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Acute cellular rejection (ACR) remains a major concern after liver transplantation. Predicting and monitoring acute rejection by non-invasive methods are very important for guiding the use of immunosuppressive drugs. Many studies have shown that exosomes and their contents are potential biomarkers for various liver diseases. Here, we identify and validate the role of exosomes and galectin-9 in ACR after liver transplantation. Exosomes were isolated from three sets of paired patients, with and without ACR, and the proteins within the exosomes were isolated and identified. Candidate proteins were then validated using a tissue microarray containing resected liver samples from 73 ACR and 63 non-rejection patients. Finally, protein expression and clinical manifestations were included in Kaplan-Meier survival and Cox regression analyses. Circulating exosomes were isolated from ACR and non-rejection patients and characterized using transmission electron microscopy and western blotting for CD63/CD81. Western blotting experiments revealed higher levels of galectin-9 protein in circulating exosomes from ACR recipients. Immunohistochemical analysis of the tissue microarray showed that the expression of galectin-9 in resected liver was significantly higher in the ACR group than in the non-rejection group (P<0.05). Higher levels of galectin-9 expression in resected livers were associated with poorer prognosis (P<0.05). Exosome-derived galectin-9 may be a novel predictor of rejection and prognosis after liver transplantation.

Aim: To isolate and characterize oral extracellular vesicles from gingival crevicular fluid at 11-14 weeks and evaluate their capacity to identify patients at risk of developing gestational diabetes mellitus., Methods: A case-control study was conducted, including patients who developed gestational diabetes mellitus (n = 11) and healthy pregnant controls (n = 23). Obstetric and periodontal histories were recorded at 11-14 weeks of gestation, and samples of gingival crevicular fluid obtained. Extracellular vesicles were isolated from gingival crevicular fluid by ExoQuick. Nanoparticle tracking analysis, ELISA and transmission electron microscopy were used to characterize extracellular vesicles., Results: Total extracellular vesicles isolated from gingival crevicular fluid were significantly higher in patients who developed gestational diabetes mellitus later in pregnancy compared to normoglycemic pregnant women (6.3x109 vs 1.7 x1010, p value = 0.0026), and the concentration of the extracellular vesicles delivered an area under the ROC curve of 0.81. The distribution size of extracellular vesicles obtained using ExoQuick was around 148 ± 57 nm. There were no significant differences in the periodontal status between cases and controls. The exosome transmembrane protein CD63 was also detected in the extracellular vesicles of gingival crevicular fluid., Conclusion: We were able to isolate extracellular vesicles from gingival crevicular fluid using a method that is suitable to be applied in a clinical setting. Our results provide an insight into the potential capacity of first trimester oral extracellular vesicles as early biomarkers for the prediction of gestational diabetes mellitus in pre-symptomatic women., Competing Interests: The authors have declared that no competing interests exist.

Weibel-Palade bodies (WPBs) are secretory granules that contain von Willebrand factor and P-selectin, molecules that regulate hemostasis and inflammation, respectively. The presence of CD63/LAMP3 in the limiting membrane of WPBs has led to their classification as lysosome-related organelles. Many lysosome-related organelles contain intraluminal vesicles (ILVs) enriched in CD63 that are secreted into the extracellular environment during cell activation to mediate intercellular communication. To date, there are no reports that WPBs contain or release ILVs. By light microscopy and live-cell imaging, we show that CD63 is enriched in microdomains within WPBs. Extracellular antibody recycling studies showed that CD63 in WPB microdomains can originate from the plasma membrane. By cryo-electron tomography of frozen-hydrated endothelial cells, we identify internal vesicles as novel structural features of the WPB lumen. By live-cell fluorescence microscopy, we directly observe the exocytotic release of EGFP-CD63 ILVs as discrete particles from individual WPBs. WPB exocytosis provides a novel route for release of ILVs during endothelial cell stimulation., (© 2019 by The American Society of Hematology.)

Cutaneous wounds, a type of soft tissue injury, are difficult to heal in aging. Differentiation, migration, proliferation, and apoptosis of skin cells are identified as key factors during wound healing processes. Mesenchymal stem cells have been documented as possible candidates for wound healing treatment because their use could augment the regenerative capacity of many tissues. However, the effects of exosomes derived from adipose-derived stem cell (ADSC-exos) on cutaneous wound healing remain to be carefully elucidated. In this present study, HaCaT cells were exposed to hydrogen peroxide (H 2 O 2 ) for the establishment of the skin lesion model. Cell Counting Kit-8 assay, migration assay, and flow cytometry assay were conducted to detect the biological function of ADSC-exos in skin lesion model. Finally, the possible mechanism was further investigated using Western blot assay. The successful construction of the skin lesion model was confirmed by results of the enhanced cell apoptosis of HaCaT cells induced by H 2 O 2 , the increased Bax expression and decreased Bcl-2 expression. CD9 and CD63 expression evidenced the existence of ADSC-exos. The results of functional experiments demonstrated that ADSC-exos could prompt cell proliferation and migration of HaCaT cells, and repress cell apoptosis of HaCaT cells. In addition, the activation of Wnt/β-catenin signaling was confirmed by the enhanced expression of β-catenin at the protein level. Collectively, our findings suggest that ADSC-exos play a positive role in cutaneous wound healing possibly via Wnt/β-catenin signaling. Our study may provide new insights into the therapeutic target for cutaneous wound healing., (© 2019 Wiley Periodicals, Inc.)

