Therapeutic potential of mesenchymal stem cell-derived extracellular vesicles in SARS-CoV-2 and H1N1 influenza-induced acute lung injury.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have shown anti-inflammatory potential in multiple inflammatory diseases. In the March 2022 issue of the Journal of Extracellular Vesicles, it was shown that EVs from human MSCs can suppress severe acute respiratory distress syndrome, coronavirus 2 (SARS-CoV-2) replication and can mitigate the production and release of infectious virions. We therefore hypothesized that MSC-EVs have an anti-viral effect in SARS-CoV-2 infection in vivo. We extended this question to ask whether also other respiratory viral infections could be treated by MSC-EVs. Adipose stem cell-derived EVs (ASC-EVs) were isolated using tangential flow filtration from conditioned media obtained from a multi-flask cell culture system. The effects of the ASC-EVs were tested  in Vero E6 cells in vitro. ASC-EVs were also given i.v. to SARS-CoV-2 infected Syrian Hamsters, and H1N1 influenza virus infected mice. The ASC-EVs attenuated SARS-CoV-2 virus replication in Vero E6 cells and reduced body weight and signs of lung injury in infected Syrian hamsters. Furthermore, ASC-EVs increased the survival rate of influenza A-infected mice and attenuated signs of lung injury. In summary, this study suggests that ASC-EVs can have beneficial therapeutic effects in models of virus-infection-associated acute lung injury and may potentially be developed to treat lung injury in humans.

Level of education, but not occupation, is differentially associated with asthma phenotypes in adults.

CONCLUSION: Education, but not occupation, was differentially associated with adult asthma phenotypes in the general population. Further research into socioeconomic status variation in various asthma phenotypes is warranted.

Proteomic profiling of tumour tissue-derived extracellular vesicles in colon cancer.

Colon cancer is one of the most commonly occurring tumours among both women and men, and over the past decades the incidence has been on the rise. As such, the need for biomarker identification as well as an understanding of the underlying disease mechanism has never been greater. Extracellular vesicles are integral mediators of cell-to-cell communication and offer a unique opportunity to study the machinery that drives disease progression, and they also function as vectors for potential biomarkers. Tumour tissue and healthy mucosal tissue from the colons of ten patients were used to isolate tissue-resident EVs that were subsequently subjected to global quantitative proteomic analysis through LC-MS/MS. In total, more than 2000 proteins were identified, with most of the common EV markers being among them. Bioinformatics revealed a clear underrepresentation of proteins involved in energy production and cellular adhesion in tumour EVs, while proteins involved in protein biosynthesis were overrepresented. Additionally, 53 membrane proteins were found to be significantly upregulated in tumour EVs. Among them were several proteins with enzymatic functions that degrade the extracellular matrix, and three of these, Fibroblast activating factor (FAP), Cell surface hyaluronidase (CEMIP2), as well as Ephrin receptor B3 (EPHB3), were validated and found to be consistent with the global quantitative results. These stark differences in the proteomes between healthy and cancerous tissue emphasise the importance of the interstitial vesicle secretome as a major player of disease development.

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.

The Anti-Inflammatory Effects of Adipose Tissue Mesenchymal Stem Cell Exosomes in a Mouse Model of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a group of chronic, relapsing inflammatory disorders that affect the gastrointestinal tract, with the primary subtypes being ulcerative colitis (UC) and Crohn's disease (CD). We aimed to evaluate the therapeutic potential of extracellular vesicles released by adipose-tissue-derived mesenchymal stem cells, which we, in this manuscript, call "exosomes" (ASC-EXOs), in a mouse model of IBD. We specifically aimed to determine the effectiveness of different treatment protocols and compare the effects with that of anti-IL-12 p40 monoclonal antibody. The addition of dextran sulfate sodium (DSS) to drinking water induced multiple signs of IBD, including weight loss, soft stool, and bloody feces. ASC-EXOs given by either intraperitoneal (IP) or intravenous (IV) routes resulted in moderate improvement in these signs of IBD. IV ASC-EXOs resulted in significantly reduced body weight loss, improved histopathological scoring, and suppressed the disease activity index (DAI) compared to the IBD control group. Also, a reduction in PCR for pro-inflammatory cytokines was observed. IV ASC treatment resulted in dose-related reduction in IBD signs, including weight loss. An increasing number of injections with ASC-EXOs reduced histopathological scores as well as DAI. Co-administration of ASC-EXOs with anti-IL-12 p40 significantly decreased DAI scores in the ASC-EXO + anti-IL-12 p40 group. In conclusion, ASC-EXOs have potential as a therapeutic agent for IBD, but the route of administration, number of injections, and dosage need to be considered to optimize the effects of ASC-EXO treatment. This study also highlights the potential benefits of combination therapies of ASC-EXOs and anti-IL-12. Our findings pave the way for further studies to unravel the underlying therapeutic mechanisms of ASC-EXOs in IBD treatment.

