detection, and biomarker analyses. OBJECTIVES: In this study, we tackled the fundamental issues of plasma EV yield and LP co-isolation and their implications in the subsequent marker analyses. METHODS: Moderate acidification of plasma was combined with size exclusion chromatography (SEC) and/or differential centrifugation (DC) to disrupt LPs and improve recovery of EVs and their subsequent detection by immunoassays and single-particle analysis methods. RESULTS: Our results demonstrate a surprisingly efficient enrichment of EVs (up to 3.3-fold higher than at pH 7) and partial depletion of LPs (up to 61.2%). Acidification of blood plasma samples enabled a quick single-step isoelectric precipitation of up to 20.4% of EVs directly from plasma, upon short low-speed centrifugation. CONCLUSION: Thus, acidification holds potential as a simple and inexpensive methodological step, which improves the efficacy of plasma EV enrichment and may have implications in future biomarker discoveries.

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility.

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Characterization of immune checkpoint inhibitor-induced cardiotoxicity reveals interleukin-17A as a driver of cardiac dysfunction after anti-PD-1 treatment.

BACKGROUND AND PURPOSE: Immune checkpoint inhibitors (ICI), such as anti-PD-1 monoclonal antibodies, have revolutionized cancer therapy by enhancing the cytotoxic effects of T-cells against tumours. However, enhanced T-cell activity also may cause myocarditis and cardiotoxicity. Our understanding of the mechanisms of ICI-induced cardiotoxicity is limited. Here, we aimed to investigate the effect of PD-1 inhibition on cardiac function and explore the molecular mechanisms of ICI-induced cardiotoxicity. EXPERIMENTAL APPROACH: C57BL6/J and BALB/c mice were treated with isotype control or anti-PD-1 antibody. Echocardiography was used to assess cardiac function. Cardiac transcriptomic changes were investigated by bulk RNA sequencing. Inflammatory changes were assessed by qRT-PCR and immunohistochemistry in heart, thymus, and spleen of the animals. In follow-up experiments, anti-CD4 and anti-IL-17A antibodies were used along with PD-1 blockade in C57BL/6J mice. KEY RESULTS: Anti-PD-1 treatment led to cardiac dysfunction and left ventricular dilation in C57BL/6J mice, with increased nitrosative stress. Only mild inflammation was observed in the heart. However, PD-1 inhibition resulted in enhanced thymic inflammatory signalling, where Il17a increased most prominently. In BALB/c mice, cardiac dysfunction was not evident, and thymic inflammatory activation was more balanced. Inhibition of IL-17A prevented anti-PD-1-induced cardiac dysfunction in C57BL6/J mice. Comparing myocardial transcriptomic changes in C57BL/6J and BALB/c mice, differentially regulated genes (Dmd, Ass1, Chrm2, Nfkbia, Stat3, Gsk3b, Cxcl9, Fxyd2, and Ldb3) were revealed, related to cardiac structure, signalling, and inflammation. CONCLUSIONS: PD-1 blockade induces cardiac dysfunction in mice with increased IL-17 signalling in the thymus. Pharmacological inhibition of IL-17A treatment prevents ICI-induced cardiac dysfunction.

The roles of extracellular vesicles in the immune system.

The twenty-first century has witnessed major developments in the field of extracellular vesicle (EV) research, including significant steps towards defining standard criteria for the separation and detection of EVs. The recent recognition that EVs have the potential to function as biomarkers or as therapeutic tools has attracted even greater attention to their study. With this progress in mind, an updated comprehensive overview of the roles of EVs in the immune system is timely. This Review summarizes the roles of EVs in basic processes of innate and adaptive immunity, including inflammation, antigen presentation, and the development and activation of B cells and T cells. It also highlights key progress related to deciphering the roles of EVs in antimicrobial defence and in allergic, autoimmune and antitumour immune responses. It ends with a focus on the relevance of EVs to immunotherapy and vaccination, drawing attention to ongoing or recently completed clinical trials that aim to harness the therapeutic potential of EVs.

Nanofiber formation as a promising technology for preservation and easy storage of extracellular vesicles.

Extracellular vesicles (EVs) are cell-derived, membrane-enclosed particles with the potential for a wide range of future therapeutic applications. However, EVs have almost always been administered by direct injection, likely hindering their efficacy because of rapid clearance from the injection site. The present study aimed to incorporate medium-sized extracellular vesicles (mEVs) into fast-dissolving electrospun polyvinylpyrrolidone-based nanofibers to explore the storage-dependent structure-activity relationship of the resulting nanofibrous formulations. Aqueous polyvinylpyrrolidone-based precursor solutions were selected for the electrospinning process. The presence of EVs in the electrospun samples was confirmed by transmission electron microscopy, flow cytometry, and confocal laser scanning microscope. The results indicate that the fibrous structure of the samples was preserved until the end of the 12-week storage period. Furthermore, regardless of the storage temperature (4 °C or room temperature), nanofibers and nanofiber-associated EVs were present throughout the experimental period. Incorporating EVs into a stable solid polymeric delivery base could preserve their stability; meanwhile, according to the characteristics of the polymer, their targeted and controlled release can be achieved.

CD3+/CD56+ NKT-like Cells Show Imbalanced Control Immediately after Exercise in Delayed-Onset Muscle Soreness.