Objective: Exosomes contain valuable biomarkers for many diseases. Tragically, standardized isolation methods and subsequent characterization criteria for exosomes remain limited. Therefore, we developed a new exosome isolation method, termed rinsing separation, and compared its advantages and weaknesses relative to the existing ultracentrifugation and ExoQuick precipitation methods., Materials and Methods: Rinsing separation utilizes heparin and glutaraldehyde as a fixative to isolate exosomes, and was developed using the culture supernatant from mesenchymal stem cells (MSCs). The isolated exosomes were characterized and compared by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blot., Results: Consistent with known exosome parameters, exosomes isolated using each method ranged in size from 30 to 150 nm and demonstrated the characteristic cup-shaped morphology. Moreover, the exosome markers CD63 and TSG101 were observed in the lysate of all exosome samples that were isolated using each method. Several advantages and drawbacks were noted for each exosome isolation method. Most notably, ultracentrifugation resulted in fewer, but highly pure, exosomes, and samples generated using the ExoQuick precipitation method contained the most contaminating debris. Samples obtained using pour rinsing separation method represented an amalgam of these two fractions, but were isolated in significantly less time., Conclusions: In this study, we propose rinsing separation as a new method of isolating exosomes. This method is convenient, and the resulting exosomes are highly pure. Moreover, rinsing separation offers time- and cost-efficiency advantages, making it a promising approach for exosome isolation for clinical applications.

Tumor-derived exosomes have emerged as promising cancer biomarkers and attracted increasing interest in non-invasive cancer diagnosis and treatment monitoring. However, the identification and quantification of exosomes in clinical samples such as blood remains challenging due to the difficulty in trade-off between recognition specificity and isolation efficiency. Here we have developed an aptamer-based fluorescence polarization assay for exosome quantification, which is a separation-free, amplification-free and sensitive approach enabling direct quantification of exosomes in human plasma. While the key specificity of this assay is based on the aptamer's inherent affinity to membrane proteins on exosomes, exosomes' inherent huge mass/volume acts as mass-based fluorescence polarization amplifier. Our assay allows quantitative analysis of exosomes in the range of 5 × 10 2 to 5 × 10 5 particles per μL with a detect limitation of 500 particles per μL for the cell line deprived exosomes. The total assay time is about 30 min with just one mix-and-read step to achieve high sensitivity. We have also demonstrated quantification of exosomes from lung cancer patients and healthy donors in clinical samples. This work describes a new and simple liquid biopsy assay to directly detect exosomes in the biological matrix, which facilitates cancer diagnosis and therapy monitoring.

Extracellular vesicles (EVs) play important roles in cell-cell communication by transferring cargo proteins and nucleic acids between cells. Due to their small size (50-150 nm) and low density, rapid capture and nondestructive release of EVs remains a technical challenge which significantly hinders study of their biofunction and biomedical application. To address this issue, we designed a DNA aptamer-based system that enabled rapid capture and nondestructive release of EVs in 90 min with similar isolation efficiency to ultracentrifugation (around 78%). Moreover, because we designed a DNA structure-switch process to release the exosomes, the isolated EVs maintained high bioactivity in cell-uptake assay and wound-healing assays. Using this method, we can isolate EVs from clinical samples and found that the amount of MUC1 positive EVs in breast cancer patient plasma sample is significantly higher than that in healthy donors. This DNA aptamer-based magnetic isolation strategy can be potentially applied for the biofunction study of EVs and EV-based point-of-care clinical tests.

Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be ∼175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.

The extracellular protein tissue inhibitor of metalloproteinase (TIMP)-1 is both a matrix metalloproteinase (MMP) inhibitor and a trophic factor. Mice lacking TIMP-1 exhibit delayed central nervous system myelination during postnatal development and impaired remyelination following immune-mediated injury in adulthood. We have previously determined that the trophic action of TIMP-1 on oligodendrocyte progenitor cells (OPCs) to mature into oligodendrocytes is independent of its MMP inhibitory function. However, the mechanism by which TIMP-1 promotes OPC differentiation is not known. To address this gap in our understanding, herein, we report that TIMP-1 signals via a CD63/β1-integrin receptor complex to activate Akt (protein kinase B) to promote β-catenin signaling in OPCs. The regulation of β-catenin by TIMP-1 to promote OPC differentiation was counteracted, but not abrogated, by canonical signaling evoked by Wnt7a. These data provide a previously uncharacterized trophic action of TIMP-1 to regulate oligodendrocyte maturation via a CD63/β1-integrin/Akt pathway mechanism. These findings contribute to our emerging understanding on the role of TIMP-1 as a growth factor expressed to promote CNS myelination during development and induced in the adult to promote myelin repair.