Three-dimensional reconstruction of interstitial extracellular vesicles in human liver as determined by electron tomography.

Extracellular vesicles (EVs) are lipid bilayer nanoparticles involved in cell-cell communication that are released into the extracellular space by all cell types. The cargo of EVs includes proteins, lipids, nucleic acids, and metabolites reflecting their cell of origin. EVs have recently been isolated directly from solid tissues, and this may provide insights into how EVs mediate communication between cells in vivo. Even though EVs have been isolated from tissues, their point of origin when they are in the interstitial space has been uncertain. In this study, we performed three-dimensional (3D) reconstruction using transmission electron tomography of metastatic and normal liver tissues with a focus on the presence of EVs in the interstitium. After chemical fixation of the samples and subsequent embedding of tissue pieces in resin, ultrathin slices (300 nm) were cut and imaged on a 120 ekV transmission electron microscopy as a tilt series (a series of subsequent images tilted at different angles). These were then computationally illustrated in a 3D manner to reconstruct the imaged tissue volume. We identified the cells delimiting the interstitial space in both types of tissues, and small distinct spherical structures with a diameter of 30-200 nm were identified between the cells. These round structures appeared to be more abundant in metastatic tissue compared to normal tissue. We suggest that the observed spherical structures in the interstitium of the metastatic and non-metastatic liver represent EVs. This work thus provides the first 3D visualization of EVs in human tissue.

Sensitization patterns to cat molecular allergens in subjects with allergic sensitization to cat dander.

BACKGROUND: The use of molecular allergology has increasingly become common in the diagnosis and management of allergic diseases. However, there is still a lack of data on cat molecular allergens in adults. Therefore, we aimed to uncover the sensitization patterns to cat molecular allergens. METHODS: Participants were recruited from the West Asthma Sweden Study, a population-based study enriched with asthma subjects aged 16-75 years. Of 1872, 361 individuals were positive for cat dander immunoglobulin E and were further analysed for cat molecular allergens (Fel d 1/2/4/7). Sensitization patterns were classified as monosensitization, polysensitization, and concomitant sensitization, and were related to demographic and clinical measurements. RESULTS: Among cat-sensitized subjects, 84.2% were sensitized to secretoglobin, while 42.4% were sensitized to lipocalins. Nearly half of the subjects were monosensitized to Fel d 1. Polysensitization was observed in 20.2%, and concomitant sensitization to protein families was seen in 7.2%. Asthma prevalence, cat exposure, and rural living were associated with poly- and concomitant sensitization to protein families. Concomitant sensitization to single allergens was more common in those with asthma than in those without, while concomitant sensitization to both Fel d 1 and Fel d 4 was the most common pattern in individuals with asthma. Sensitization patterns also differed according to cat ownership and the degree of urbanization. CONCLUSION: Sensitization to molecular allergens was observed in 90.9% of cat-sensitized subjects and showed variations across participants' background characteristics and the presence of asthma. Identification of sensitization patterns to cat allergens might provide better characterization of cat-allergic subjects.

Detoxified synthetic bacterial membrane vesicles as a vaccine platform against bacteria and SARS-CoV-2.

The development of vaccines based on outer membrane vesicles (OMV) that naturally bud off from bacteria is an evolving field in infectious diseases. However, the inherent inflammatory nature of OMV limits their use as human vaccines. This study employed an engineered vesicle technology to develop synthetic bacterial vesicles (SyBV) that activate the immune system without the severe immunotoxicity of OMV. SyBV were generated from bacterial membranes through treatment with detergent and ionic stress. SyBV induced less inflammatory responses in macrophages and in mice compared to natural OMV. Immunization with SyBV or OMV induced comparable antigen-specific adaptive immunity. Specifically, immunization with Pseudomonas aeruginosa-derived SyBV protected mice against bacterial challenge, and this was accompanied by significant reduction in lung cell infiltration and inflammatory cytokines. Further, immunization with Escherichia coli-derived SyBV protected mice against E. coli sepsis, comparable to OMV-immunized group. The protective activity of SyBV was driven by the stimulation of B-cell and T-cell immunity. Also, SyBV were engineered to display the SARS-CoV-2 S1 protein on their surface, and these vesicles induced specific S1 protein antibody and T-cell responses. Collectively, these results demonstrate that SyBV may be a safe and efficient vaccine platform for the prevention of bacterial and viral infections.

