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Abstract Background Exosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages. Method Exosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy. Results RNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages. Conclusions Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.

Background: Postoperative neurocognitive decline is a frequent complication triggered by unclear signalling mechanisms. This observational case-control study investigated the effects of hip or knee replacement surgery on the composition of circulating extracellular vesicles (EVs), potential periphery-to-brain messengers, and their association with neurocognitive outcomes., Methods: We mapped the microRNAome and proteome of plasma-derived EVs from 12 patients (six with good and six with poor neurocognitive outcomes at 3 months after surgery) at preoperative and postoperative timepoints (4, 8, 24, and 48 h). Complement C3-EV association was confirmed by flow cytometry in plasma- and cerebrospinal fluid (CSF)-derived EVs, with total plasma and CSF C3 and C3a concentrations determined using enzyme-linked immunosorbent assay., Results: Differential expression analysis found eight dysregulated EV microRNAs (miRNAs) exclusively in the poor neurocognitive outcomes group. Pathway analysis suggested potential downregulation of proliferative pathways and activation of extracellular matrix and inflammatory response pathways in EV target tissues. Proteome analysis revealed a time-dependent increase in immune-related EV proteins, including complement system proteins, notably EV surface-associated C3. Such upward kinetics was detected earlier in the poor neurocognitive outcomes group. Interestingly, CSF-derived EVs from the same group showed a drastic drop of C3 at 48 h with unchanged concentrations in the good neurocognitive outcomes group. Functionally, the complement system was activated in both patient groups in plasma, but only in the poor neurocognitive outcomes group in CSF., Conclusions: Our findings highlight the impact of surgery on plasma- and CSF-derived EVs, particularly in patients with poor neurocognitive outcomes, indicating a potential role for EVs. The small sample size necessitates verification with a larger patient cohort., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Extracellular vesicles (EVs), secreted by almost all living cells, have gained significant attention for their role in intercellular communication and their potential as versatile carriers for biotherapeutics. However, the clinical translation of EV-based therapies faces significant challenges, primarily due to the lack of efficient methods for loading biotherapeutic agents into EVs. This study introduces a simple, reproducible strategy for the simultaneous incorporation of various biotherapeutics within EVs. The process is gentle and preserves the essential physicochemical and biological characteristics of EVs, thereby protecting labile ligands from premature degradation and elimination. The binding and uptake efficiency of EVs by target cells reached approximately 97 % within 24 h of incubation. Administration of EVs loaded with oligodeoxynucleotides (ODN) resulted in a 4-fold increase in IFNγ + CD4 + T cells and a 5-fold increase in IFNγ + CD8 + T cells in the spleens and lymph nodes. Additionally, the co-administration of EVs with ODN and ovalbumin (OVA) induced elevated Th1-biased antibody responses and antigen-specific cytotoxic T-cell responses, providing long-lasting complete protection in 60 % of mice against T-cell thymoma challenge. Furthermore, EVs associated with three different ligands (OVA, CpG-ODN, and α-GalCer) effectively regressed established murine melanoma and significantly improved survival rates in mice. This study presents a powerful and promising approach to overcoming the limitations of EV-based cancer vaccines, advancing the development of effective cancer immunotherapies. SUMMARY: Immunization with EVs that are co-associated with antigen and biotherapeutic cargo through a lyophilization-based technique elicits potent anti-cancer immunity., Competing Interests: Declaration of competing interest The funding bodies played no role in study design, data collection, decision to publish, or preparation of the manuscript. The authors (I.G. & M.G.) declare that they have the inventor rights on CpG ODN related patents., (Copyright © 2024. Published by Elsevier B.V.)