Understanding the biological mechanisms behind aging, lifespan and healthspan is becoming increasingly important as the proportion of the world's population over the age of 65 grows, along with the cost and complexity of their care. BigData oriented approaches and analysis methods enable current and future bio-gerontologists to synthesize, distill and interpret vast, heterogeneous data from functional genomics studies of aging. GeneWeaver is an analysis system for integration of data that allows investigators to store, search, and analyze immense amounts of data including user-submitted experimental data, data from primary publications, and data in other databases. Aging related genome-wide gene sets from primary publications were curated into this system in concert with data from other model-organism and aging-specific databases, and applied to several questions in genrontology using. For example, we identified Cd63 as a frequently represented gene among aging-related genome-wide results. To evaluate the role of Cd63 in aging, we performed RNAi knockdown of the C. elegans ortholog, tsp-7, demonstrating that this manipulation is capable of extending lifespan. The tools in GeneWeaver enable aging researchers to make new discoveries into the associations between the genes, normal biological processes, and diseases that affect aging, healthspan, and lifespan., Competing Interests: The authors have declared that no competing interests exist.

The presence of mast cells in human atherosclerotic plaques has been associated with adverse cardiovascular events. Mast cell activation, through the classical antigen sensitized-IgE binding to their characteristic Fcε-receptor, causes the release of their cytoplasmic granules. These granules are filled with neutral proteases such as tryptase, but also with histamine and pro-inflammatory mediators. Mast cells accumulate in high numbers within human atherosclerotic tissue, particularly in the shoulder region of the plaque. These findings are largely based on immunohistochemistry, which does not allow for the extensive characterization of these mast cells and of the local mast cell activation mechanisms. In this study, we thus aimed to develop a new flow-cytometry based methodology in order to analyze mast cells in human atherosclerosis. We enzymatically digested 22 human plaque samples, collected after femoral and carotid endarterectomy surgery, after which we prepared a single cell suspension for flow cytometry. We were able to identify a specific mast cell population expressing both CD117 and the FcεR, and observed that most of the intraplaque mast cells were activated based on their CD63 protein expression. Furthermore, most of the activated mast cells had IgE fragments bound on their surface, while another fraction showed IgE-independent activation. In conclusion, we are able to distinguish a clear mast cell population in human atherosclerotic plaques, and this study establishes a strong relationship between the presence of IgE and the activation of mast cells in advanced atherosclerosis. Our data pave the way for potential therapeutic intervention through targeting IgE-mediated actions in human atherosclerosis., Competing Interests: The authors declare no conflict of interest.

Gut microbiota plays a pivotal role in not only the gastrointestinal (GI) immune system but also GI motility and metabolism. Antibiotic treatments are likely to affect the gut flora and GI immune system, subsequently disturbing GI motility and body metabolism. In the present study, we investigated antibiotic‑induced alterations of body metabolism and GI motility in association with the macrophage profile in the colon. Specific pathogen‑free (SPF) mice (ICR; 6 weeks old; female) were orally administered vancomycin (0.2 mg/ml) in drinking water for 5 weeks, and subsequent changes in pathophysiology were observed. The expression of CD80 and CD163 was examined by immunohistochemistry and the expression of cytokines in colonic tissues was evaluated by reverse transcription‑quantitative polymerase chain reaction. The gastrointestinal transit time (GITT) was measured by administration of carmine red (6% w/v) solution. In the vancomycin‑treated SPF mice, significant increases in body weight, cecum weight and GITT were observed compared with the controls. The number of CD80‑positive M1 macrophages and the expression of interferon‑γ and interleukin‑12 were significantly increased, whereas, the numbers of CD163‑positive M2 macrophages in the mucosal and muscular layers were decreased in the colon of vancomycin‑treated mice. GITT was positively correlated with the number of CD80‑positive M1 macrophages in the colonic mucosa; however, was negatively correlated with the number of CD163‑positive M2 macrophages in the mucosal and muscular layers. Therefore, it was suggested that antibiotic treatment affects body metabolism and GI motility, accompanied by alterations in macrophage polarization and cytokine profiles in the colon.

Extracellular vesicles (EV) that are released by immune cells are studied intensively for their functions in immune regulation and are scrutinized for their potential in human immunotherapy, for example against cancer. In our search for signals that stimulate the release of functional EV by dendritic cells we observed that LPS-activated human monocyte-derived dendritic cells (moDC) changed their morphological characteristics upon contact with non-cognate activated bystander T-cells, while non-activated bystander T-cells had no effect. Exposure to activated bystander T-cells also stimulated the release of EV-associated proteins by moDC, particularly CD63, and ICAM-1, although the extent of stimulation varied between individual donors. Stimulation of moDC with activated bystander T-cells also increased the release of EV-associated miR155, which is a known central modulator of T-cell responses. Functionally, we observed that EV from moDC that were licensed by activated bystander T-cells exhibited a capacity for antigen-specific T-cell activation. Taken together, these results suggest that non-cognatei interactions between DC and bystander T-cells modulates third party antigen-specific T-cell responses via EV.