Comparison of clinician diagnosis of COVID-19 with real time polymerase chain reaction in an adult-representative population in Sweden.

BACKGROUND: Due to the high transmissibility of SARS-CoV-2, accurate diagnosis is essential for effective infection control, but the gold standard, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), is costly, slow, and test capacity has at times been insufficient. We compared the accuracy of clinician diagnosis of COVID-19 against RT-PCR in a general adult population. METHODS: COVID-19 diagnosis data by 30th September 2021 for participants in an ongoing population-based cohort study of adults in Western Sweden were retrieved from registers, based on positive RT-PCR and clinician diagnosis using recommended ICD-10 codes. We calculated accuracy measures of clinician diagnosis using RT-PCR as reference for all subjects and stratified by age, gender, BMI, and comorbidity collected pre-COVID-19. RESULTS: Of 42,621 subjects, 3,936 (9.2%) and 5705 (13.4%) had had COVID-19 identified by RT-PCR and clinician diagnosis, respectively. Sensitivity and specificity of clinician diagnosis against RT-PCR were 78% (95%CI 77-80%) and 93% (95%CI 93-93%), respectively. Positive predictive value (PPV) was 54% (95%CI 53-55%), while negative predictive value (NPV) was 98% (95%CI 98-98%) and Youden's index 71% (95%CI 70-72%). These estimates were similar between men and women, across age groups, BMI categories, and between patients with and without asthma. However, while specificity, NPV, and Youden's index were similar between patients with and without chronic obstructive pulmonary disease (COPD), sensitivity was slightly higher in patients with (84% [95%CI 74-90%]) than those without (78% [95%CI 77-79%]) COPD. CONCLUSIONS: The accuracy of clinician diagnosis for COVID-19 is adequate, regardless of gender, age, BMI, and asthma, and thus can be used for screening purposes to supplement RT-PCR.

Sensitization to molecular dog allergens in an adult population: Results from the West Sweden Asthma Study.

BACKGROUND: As the prevalence of dog allergy rises, component resolved diagnosis might improve the diagnosis, understanding of the clinical outcomes and the effectiveness of immunotherapy. Considering the paucity of data in adults, the current study characterized the patterns of sensitization to dog molecular allergens in an adult population. METHODS: Data were derived from the West Sweden Asthma Study, a population-based and representative sample of adults from western Sweden. Of the 2006 subjects clinically examined, 313 participants sensitized to whole dog allergen extract were measured for specific immunoglobulin E (sIgE) levels to Can f 1, Can f 2, Can f 3, Can f 4, Can f 5 and Can f 6 using ImmunoCAP™. Polysensitization was defined as sensitization to ≥3 components. Overlapping sensitization was defined as having concomitant sensitization to at least two dog molecular allergen families (lipocalin, albumin or prostatic kallikrein). RESULTS: Of 313, 218 (70%) subjects tested positive to at least one dog allergen component. Sensitization to Can f 1 (43%) was the most common, followed by Can f 5 (33%) among molecular allergens, while sensitization to lipocalins (56%) was the most common among component families. Polysensitization was found in 22% of all participants and was more common in participants with than in those without asthma. Subjects with asthma were less likely to be monosensitized to Can f 5 than those without asthma. Subjects with asthma had higher IgE levels of Can f 3, Can f 4 and Can f 6 than those without asthma. Overlapping sensitizations also differed between those with asthma and allergic rhinitis and those without. CONCLUSION: Increased knowledge about the sensitization patterns of dog allergen components can aid in defining their role in asthma and rhinitis. In complex clinical cases of dog allergy, a detailed analysis of dog allergen components can provide additional information on the nature of sensitization.

Extracellular vesicle DNA from human melanoma tissues contains cancer-specific mutations.