Background: The α-Gal syndrome (AGS) is an emerging allergy to mammalian food caused by IgE-mediated reactions to the carbohydrate galactose-α-1,3-galactose (α-Gal). Mammalian food sources contain α-Gal, but the amount differs. The objective of this study was to investigate the allergenic potency of various foods of mammalian origin among AGS patients., Methods: Twenty-six AGS patients were included. Food extracts from innards, lean meats, processed meat products, milk, and whey were analyzed. Immunoblot, ELISA, immunofluorescence, and basophil activation test were used to determine the α-Gal content, characterize IgE binding, and assess foods' allergenicity., Results: The determined amount of α-Gal, IgE reactivity to food extracts, and food extract potencies to activate patients' basophils correlated well with each other. Pork and beef kidney showed the highest allergenicity. Beef liver and bacon showed allergenicity comparable to that of lean meats. Game meat seemed to have a higher allergenic potency than meats from farm-raised animals. The processed meat products liver pâté and black pudding, despite lower α-Gal content, demonstrated moderate allergenicity. Milk showed the lowest allergenicity. IgE reactivity to food extracts was highly similar for all patients and strongly dominated by the α-Gal epitope., Conclusions: The allergenic potency of mammalian meat depends on the origin of the meat, the different cuts, and type of processing, with innards posing the greatest risk to AGS patients. Even processed mammalian meat constitutes a risk. Dairy products show the lowest risk. This study highlights the importance of analyzing even more foods to improve the management of AGS., (© 2024 The Author(s). Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

The galactose-α-1,3-galactose (α-Gal) epitope is the cause of a global allergic disease, the α-Gal syndrome (AGS). It is a severe form of allergy to food and products of mammalian origin where IgE against the mammalian carbohydrate, α-Gal, is the cause of the allergic reactions. Allergic reactions triggered by parenterally administered α-Gal sources appear immediately, but those triggered via the oral route appear with a latency of several hours. The α-Gal epitope is highly immunogenic to humans, apes and old-world monkeys, all of which produce anti-α-Gal antibodies of the IgM, IgA and IgG subclasses. Strong evidence suggests that in susceptible individuals, class switch to IgE occurs after several tick bites. In this review, we discuss the strong immunogenic role of the α-Gal epitope and its structural resemblance to the blood type B antigen. We emphasize the broad abundance of α-Gal in different foods and pharmaceuticals and the allergenicity of various α-Gal containing molecules. We give an overview of the association of tick bites with the development of AGS and describe innate and adaptive immune response to tick saliva that possibly leads to sensitization to α-Gal. We further discuss a currently favored hypothesis explaining the mechanisms of the delayed effector phase of the allergic reaction to α-Gal. We highlight AGS from a clinical point of view. We review the different clinical manifestations of the disease and the prevalence of sensitization to α-Gal and AGS. The usefulness of various diagnostic tests is discussed. Finally, we provide different aspects of the management of AGS. With climate change and global warming, the tick density is increasing, and their geographic range is expanding. Thus, more people will be affected by AGS which requires more knowledge of the disease., Competing Interests: MvH has received lecture fees from Thermo Fisher Scientific and Astra Zeneca outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Perusko, Grundström, Eldh, Hamsten, Apostolovic and van Hage.)

The mechanism by which pancreatic beta cells are destroyed in type 1 diabetes (T1D) remains to be fully understood. Recent observations indicate that the disease may arise because of different pathobiological mechanisms (endotypes). The discovery of one or several protein biomarkers measurable in readily available liquid biopsies (e.g. blood plasma) during the pre-diabetic period may enable personalized disease interventions. Recent studies have shown that extracellular vesicles (EVs) are a source of tissue proteins in liquid biopsies. Using plasma samples collected from pre-diabetic non-obese diabetic (NOD) mice (an experimental model of T1D) we addressed if combined analysis of whole plasma samples and plasma-derived EV fractions increases the number of unique proteins identified by mass spectrometry (MS) compared to the analysis of whole plasma samples alone. LC-MS/MS analysis of plasma samples depleted of abundant proteins and subjected to peptide fractionation identified more than 2300 proteins, while the analysis of EV-enriched plasma samples identified more than 600 proteins. Of the proteins detected in EV-enriched samples, more than a third were not identified in whole plasma samples and many were classified as either tissue-enriched or of tissue-specific origin. In conclusion, parallel profiling of EV-enriched plasma fractions and whole plasma samples increases the overall proteome depth and facilitates the discovery of tissue-enriched proteins in plasma. If applied to plasma samples collected longitudinally from the NOD mouse or from models with other pathobiological mechanisms, the integrated proteome profiling scheme described herein may be useful for the discovery of new and potentially endotype specific biomarkers in T1D., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Diaz Lozano, Sork, Stone, Eldh, Cao, Pernemalm, Gabrielsson and Flodström-Tullberg.)

Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 μl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget., Competing Interests: Susanne Gabrielsson has a patent on B cell derived exosomes in immune therapy and is part of the Scientific Advisory Board of Anjarium Biosciences., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Invasive urothelial bladder cancer (UBC) has high recurrence rates even after radical cystectomy (RC). Exosomes are membrane-bound nanovesicles, which have been shown to contribute to carcinogenesis and metastasis. We previously showed that urinary exosomes display a malignant profile in UBC patients despite the absence of detectable tumour. Here, we investigated exosomes from sampling sites close to or distant from the former tumour, aiming to understand the effect of the tumour on the local milieu. Ten patients scheduled for cystectomy after transurethral bladder resection (TUR-B), without remaining detectable tumour, were included. Exosomes were isolated from tissue explants of both the previous tumour site and distant bladder tissue. Proteins were quantified by mass spectrometry in seven patients. Exosomes from the previous tumour site were enriched in inflammatory but not cancer-related pathways compared to distant tissue. However, the 69 most abundant proteins in tissue-derived exosomes regardless of site, 20 of which were also found in urinary exosomes from our previous study, were enriched for cancer-related metabolic pathways and associated with poor prognosis in an external mRNA dataset. The enrichment of cancer-related pathways in the most abundant proteins, regardless of sampling site, confirms our hypothesis that despite the absence of detectable tumour, the entire bladder releases exosomes that contribute to metastasis and highlights the need for early RC.

Invasive urinary bladder cancer shows high recurrence rates after cystectomy even with apparent complete downstaging at cystectomy. Exosomes are nano-sized vesicles important in cell-cell communication, which have been hypothesized to contribute to cancer dissemination and recurrence. The aim of this study was to investigate if pro-carcinogenic exosomes could be detected in urine from histologically downstaged bladder cancer patients. 13 Patients were included in this study. Paired ureter and urine samples from nine patients underwent mass spectrometry, while samples from the remaining patients were used for exosome characterization. At cystectomy, exosomes were isolated from bladder and ureter urine, whereafter quantitative proteome profiling was performed. Urinary exosomes clustered based on whether they came from the bladder, with tumour contact, or the ureters, without tumour contact, even though all came from completely downstaged patients. Proteins overexpressed in exosomes derived from bladder urine contained several oncogenes and were mainly associated with tumour metabolism pathways. Although patients were histologically tumour-free at cystectomy, the bladder urine contained exosomes with a carcinogenic metabolic profile. This suggests a continuous release of exosomes from the bladder, which may promote recurrence at distant sites through metabolic rewiring, even after apparent complete downstaging. These exosomes, coming from either undetected cancer cells or partly transformed cells, are likely to increase the risk of metastasis and encourages cystectomy even in completely downstaged patients.

Background: The malaria parasite Plasmodium falciparum holds an extensive genetic polymorphism. In this pooled analysis, we investigate how the multiplicity in asymptomatic P. falciparum infections-that is, the number of coinfecting clones-affects the subsequent risk of clinical malaria in populations living under different levels of transmission., Methods: A systematic search of the literature was performed to identify studies in which P. falciparum infections were genotyped in asymptomatic individuals who were followed up prospectively regarding the incidence of clinical malaria. Individual participant data were pooled from 15 studies (n = 3736 individuals)., Results: Multiclonal asymptomatic infections were associated with a somewhat increased subsequent risk of clinical malaria in the youngest children, followed by an initial declining risk with age irrespective of transmission intensity. At approximately 5 years of age, the risk continued the gradual decline with age in high-transmission settings. However, in older children in moderate-, low-, and seasonal-transmission settings, multiclonal infections were either not significantly associated with the risk of subsequent febrile malaria or were associated with an increased risk., Conclusions: The number of clones in asymptomatic P. falciparum infections is associated with different risks of subsequent clinical malaria depending on age and transmission intensity., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Extracellular vesicles (EVs) are nano-sized vesicles with the capacity to transfer nucleic acids, proteins, and lipids, and are released by all cells. EV is an umbrella term for exosomes originating from the endosomal compartment, microvesicles from the cell membrane, and apoptotic bodies released during apoptosis. EVs are being investigated for their role in health and disease, and as potential biomarkers, with newly developed FDA-approved assays reaching the market. Currently, both academic institutions and industrial ventures are developing clinical trials to use EVs in diseases such as cancer, graft-versus-host disease, and neurodegenerative diseases. This review describes and discusses current understanding of the functions of immune cell-derived exosomes and microvesicles, and how they might be explored for immunotherapy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.