Despite intense interests in developing blood measurements of Alzheimer's disease (AD), the progress has been confounded by limited sensitivity and poor correlation to brain pathology. Here, we present a dedicated analytical platform for measuring different populations of circulating amyloid β (Aβ) proteins - exosome-bound vs. unbound - directly from blood. The technology, termed amplified plasmonic exosome (APEX), leverages in situ enzymatic conversion of localized optical deposits and double-layered plasmonic nanostructures to enable sensitive, multiplexed population analysis. It demonstrates superior sensitivity (~200 exosomes), and enables diverse target co-localization in exosomes. Employing the platform, we find that prefibrillar Aβ aggregates preferentially bind with exosomes. We thus define a population of Aβ as exosome-bound (Aβ42+ CD63+) and measure its abundance directly from AD and control blood samples. As compared to the unbound or total circulating Aβ, the exosome-bound Aβ measurement could better reflect PET imaging of brain amyloid plaques and differentiate various clinical groups.

Exosomes are micro messengers encapsulating RNA, DNA, and proteins for intercellular communication associated with various physiological and pathological reactions. Several viral infection processes have been reported to pertain to exosomal pathways. However, because of the difficulty in obtaining avian-sourced exosomes, avian virus-related exosomes are scarcely investigated. In this study, we developed a protein A/G-correlated method and successfully obtained the Newcastle disease virus-related exosome (NDV Ex). These exosomes promoted NDV propagation, proven by both GW4869-mediated deprivation and exosomal supplementation. Viral structural proteins NP and F were detected in the NDV Ex and further investigation indicated that the NP protein can be transferred to DF-1 cells through exosomes. The intracellular NP protein exhibited viral replication-promoting and cytokine-suppressing abilities. Therefore, NDV infection produces exosomes, which transfer viral NP protein and promote NDV infection, emphasizing the importance of exosomes in an NDV infection., (Copyright © 2018. Published by Elsevier Ltd.)

The BRAF inhibitor PLX4032 is effective in treating BRAF-mutated melanoma; however, because drug resistance develops in most cases, it is critical to develop a new strategy for inhibiting drug-resistant melanoma growth. The melanoma-associated membrane glycoprotein CD63 is involved in cell proliferation and metastasis. Here, we found that cell surface CD63 suppresses the proliferation of human melanoma cells and PLX4032-resistant cells. Endogenous CD63 protein levels were negatively correlated with PLX4032 resistance of human melanoma cell lines. CD63 overexpression in these cells, in which endogenous CD63 levels are low, suppressed cell proliferation under PLX4032 treatment. The cell surface levels and average molecular mass of CD63 were increased with PLX4032 treatment because of the up-regulated polylactosamine modification caused by induced β1,3- N-acetylglucosaminyltransferase 2 expression, which is involved in polylactosamine synthesis. Forced cell surface localization of CD63 led to reduced melanoma cell proliferation without PLX4032 treatment. CD63 overexpression in PLX4032-resistant cells, in which CD63 levels were lower and cell surface polylactosamine levels were higher than those in parental cells, effectively suppressed proliferation. Our study shows the potential of CD63 to sensitize melanoma cells to PLX4032 and to reduce the proliferation of PLX4032-resistant cells.-Kudo, K., Yoneda, A., Sakiyama, D., Kojima, K., Miyaji, T., Yamazaki, M., Yaita, S., Hyodo, T., Satow, R., Fukami, K. Cell surface CD63 increased by up-regulated polylactosamine modification sensitizes human melanoma cells to the BRAF inhibitor PLX4032.

We have proposed a new exosomes-zirconium-liposomes sandwich structure to detect exosomes by using zirconium-phosphate coordination chemistry. The combined use of the intrinsic property of phosphate in both exosomes and liposomes as well as zirconium ions can endow the method lower cost, no modified label, simplicity and high efficiency.

Background: As is known, exosomes play an important role in promoting progression of cancers by increasing its invasive potential. The aim of this study was to evaluate the levels of tetraspanine-associated (ADAM-10) and tetraspanine-nonassociated proteases (20S proteasomes) in exosomes from culture medium, plasma exosomes of patients with breast tumors and plasma and ascites of ovarian tumor patients. Methods: MCF-7 and SVO-3 culture mediums and blood samples from healthy females (n = 30, HFs), patients with diffuse dyshormonal dysplasia of the breast (n=28, BBTPs), breast cancer patients (n=32, BCPs), borderline ovarian tumor patients (n=20, BOTPs) and blood and ascites samples ovarian cancer patients (n=35, OCPs) were included in the study. Exosomes from plasma, ascites and culture mediums were isolated and characterized in according to Extracellular Vesicles Society. The expression levels of 20S proteasome and ADAM-10 in exosomes were determined using flow cytometry and western blot analysis, correspondingly. Results: The subpopulation composition of the exosomes from MCF-7 culture medium and from blood plasma of HFs and breast diseases patients is similar, however CD9/CD24 subpopulation significantly increased at cell supernatant. The similar results was obtained for exosomes from SVO-3 medium and blood plasma and ascites of ovary tumor patients, but CD9/CD24 subpopulation significantly decreased at cells and illness samples, however CD63/CD24 exosomes increased significantly from cell supernatant. 20S proteasome level is significantly increased in exosomes from MCF-7 and SVO-3 culture medium, breast tumor patients and OCPs plasma in comparison to HUVEC culture medium and HFs plasma samples. At CD9-positive exosomes from BCPs plasma and MCF-7 was reveal a high expression of ADAM-10 and low expression is from BBDPs plasma and ovarian tumor patients plasma/ ascites samples. Exosomes from ascites OCP had high expression of ADAM-10 in the CD24-positive subpopulation. Conclusion: Breast and ovarian cancer development is connected with functioning of immune proteasome forms in plasma and ascites exosomes, while increased ADAM10 expression at CD9-positive exosome was associated with breast cancer and at CD24-positive subpopulation – with ovarian cancer. Obtained data confirm role of exosomal proteases in tumor progression., (Creative Commons Attribution License)