Liquid biopsies are promising tools for early diagnosis and residual disease monitoring in patients with cancer, and circulating tumor DNA isolated from plasma has been extensively studied as it has been shown to contain tumor-specific mutations. Extracellular vesicles (EVs) present in tumor tissues carry tumor-derived molecules such as proteins and nucleic acids, and thus EVs can potentially represent a source of cancer-specific DNA. Here we identified the presence of tumor-specific DNA mutations in EVs isolated from six human melanoma metastatic tissues and compared the results with tumor tissue DNA and plasma DNA. Tumor tissue EVs were isolated using enzymatic treatment followed by ultracentrifugation and iodixanol density cushion isolation. A panel of 34 melanoma-related genes was investigated using ultra-sensitive sequencing (SiMSen-seq). We detected mutations in six genes in the EVs (BRAF, NRAS, CDKN2A, STK19, PPP6C, and RAC), and at least one mutation was detected in all melanoma EV samples. Interestingly, the mutant allele frequency was higher in DNA isolated from tumor-derived EVs compared to total DNA extracted directly from plasma DNA, supporting the potential role of tumor EVs as future biomarkers in melanoma.

Targeting Myd88 using peptide-loaded mesenchymal stem cell membrane-derived synthetic vesicles to treat systemic inflammation.

Mesenchymal stem cells (MSC) secrete extracellular vesicles (EV) with a regenerative profile, and an increasing number of studies have focused on the utilization of MSC-EV for therapeutic drug delivery. However, EV are usually produced by cells in low quantities and are packed with numerous cytoplasmic components, which may be unfavorable for further drug loading. In this study, we developed a simple process for generating membrane vesicles directly from the cells, which we refer to as synthetic eukaryotic vesicles (SyEV). We hypothesized that MSC-derived SyEV can be efficiently loaded with an anti-inflammatory drug and the loaded vesicles can strongly suppress the systemic inflammation induced by bacterial outer membrane vesicles (OMV). SyEV were generated from MSC membranes through serial extrusion of the cells, ionic stress, and subsequent vesiculation of the membrane sheets, leading to high yield and purity of the SyEV with few cytosolic components remaining. When these SyEV were given to macrophages or mice exposed to OMV, the release of pro-inflammatory cytokines was similarly attenuated comparable to treatment with natural EV. We then loaded the SyEV with large numbers of peptides targeting Myd88 and observed enhanced therapeutic potential of the loaded vesicles in OMV-induced macrophages. Further, in vivo experiments showed that the peptide-encapsulated MSC-SyEV suppressed cytokine production synergistically. Taken together, these findings suggest that SyEV-based therapeutics is a highly interesting platform for delivering an advanced therapeutic drug for the treatment of systemic inflammation without severe side effects.

Tetraspanins distinguish separate extracellular vesicle subpopulations in human serum and plasma - Contributions of platelet extracellular vesicles in plasma samples.

BACKGROUND: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. METHOD: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. RESULTS: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. CONCLUSION: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail.

A brief history of nearly EV-erything - The rise and rise of extracellular vesicles.

Extracellular vesicles (EVs) are small cargo-bearing vesicles released by cells into the extracellular space. The field of EVs has grown exponentially over the past two decades; this growth follows the realisation that EVs are not simply a waste disposal system as had originally been suggested by some, but also a complex cell-to-cell communication mechanism. Indeed, EVs have been shown to transfer functional cargo between cells and can influence several biological processes. These small biological particles are also deregulated in disease. As we approach the 75th anniversary of the first experiments in which EVs were unknowingly isolated, it seems right to take stock and look back on how the field started, and has since exploded into its current state. Here we review the early experiments, summarise key findings that have propelled the field, describe the growth of an organised EV community, discuss the current state of the field, and identify key challenges that need to be addressed.

Updating MISEV: Evolving the minimal requirements for studies of extracellular vesicles.

The minimal information for studies of extracellular vesicles (EVs, MISEV) is a field-consensus rigour initiative of the International Society for Extracellular Vesicles (ISEV). The last update to MISEV, MISEV2018, was informed by input from more than 400 scientists and made recommendations in the six broad topics of EV nomenclature, sample collection and pre-processing, EV separation and concentration, characterization, functional studies, and reporting requirements/exceptions. To gather opinions on MISEV and ideas for new updates, the ISEV Board of Directors canvassed previous MISEV authors and society members. Here, we share conclusions that are relevant to the ongoing evolution of the MISEV initiative and other ISEV rigour and standardization efforts.