Objective: To examine whether the use of a surgical safety checklist (SSC) could reduce the incidence of complications after small animal surgery., Study Design: Prospective clinical study., Animals: Client-owned dogs and cats (n = 520)., Methods: Consecutive cases were enrolled in the study, the first 300 cases without implementation of the surgical checklist (SSC-), followed by 220 cases with implementation of the checklist (SSC+). The checklist was adapted from the WHO surgical checklist and consisted of three different check points: (1) before induction of anaesthesia (sign in), (2) before surgical incision (time out), and (3) before recovery (sign out). In-hospital outcomes were prospectively recorded, and complications within 6 weeks were retrospectively recorded by reviewing medical records and by telephone interviews with owners. The severity of each recorded complication was graded as minor, moderate, or severe. Comparisons were made between SSC- and SSC+ outcomes., Results: There were significantly more complications in SSC- animals than SSC+ animals (SSC- 52/300 vs. SSC+ 15/220, P = .0003). There was a significantly higher frequency of SSI (P = .045) and wound healing complications (P = .0006) for SSC- animals than SSC+ animals., Conclusion: The frequency and severity of postoperative complications was significantly decreased after introduction of a surgical checklist. All veterinary hospitals should consider using a surgical checklist. Compliance with implementation of the checklist is important for success., (© Copyright 2016 by The American College of Veterinary Surgeons.)

Peptide-loaded exosomes are promising cancer treatment vehicles; however, moderate T cell responses in human clinical trials indicate a need to further understand exosome-induced immunity. We previously demonstrated that antigen-loaded exosomes carry whole protein antigens and require B cells for inducing antigen-specific T cells. Therefore, we investigated the relative importance of exosomal major histocompatibility complex (MHC) class I for the induction of antigen-specific T cell responses and tumour protection. We show that ovalbumin-loaded dendritic cell-derived exosomes from MHCI-/- mice induce antigen-specific T cells at the same magnitude as wild type exosomes. Furthermore, exosomes lacking MHC class I, as well as exosomes with both MHC class I and II mismatch, induced tumour infiltrating T cells and increased overall survival to the same extent as syngeneic exosomes in B16 melanoma. In conclusion, T cell responses are independent of exosomal MHC/peptide complexes if whole antigen is present. This establishes the prospective of using impersonalised exosomes, and will greatly increase the feasibility of designing exosome-based vaccines or therapeutic approaches in humans., Competing Interests: S.G. has a patent on B-cell exosomes for immune therapy, US patent no. US 8932855 B2.

After birth, the intestinal immune system enters a critical developmental stage, in which tolerogenic and pro-inflammatory cells emerge to contribute to the overall health of the host. The neonatal health is continuously challenged by microbial colonization and food intake, first in the form of breast milk or formula and later in the form of solid food. The microbiota and dietary compounds shape the newborn immune system, which acquires the ability to induce tolerance against innocuous antigens or induce pro-inflammatory immune responses against pathogens. Disruption of these homeostatic mechanisms might lead to undesired immune reactions, such as food allergies and inflammatory bowel disease. Hence, a proper education and maturation of the intestinal immune system is likely important to maintain life-long intestinal homeostasis. In this review, the most recent literature regarding the effects of dietary compounds in the development of the intestinal immune system are discussed.