The interferon-induced transmembrane proteins (IFITMs) are a family of highly related antiviral factors that affect numerous viruses at two steps: in target cells by sequestering incoming viruses in endosomes and in producing cells by leading to the production of virions that package IFITMs and exhibit decreased infectivity. While most studies have focused on the former, little is known about the regulation of the negative imprinting of virion particle infectivity by IFITMs and about its relationship with target cell protection. Using a panel of IFITM3 mutants against HIV-1, we have explored these issues as well as others related to the biology of IFITM3, in particular virion packaging, stability, the relation to CD63/multivesicular bodies (MVBs), the modulation of cholesterol levels, and the relationship between negative imprinting of virions and target cell protection. The results that we have obtained exclude a role for cholesterol and indicate that CD63 accumulation does not directly relate to an antiviral behavior. We have defined regions that modulate the two antiviral properties of IFITM3 as well as novel domains that modulate protein stability and that, in so doing, influence the extent of its packaging into virions. The results that we have obtained, however, indicate that, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for negative imprinting. Finally, while most mutations concomitantly affect target cell protection and negative imprinting, a region in the C-terminal domain (CTD) exhibits a differential behavior, potentially highlighting the regulatory role that this domain may play in the two antiviral activities of IFITM3. IMPORTANCE IFITM proteins have been associated with the sequestration of incoming virions in endosomes (target cell protection) and with the production of virion particles that incorporate IFITMs and exhibit decreased infectivity (negative imprinting of virion infectivity). How the latter is regulated and whether these two antiviral properties are related remain unknown. By examining the behavior of a large panel of IFITM3 mutants against HIV-1, we determined that IFITM3 mutants are essentially packaged into virions proportionally to their intracellular levels of expression. However, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for the antiviral effects. Most mutations were found to concomitantly affect both antiviral properties of IFITM3, but one CTD mutant exhibited a divergent behavior, possibly highlighting a novel regulatory role for this domain. These findings thus advance our comprehension of how this class of broad antiviral restriction factors acts., (Copyright © 2019 American Society for Microbiology.)

CD63 has been suggested to participate in tumorigenesis, invasion, and metastasis. In this study, we aimed to investigate the prognostic significance of CD63 expression in non-small cell lung cancer (NSCLC). CD63 expression was evaluated in 133 cases of NSCLC via immunohistochemical staining using tissue microarray blocks. We assessed the relationship between CD63 expression and clinicopathological characteristics, as well as the prognostic significance of CD63 expression in NSCLC. CD63 expression was significantly correlated with patient gender (p < 0.001), smoking history (p = 0.020), histologic type (p < 0.001), tumour stage (p = 0.016), lymph node metastasis (p = 0.034), and TNM stage (p = 0.007). A multivariate Cox proportional hazards regression analysis determined low CD63 expression to be an independent factor for unfavourable disease-free survival (DFS) (HR = 2.043, 95% CI: 1.035-4.035, p = 0.040), and Kaplan-Meier analysis indicated that the low CD63 expression group showed significantly lower DFS than the high CD63 expression group of patients with NSCLC (p = 0.019). Low CD63 expression might be an unfavourable prognostic factor in patients with NSCLC.

Platelet transfusion has become essential therapy in modern medicine. Although the clinical advantage of platelet transfusion has been well established, adverse reactions upon transfusion, especially transmission of bacterial infection, still represent a major challenge. While bacterial contamination is favored by the storage of platelets at room temperature, cold storage may represent a solution for this important clinical issue. In this study, we aimed to clarify whether plasma has protective or detrimental effects on cold-stored platelets. We investigated the impact of different residual plasma contents in apheresis-derived platelet concentrates, stored at 4°C or room temperature, on platelet function and survival. We found that platelets stored at 4°C have higher expression of apoptosis marker compared to platelets stored at room temperature, leading to accelerated clearance from the circulation in a humanized animal model. While cold-induced apoptosis was independent of the residual plasma concentration, cold storage was associated with better adhesive properties and higher response to activators. Interestingly, delta (δ) granule-related functions, such as ADP-mediated aggregation and CD63 release, were better preserved at 4°C, especially in 100% plasma. An extended study to assess cold-stored platelet concentrates produced under standard care Good Manufacturing Practice conditions showed that platelet function, metabolism and integrity were better compared to those stored at room temperature. Taken together, our results show that residual plasma concentration does not have a cardinal impact on the cold storage lesions of apheresis-derived platelet concentrates and indicate that the current generation of additive solutions represent suitable substitutes for plasma to store platelets at 4°C., (Copyright© 2019 Ferrata Storti Foundation.)