Synthetic bacterial vesicles combined with tumour extracellular vesicles as cancer immunotherapy.

Bacterial outer membrane vesicles (OMV) have gained attention as a promising new cancer vaccine platform for efficiently provoking immune responses. However, OMV induce severe toxicity by activating the innate immune system. In this study, we applied a simple isolation approach to produce artificial OMV that we have named Synthetic Bacterial Vesicles (SyBV) that do not induce a severe toxic response. We also explored the potential of SyBV as an immunotherapy combined with tumour extracellular vesicles to induce anti-tumour immunity. Bacterial SyBV were produced with high yield by a protocol including lysozyme and high pH treatment, resulting in pure vesicles with very few cytosolic components and no RNA or DNA. These SyBV did not cause systemic pro-inflammatory cytokine responses in mice compared to naturally released OMV. However, SyBV and OMV were similarly effective in activation of mouse bone marrow-derived dendritic cells. Co-immunization with SyBV and melanoma extracellular vesicles elicited tumour regression in melanoma-bearing mice through Th-1 type T cell immunity and balanced antibody production. Also, the immunotherapeutic effect of SyBV was synergistically enhanced by anti-PD-1 inhibitor. Moreover, SyBV displayed significantly greater adjuvant activity than other classical adjuvants. Taken together, these results demonstrate a safe and efficient strategy for eliciting specific anti-tumour responses using immunotherapeutic bacterial SyBV.

[High correlation between the different measures suggests that mortality is relatively correctly reported in Sweden]. / God samstämmighet mellan olika svenska mått på avlidna i covid-19.

Infection fatality ratio (IFR) in covid-19 is highly debated in international and Swedish press. In Sweden, three different estimates have been used to estimate mortality, based on statistics either from the Swedish National Board of Health and Welfare, and the Public Health Agency of Sweden, whereas excess mortality calculated by EuroMOMO. Mortality is based on death certificates, which can be accurate or erroneous, but previous analyses have suggested that over- and underdiagnosis usually even out. EuroMOMO on the other hand reports all-cause mortality compared to an estimated baseline. In view of high correlation between the different measures, we suggest that mortality is relatively correctly reported in Sweden. We discuss IFR internationally  and in Sweden, and suggest that IFR in the Western world is approximately 0.5-1%. However, these numbers will change over time depending on immunity induced by vaccination efforts, but also the potential spread of new virus variants.

Immune-Associated Proteins Are Enriched in Lung Tissue-Derived Extracellular Vesicles during Allergen-Induced Eosinophilic Airway Inflammation.

Studying the proteomes of tissue-derived extracellular vesicles (EVs) can lead to the identification of biomarkers of disease and can provide a better understanding of cell-to-cell communication in both healthy and diseased tissue. The aim of this study was to apply our previously established tissue-derived EV isolation protocol to mouse lungs in order to determine the changes in the proteomes of lung tissue-derived EVs during allergen-induced eosinophilic airway inflammation. A mouse model for allergic airway inflammation was used by sensitizing the mice intraperitoneal with ovalbumin (OVA), and one week after the final sensitization, the mice were challenged intranasal with OVA or PBS. The animals were sacrificed 24 h after the final challenge, and their lungs were removed and sliced into smaller pieces that were incubated in culture media with DNase I and Collagenase D for 30 min at 37 °C. Vesicles were isolated from the medium by ultracentrifugation and bottom-loaded iodixanol density cushions, and the proteomes were determined using quantitative mass spectrometry. More EVs were present in the lungs of the OVA-challenged mice compared to the PBS-challenged control mice. In total, 4510 proteins were quantified in all samples. Among them, over 1000 proteins were significantly altered (fold change >2), with 614 proteins being increased and 425 proteins being decreased in the EVs from OVA-challenged mice compared to EVs from PBS-challenged animals. The associated cellular components and biological processes were analyzed for the altered EV proteins, and the proteins enriched during allergen-induced airway inflammation were mainly associated with gene ontology (GO) terms related to immune responses. In conclusion, EVs can be isolated from mouse lung tissue, and the EVs' proteomes undergo changes in response to allergen-induced airway inflammation. This suggests that the composition of lung-derived EVs is altered in diseases associated with inflammation of the lung, which may have implications in type-2 driven eosinophilic asthma pathogenesis.