Background: Uveal melanoma is a tumour arising from melanocytes of the eye, and 30 per cent of these patients develop liver metastases. Exosomes are small RNA containing nano-vesicles released by most cells, including malignant melanoma cells. This clinical translational study included patients undergoing isolated hepatic perfusion (IHP) for metastatic uveal melanoma, from whom exosomes were isolated directly from liver perfusates. The objective was to determine whether exosomes are present in the liver circulation, and to ascertain whether these may originate from melanoma cells., Methods: Exosomes were isolated from the liver perfusate of twelve patients with liver metastases from uveal melanoma undergoing IHP. Exosomes were visualised by electron microscopy, and characterised by flow cytometry, Western blot and real-time PCR. Furthermore, the concentration of peripheral blood exosomes were measured and compared to healthy controls., Results: The liver perfusate contained Melan-A positive and RNA containing exosomes, with similar miRNA profiles among patients, but dissimilar miRNA compared to exosomes isolated from tumor cell cultures. Patients with metastatic uveal melanoma had a higher concentration of exosomes in their peripheral venous blood compared to healthy controls., Conclusions: Melanoma exosomes are released into the liver circulation in metastatic uveal melanoma, and is associated with higher concentrations of exosomes in the systemic circulation. The exosomes isolated directly from liver circulation contain miRNA clusters that are different from exosomes from other cellular sources.

Exosomes are cell-secreted nanometer-sized extracellular vesicles that have been reported to play an important role in intercellular communication. They are also considered potential diagnostic markers for various health disorders, and intense investigations are presently directed toward their use as carriers in drug-delivery and gene-therapy applications. This has generated a growing need for sensitive methods capable of accurately and specifically determining the concentration of exosomes in complex biological fluids. Here, we explore the use of label-free surface-based sensing with surface plasmon resonance (SPR) read-out to determine the concentration of exosomes in solution. Human mast cell secreted exosomes carrying the tetraspanin membrane protein CD63 were analyzed by measuring their diffusion-limited binding rate to an SPR sensor surface functionalized with anti-CD63 antibodies. The concentration of suspended exosomes was determined by first converting the SPR response into the surface-bound mass. The increase in mass uptake over time was then related to the exosome concentration in solution using a formalism describing diffusion-limited binding under controlled flow conditions. The proposed quantification method is based on a calibration and control measurements performed with proteins and synthetic lipid vesicles and takes into account (i) the influence of the broad size distribution of the exosomes on the surface coverage, (ii) the fact that their size is comparable to the ∼150 nm probing depth of SPR, and (iii) possible deformation of exosomes upon adsorption. Under those considerations, the accuracy of the concentration determination was estimated to be better than ±50% and significantly improve if the exosome deformation is negligible.

Background: Allergic asthma is a chronic disease of the conducting airways characterized by T(H)2 inflammation and tissue remodeling after exposure to inhaled allergens. Although the T(H)2 profile is undisputed, the underlying molecular mechanisms leading to this abnormal T(H)2 profile remain largely unclear. MicroRNAs (miRNAs) are short noncoding RNAs that are important regulators of gene expression in the immune system. However, the role of miRNAs, specifically miR-155, in the regulation of allergic airway inflammation is unexplored., Objectives: We sought to assess the contribution of miR-155 in a mouse model of allergic airway inflammation., Methods: To investigate a role for miR-155 in the regulation of allergic inflammation in vivo, we used miR-155 knockout (KO) and wild-type (WT) mice sensitized and exposed to ovalbumin., Results: miR-155 deficiency resulted in diminished eosinophilic inflammation and mucus hypersecretion in the lungs of allergen-sensitized and allergen-challenged mice compared with WT control animals. This was supported by a reduction in T(H)2 cell numbers and airway T(H)2 cytokine levels and complete abrogation of allergen-induced airway eotaxin-2/CCL24 and periostin levels in miR-155 KO mice. Intranasal instillation of eotaxin-2/CCL24 before allergen challenge partially restored airway eosinophilia in miR-155 KO mice, and adoptive transfer of CD4(+) T cells resulted in a similar degree of airway eosinophilia in miR-155 KO and WT mice. Furthermore, the transcription factor PU.1, a negative regulator of T(H)2 cytokine production, was upregulated in the airways of allergen-challenged miR-155 KO mice compared with WT mice., Conclusions: Our data provides evidence that miR-155 contributes to the regulation of allergic airway inflammation by modulating T(H)2 responses through the transcription factor PU.1., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Introduction: In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination., Method: EVS RELEASED FROM THREE DIFFERENT KINDS OF CELL LINES: HMC-1, TF-1 and BV-2 were isolated using two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer(®)., Results: RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF-1-derived vesicles, as these cells do not express CD9., Conclusions: Our results demonstrate that centrifugation-based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and morphological characteristics, but they are indistinguishable using CD63-coated beads for flow cytometry analysis.