The purpose of the study was to carry out an immunophenotypical characterization with a special focus on natural killer cells of junior swimmers from the Hungarian National Swim Team before and after an intensive acute exercise. Nineteen swimmers, ten females and nine males, completed the exercise protocol. Sixteen swimmers experienced delayed-onset muscle soreness. Most of our findings substantiated earlier results, such as the increase in the percentage of the CD3-/CD56+ natural killer cells and the CD3-/CD56dim+ NK cells, and the decrease in the percentage of CD3+ T cells among lymphocytes after the exercise protocol. The drop of natural killer cell activity back to the pre-exercise level was in line with earlier findings. Interestingly, the percentage of CD3+/CD56+ NKT-like cells did not change significantly in those three swimmers who did not report delayed-onset muscle soreness. On the contrary, the percentage of CD3+/CD56+ NKT-like cells among lymphocytes increased in fourteen and decreased in two swimmers reporting delayed-onset muscle soreness. This study for the first time demonstrated a link between the delayed-onset muscle soreness and the imbalanced control of CD3+/CD56+ NKT-like cells among lymphocytes. However, validation of this association in a larger sample size study will be necessary.

Extracellular Vesicle-Mediated Metastasis Suppressors NME1 and NME2 Modify Lipid Metabolism in Fibroblasts.

Nowadays, extracellular vesicles (EVs) raise a great interest as they are implicated in intercellular communication between cancer and stromal cells. Our aim was to understand how vesicular NME1 and NME2 released by breast cancer cells influence the tumour microenvironment. As a model, we used human invasive breast carcinoma cells overexpressing NME1 or NME2, and first analysed in detail the presence of both isoforms in EV subtypes by capillary Western immunoassay (WES) and immunoelectron microscopy. Data obtained by both methods showed that NME1 was present in medium-sized EVs or microvesicles, whereas NME2 was abundant in both microvesicles and small-sized EVs or exosomes. Next, human skin-derived fibroblasts were treated with NME1 or NME2 containing EVs, and subsequently mRNA expression changes in fibroblasts were examined. RNAseq results showed that the expression of fatty acid and cholesterol metabolism-related genes was decreased significantly in response to NME1 or NME2 containing EV treatment. We found that FASN (fatty acid synthase) and ACSS2 (acyl-coenzyme A synthetase short-chain family member 2), related to fatty acid synthesis and oxidation, were underexpressed in NME1/2-EV-treated fibroblasts. Our data show an emerging link between NME-containing EVs and regulation of tumour metabolism.

Small extracellular vesicle DNA-mediated horizontal gene transfer as a driving force for tumor evolution: Facts and riddles.

The basis of the conventional gene-centric view on tumor evolution is that vertically inherited mutations largely define the properties of tumor cells. In recent years, however, accumulating evidence shows that both the tumor cells and their microenvironment may acquire external, non-vertically inherited genetic properties via horizontal gene transfer (HGT), particularly through small extracellular vesicles (sEVs). Many phases of sEV-mediated HGT have been described, such as DNA packaging into small vesicles, their release, uptake by recipient cells, and incorporation of sEV-DNA into the recipient genome to modify the phenotype and properties of cells. Recent techniques in sEV separation, genome sequencing and editing, as well as the identification of new secretion mechanisms, shed light on a number of additional details of this phenomenon. Here, we discuss the key features of this form of gene transfer and make an attempt to draw relevant conclusions on the contribution of HGT to tumor evolution.

Activated polymorphonuclear derived extracellular vesicles are potential biomarkers of periprosthetic joint infection.

BACKGROUND: Extracellular vesicles (EVs) are considered as crucial players in a wide variety of biological processes. Although their importance in joint diseases or infections has been shown by numerous studies, much less is known about their function in periprosthetic joint infection (PJI). Our aim was to investigate activated polymorphonuclear (PMN)-derived synovial EVs in patients with PJI. QUESTIONS/PURPOSES: (1) Is there a difference in the number and size of extracellular vesicles between periprosthetic joint aspirates of patients with PJI and aseptic loosening? (2) Are these vesicles morphologically different in the two groups? (3) Are there activated PMN-derived EVs in septic samples evaluated by flow cytometry after CD177 labelling? (4) Is there a difference in the protein composition carried by septic and aseptic vesicles? METHODS: Thirty-four patients (n = 34) were enrolled into our investigation, 17 with PJI and 17 with aseptic prosthesis loosening. Periprosthetic joint fluid was aspirated and EVs were separated. Samples were analysed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) and flow cytometry (after Annexin V and CD177 labelling). The protein content of the EVs was studied by mass spectrometry (MS). RESULTS: NTA showed particle size distribution in both groups between 150 nm and 450 nm. The concentration of EVs was significantly higher in the septic samples (p = 0.0105) and showed a different size pattern as compared to the aseptic ones. The vesicular nature of the particles was confirmed by TEM and differential detergent lysis. In the septic group, FC analysis showed a significantly increased event number both after single and double labelling with fluorochrome conjugated Annexin V (p = 0.046) and Annexin V and anti-CD177 (p = 0.0105), respectively. MS detected a significant difference in the abundance of lactotransferrin (p = 0.00646), myeloperoxidase (p = 0.01061), lysozyme C (p = 0.04687), annexin A6 (p = 0.03921) and alpha-2-HS-glycoprotein (p = 0.03146) between the studied groups. CONCLUSIONS: An increased number of activated PMN derived EVs were detected in the synovial fluid of PJI patients with a characteristic size distribution and a specific protein composition. The activated PMNs-derived extracellular vesicles can be potential biomarkers of PJI.