Cystic fibrosis (CF) is the most common life-shortening genetic disease and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Several current therapies aim at improving availability and/or function of the mutant CFTR proteins. The combination therapeutic lumacaftor/ivacaftor (Orkambi, luma/iva) partially corrects folding and potentiates CFTR function impaired by the F508del mutation. Despite the potential for clinical benefit, a substantial number of patients discontinue treatment due to intolerable adverse effects. The aim of the present study is to identify differences between individuals who continued treatment and those who discontinued due to adverse respiratory effects to potentially inform treatment decisions. Clinical data from the year prior to treatment initiation were analyzed from 82 patients homozygous for the F508del mutation treated at the Colorado Adult CF Program. Blood samples were collected from 30 of these subjects before initiation of treatment to examine expression of circulating leukocyte surface antigens and cytokines. Clinical and demographic characteristics were analyzed along with inflammatory markers to determine biomarkers of drug discontinuation. The use of oral prednisone and/or nasal budesonide in the year prior to luma/iva initiation was more prevalent in CF subjects who did not tolerate luma/iva (82% vs. 43%). Increased age, but not gender or initial lung function, was associated with higher probability of discontinuing treatment due to side effects overall. Worse lung function (lower ppFEV1, ppFEF25-75 ≤ 60%) was associated with higher incidence of discontinuing treatment due to pulmonary adverse effects. In a nested cohort of patients, increased surface levels of CXCR2 on CD14+CD16- monocytes were associated with discontinuation. Overall, the patients who tolerated luma/iva were distinguishable from those who did not tolerate the drug based on clinical and cellular markers obtained prior to treatment initiation., Competing Interests: DPN has received grant funding from Vertex Pharmaceuticals. JLT has received grant funding from Vertex Pharmaceuticals, Gilead, Bayer, Proteostasis, and Novalis, reports personal fees from participation in advisory boards for Vertex Pharmaceuticals, Gilead, Novartis, Genentech and Proteostasis, and reports personal fees for speaking on behalf of Vertex Pharmaceuticals and Gilead. Commercial funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Human mesenchymal stem cells were exposed to diabetic sera for 7 days. Cell viability and apoptosis rate were detected by MTT and flow cytometry assays. The expression of key genes such as CD63, Alix, Rab27a, Rab27b, and Rab8b was monitored by real-time PCR. We also measured acetylcholinesterase activity and size and zeta potential of exosomes in the supernatant form diabetic cells and control. The cellular distribution of CD63 was shown by immunofluorescence imaging and western blotting. Any changes in the ultrastructure of cells were visualized by electron microscopy. Data showed a slight decrease in survival rate and an increased apoptosis in diabetic cells as compared to control (p < 0.05). By exposing cells to diabetic sera, a significant increase in the level of all genes CD63, Alix, Rab27a, Rab27b, and Rab8b was observed (p < 0.05). Flow cytometry analysis and immunofluorescence imaging confirmed increasing CD63 protein content upon treatment with diabetic sera (p < 0.05). We found an enhanced acetylcholinesterase activity in a diabetic condition which coincided with the increasing size of exosomes and decrease in zeta potential (p < 0.05). The fatty acid profile was not significantly affected by diabetic sera. Ultrastructural examination detected more accumulated cytoplasmic lipid vacuoles in diabetic cells.

Hypoxia is the most common characteristic of solid tumours driving cancer metastasis. Cancer cells release exosomes with various functions into the tumour microenvironment during cancer progression. However, the roles and associated mechanisms of hypoxic colorectal cancer (CRC) cell-derived exosomes remain poorly understood. Here, we found that exosomes secreted by hypoxic CRC cells promoted the migration and invasion abilities of normoxic CRC cells. Inhibition of exosome secretion by GW4869 reduced hypoxic exosome-mediated migration and invasion of normoxic CRC cells. Furthermore, we found that these hypoxic exosomes contained Wnt4 depending on HIF1α. Exosomal Wnt4 mediated hypoxic exosome-mediated migration and invasion of normoxic CRC cells. Moreover, exosomal Wnt4 enhanced β-catenin translocation to the nucleus in normoxic CRC cells. The activation of β-catenin signalling was important for the migration and invasion of normoxic CRC cells, which was eliminated by treatment with the β-catenin inhibitor ICG-001. Taken together, the results of our study indicate that hypoxia may stimulate tumour cells to release Wnt4-rich exosomes that are delivered to normoxic cells to enhance prometastatic behaviours, which might provide new targets for CRC treatment., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Exosomes as nanosized biomarkers hold great potential for the diagnosis of cancer. However, the low concentration of cancer-derived exosomes present in biofluids makes early diagnosis strenuous. Here, we developed a fluorescent biosensing platform, namely a dual signal amplification, for the ultrasensitive detection of leukemia cell-derived exosomes. The protocol consists of three steps: first, leukemia-derived exosomes containing CD63 and nucleolin were captured by anti-CD63 antibody modified magnetic bead conjugates (MB-CD63); then, a DNA primer comprising a nucleolin-recognition aptamer (AS1411) was applied to bind the exosomes which further initiated a rolling circle amplification (RCA) reaction to generate many repeat sequences for hybridization with gold nanoparticle (GNP)-DNA-fluorescent dye (FAM) conjugates (GNP-DNA-FAM); finally, nicking endonuclease (Nb·BbvCI) assisted target recycling was introduced. As a result, FAM was released from GNP-DNA-FAM conjugates, transformed from the quenching state to the emission state and thus fluorescence signals continuously accumulated. With this dual signal amplification platform, as low as 1 × 102 particles per μL exosomes could be detected. Furthermore, we have successfully applied this method for the detection of exosomes in spiked serum samples, indicating a promising tool for clinical application.