Background: Exosomes are nanosized vesicles of endocytic origin that are released into the extracellular environment by many different cells. It has been shown that exosomes from various cellular origins contain a substantial amount of RNA (mainly mRNA and microRNA). More importantly, exosomes are capable of delivering their RNA content to target cells, which is a novel way of cell-to-cell communication. The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34(+) progenitor cells., Methods: The mRNA and microRNA content of exosomes from a human mast cell line, HMC-1, was analysed by using microarray technology. Co-culture experiments followed by flow cytometry analysis and confocal microscopy as well as radioactive labeling experiments were performed to examine the uptake of these exosomes and the shuttle of the RNA to other mast cells and CD34(+) progenitor cells., Results: In this study, we show that human mast cells release RNA-containing exosomes, with the capacity to shuttle RNA between cells. Interestingly, by using microRNA microarray analysis, 116 microRNAs could be identified in the exosomes and 134 microRNAs in the donor mast cells. Furthermore, DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. In addition, transfer experiments revealed that exosomes can shuttle RNA between human mast cells and to CD34(+) hematopoietic progenitor cells., Conclusion: These findings suggest that exosomal shuttle RNA (esRNA) can play a role in the communication between cells, including mast cells and CD34(+) progenitor cells, implying a role in cells maturation process.

Exosomes are small RNA containing vesicles of endocytic origin, which can take part in cell-to-cell communication partly by the transfer of exosomal RNA between cells. Exosomes are released by many cells and can also be found in several biological fluids including blood plasma and breast milk. Exosomes differ compared to their donor cells not only in size but also in RNA, protein and lipid composition. The aim of the current study was to determine the optimal RNA extraction method for analysis of exosomal RNA, to support future studies determining the biological roles of the exosomal RNA. Different methods were used to extract exosomal and cellular RNA. All methods evaluated extracted high quality and purity RNA as determined by RNA integrity number (RIN) and OD values for cellular RNA using capillary electrophoresis and spectrophotometer. Interestingly, the exosomal RNA yield differed substantially between the different RNA isolation methods. There was also a difference in the exosomal RNA patterns in the electropherograms, indicating that the tested methods extract exosomal RNA with different size distribution. A pure column based approach resulted in the highest RNA yield and the broadest RNA size distribution, whereas phenol and combined phenol and column based approaches lost primarily large RNAs. Moreover, the use of phenol and combined techniques resulted in reduced yield of exosomal RNA, with a more narrow size distribution pattern resulting in an enrichment of small RNA including microRNA. In conclusion, the current study presents a unique comparison of seven different methods for extraction of exosomal RNA. As the different isolation methods give extensive variation in exosomal RNA yield and patterns, it is crucial to select an isolation approach depending on the research question at hand., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen, eradication of established tumors in mice, inhibition and activation of natural killer cells, promotion of differentiation into T regulatory cells, stimulation of T cell proliferation and induction of T cell apoptosis. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer., (Copyright © 2012 Creative Commons Attribution License)

Background: Exosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages., Method: Exosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy., Results: RNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages., Conclusions: Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.