Exosomes, as novel noninvasive biomarkers for disease prediction and diagnosis, have shown fascinating prospects in monitoring cancer-linked public health issues. Herein, a unique Cy3 labeled CD63 aptamer (Cy3-CD63 aptamer)/Ti 3 C 2 MXenes nanocomplex was constructed as a self-standard ratiometric fluorescence resonance energy transfer (FRET) nanoprobe for quantitative detection of exosomes. The Cy3-CD63 aptamer can be selectively adsorbed onto the Ti 3 C 2 MXene nanosheets by hydrogen bond and metal chelate interaction between the aptamer and MXenes, and the fluorescence signal from Cy3-CD63 aptamer was quenched quickly owing to the FRET between the Cy3 and MXenes. The fluorescence of Cy3 greatly recovered after the addition of the exosomes which can specifically combine with the aptamer and release from the surface of Ti 3 C 2 MXenes due to the high affinity between the aptamer and CD63 protein on exosome surface. Meanwhile, the self-fluorescence signal of MXenes in the whole process showed little change, which can be used as a standard reference. Based on the self-standard turn-on FRET biosensing platform the detection limit of exosome was determined as 1.4 × 10 3 particles mL -1 , which was over 1000× lower than that of conventional ELISA method. This fluorescence sensor can also be used for the identification of multiple biomarkers on the exosome surface and different kinds of exosomes, combining with the fluorescent confocal scanning microscope image. The proposed strategy not only provides a universal nanoplatform for exosomes, but also can be extensively expanded to multiple biomarkers detection, which may promise the prospect of MXenes as robust candidates in biological fields.

The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detection and reproducible protein quantitation, which is a considerable advantage for biomarker study of urinary extracellular vesicles. We developed a SWATH-MS workflow with a curated spectral library of 1,145 targets. Application of the workflow across nine replicates of three sample types (exosome-like vesicles (ELVs), microvesicles (MVs) and urine proteins (UPs)) resulted in the quantitation of 888 proteins at FDR <1%. The median-coefficient of variation of the 888 proteins in the ELV sample was 7.7%, indicating excellent reproducibility. Data analysis showed common exosome markers, (i.e. CD9, CD63, ALIX, TSG101 and HSP70) were enriched in urinary ELVs as compared to MVs and UPs. The use of a multiplex biomarker screening assay focused on ELVs was investigated, and perspectives in future applications are discussed.