Chronic exposure to tobacco smoke leads to an increase in the frequency of infections and in the number of CD8(+) and CD4(+) cells as well as the CD4(+) chemoattractant cytokine IL-16 in the airways. Here, we investigated whether tobacco smoke depletes intracellular IL-16 protein and inhibits de novo production of IL-16 in CD8(+) cells from human airways and blood while increasing extracellular IL-16 and whether oxygen free radicals (OFR) are involved. Intracellular IL-16 protein in CD8(+) cells and mRNA in all cells was decreased in bronchoalveolar lavage (BAL) samples from chronic smokers. This was also the case in human blood CD8(+) cells exposed to water-soluble tobacco smoke components in vitro, in which oxidized proteins were markedly increased. Extracellular IL-16 protein was increased in cell-free BAL fluid from chronic smokers and in human blood CD8(+) cells exposed to water-soluble tobacco smoke components in vitro. This was not observed in occasional smokers after short-term exposure to tobacco smoke. A marker of activation (CD69) was slightly increased, whereas other markers of key cellular functions (membrane integrity, apoptosis, and proliferation) in human blood CD8(+) cells in vitro were negatively affected by water-soluble tobacco smoke components. An OFR scavenger prevented these effects, whereas a protein synthesis inhibitor, a β-adrenoceptor, a glucocorticoid receptor agonist, a phosphodiesterase, a calcineurin phosphatase, and a caspase-3 inhibitor did not. In conclusion, tobacco smoke depletes preformed intracellular IL-16 protein, inhibits its de novo synthesis, and distorts key cellular functions in human CD8(+) cells. OFR may play a key role in this context.

Background: Exosomes are small extracellular nanovesicles of endocytic origin that mediate different signals between cells, by surface interactions and by shuttling functional RNA from one cell to another. Exosomes are released by many cells including mast cells, dendritic cells, macrophages, epithelial cells and tumour cells. Exosomes differ compared to their donor cells, not only in size, but also in their RNA, protein and lipid composition., Methodology/principal Findings: In this study, we show that exosomes, released by mouse mast cells exposed to oxidative stress, differ in their mRNA content. Also, we show that these exosomes can influence the response of other cells to oxidative stress by providing recipient cells with a resistance against oxidative stress, observed as an attenuated loss of cell viability. Furthermore, Affymetrix microarray analysis revealed that the exosomal mRNA content not only differs between exosomes and donor cells, but also between exosomes derived from cells grown under different conditions; oxidative stress and normal conditions. Finally, we also show that exposure to UV-light affects the biological functions associated with exosomes released under oxidative stress., Conclusions/significance: These results argue that the exosomal shuttle of RNA is involved in cell-to-cell communication, by influencing the response of recipient cells to an external stress stimulus.

Objective: Individually applied urotherapy is first-line treatment in children with bladder dysfunction. A new concept of treatment for small groups of children was applied and evaluated., Patients and Methods: Two hundred children, 116 of them girls, aged 3-14 years (median 7.2) with bladder dysfunction and incontinence received urotherapy in small groups (2-5), called voiding school (VS). Outcome was evaluated after 3 and 12 months by voiding/leakage diary and questionnaire, and at 3 months by uroflow and post-void residual urine as well., Results: The outcome of VS was independent of age and gender. At follow up at 3 and 12 months, respectively, 35% and 40% of the children were cured and another 30% and 34% improved (P≤0.0001). Compared with the year before start of VS, urinary tract infections decreased from 34% to 6% (P<0.0001). Median residual urine decreased from 15 ml before VS to 6 ml after 3 months (P<0.001)., Conclusion: The concept of VS is a good alternative to individual urotherapy, with the outcome of fewer urinary tract infections and improved continence. Urotherapy for groups of children compared to individual treatment is also expected to have financial benefits., (Copyright © 2009 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Aim: To bring forward the arguments for active treatment of urine incontinence in otherwise healthy children, a quality-of-life (QoL) study was performed., Subjects and Methods: A self-rating QoL questionnaire, child-adjusted and validated, was completed by 120 neurologically healthy children, aged 6-16 y, with urinary incontinence. Another 239 age-matched children made up a control group. The two groups were compared both totally and in age-related subgroups (6-8, 9-12, >12 y) concerning the index for all questions, for universal parts (without questions dealing with incontinence) as well as for specific key domains., Results: The patient group had a significantly lower index than the control group both with and without items related to incontinence (p<0.0001). Social situation, self-esteem and self-confidence were most influenced, particularly in the youngest children. Thirty-one children (13%) of the control group reported incontinence and did not score their QoL as good as their continent peers but better than the study patients., Conclusion: From the quality-of-life aspects, the study supports active treatment of urinary incontinence in children already at younger ages.