Intracellular trafficking is a tightly regulated cellular process, mediated in part by Rab GTPases and their corresponding effector proteins. Viruses have evolved mechanisms to hijack these processes to promote their lifecycles. Here we describe a mechanism by which cleavage of the Rab7 adaptor protein, RILP (Rab interacting lysosomal protein) is induced by viral infection. We report that RILP is directly cleaved by caspase-1 and we have identified a novel caspase-1 recognition site at aspartic acid 75 within the RILP sequence. Alanine substitution at D75 blocks caspase-1-mediated RILP cleavage. Full-length RILP localizes in a tight vesicular structure near the perinuclear region while the cleaved form of RILP re-distributes throughout the cytoplasm. However, cleavage alone was insufficient to re-localize RILP to the cellular periphery and re-localization required specific phosphorylation events near the caspase-1 recognition site. The combination of cleavage and phosphorylation were both needed for release from the dynein component p150 Glued and redistribution of CD63 +ve intracellular vesicles., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lysosomes are not merely degradative organelles but play a central role in nutrient sensing, metabolism and cell-growth regulation. Our ability to study their function in living cells strictly relies on the use of lysosome-specific fluorescent probes tailored to optical microscopy applications. Still, no report thus far quantitatively analyzed the effect of labeling strategies/procedures on lysosome properties in live cells. We tackle this issue by a recently developed spatiotemporal fluctuation spectroscopy strategy that extracts structural (size) and dynamic (diffusion) properties directly from imaging, with no a-priori knowledge of the system. We highlight hitherto neglected alterations of lysosome properties upon labeling. In particular, we demonstrate that Lipofectamine reagents, used to transiently express lysosome markers fused to fluorescent proteins (FPs) (e.g. LAMP1-FP or CD63-FP), irreversibly alter the organelle structural identity, inducing a ∼2-fold increase of lysosome average size. The organelle structural identity is preserved, instead, if electroporation or Effectene are used as transfection strategies, provided that the expression levels of the recombinant protein marker are kept low. This latter condition can be achieved also by generating cell lines stably expressing the desired FP-tagged marker. Reported results call into question the interpretation of a massive amount of data collected so far using fluorescent protein markers and suggest useful guidelines for future studies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Proton pump inhibitors (PPIs) are commonly used to treat gastrointestinal diseases. Incidence of hypersensitivity reactions to PPIs has risen, likely because of increased consumption. Their diagnosis is difficult, with skin tests (STs) presenting low sensitivity, making it necessary to perform drug provocation tests (DPTs). The value of in vitro tests for the diagnosis of immediate reaction to PPI is unclear., Objective: To analyze the diagnostic value of the basophil activation test (BAT) in a group of patients diagnosed with immediate allergy to omeprazole., Methods: The study included 42 patients with confirmed immediate allergic reactions to omeprazole confirmed by positive ST results or DPT results and 22 age- and sex-matched subjects tolerant to PPIs. BAT was performed with omeprazole, pantoprazole, and lansoprazole using CD63 and CD203c as activation markers., Results: ST sensitivity was 66.7% with a specificity of 100%. BAT using CD63 with a stimulation index of more than 2 as positive revealed a sensitivity of 73.8%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 66.7%. BAT was positive in 57.1% of patients with negative ST result, and thus by combining ST and BAT we can correctly diagnose 85.7% of patients with immediate allergy to omeprazole., Conclusion: BAT represents a complementary tool for inclusion in the allergological workup for patients allergic to omeprazole. When combined with ST, it can be of value to guide the clinician as to whether to perform a DPT., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Circulating exosomes from prostate cancer (PCa) patients undergoing radiotherapy are attractive candidate biomarkers for monitoring treatment response. Multiple workflows for isolation and content characterization of exosomes in biofluids have been attempted. We report a protocol to isolate and characterize exosomal miRNAs content and assess radiation-induced changes., Methods: In this pilot study, we performed targeted exosomal miRNA profiling of 25 serum samples obtained from PCa patients with intermediate- and high-risk disease treated with curative radiotherapy (RT), and controls. Post-treatment blood samples were collected at least 28 days after radiation therapy as a paired follow-up sample. The complete workflow consisted of two phases: I) filtration and polyethylene glycol salt precipitation phase which enriched particles below 200 nm in size followed by characterization using electron microscopy, and II) flow cytometry. Finally, miRNA expression analysis between untreated and treated patient samples was performed using RNA extraction kit, and qRT-PCR., Results: In our preliminary data, 1 ml of serum from PCa patients showed higher exosomal concentration (3.68E+10) compared to controls (6.07E+08). The overall expression of exosomes after RT was found to be higher compared to untreated samples; the median value changed from 3.68E+10 to 5.40E+10; p = 0.52. Using electron microscopy, we were able to visualize cup-shaped vesicles with morphology and size compatible with exosomes. The bead-based flow cytometry showed positivity for exosomal tetraspanins surface markers CD63 and CD9. All five miRNAs (hsa-let-7a-5p, hsa-miR-141-3p, hsa-miR-145-5p, hsa-miR-21-5p, hsa-miR-99b-5p) have been identified in exosomes. Despite overall changes in hsa-let-7a-5p expression after radiation, the difference was significant only in the high-risk group (p = 0.037). In addition, the radiation response to hsa-miR-21-5p was elevated in the high-risk group compared to the intermediate group (p = 0.036)., Conclusions: Herewith, we demonstrated a protocol for isolation of serum exosomes and exosomal miRNA amplification. The recovery of exosomal miRNAs and their differential expression after radiation treatment suggests promising biomarker potential that requires further investigation in larger patient cohorts.

Cancer cells secrete copious amounts of exosomes, and elevated intracellular Ca 2+ is critical for tumor progression and metastasis, but the underlying cellular mechanisms are unknown. Munc13-4 is a Ca 2+ -dependent SNAP receptor- and Rab-binding protein required for Ca 2+ -dependent membrane fusion. Here we show that acute elevation of Ca 2+ in cancer cells stimulated a fivefold increase in CD63 + , CD9 + , and ALIX + exosome release that was eliminated by Munc13-4 knockdown and not restored by Ca 2+ binding-deficient Munc13-4 mutants. Direct imaging of CD63-pHluorin exosome release confirmed its Munc13-4 dependence. Depletion of Munc13-4 in highly aggressive breast carcinoma MDA-MB-231 cells reduced the size of CD63 + multivesicular bodies (MVBs), indicating a role for Munc13-4 in MVB maturation. Munc13-4 used a Rab11-dependent trafficking pathway to generate MVBs competent for exosome release. Membrane type 1 matrix metalloproteinase trafficking to MVBs by a Rab11-dependent pathway was also Munc13-4 dependent, and Munc13-4 depletion reduced extracellular matrix degradation. These studies identify a novel Ca 2+ - and Munc13-4-dependent pathway that underlies increased exosome release by cancer cells., (© 2018 Messenger et al.)

The exploration of effective spinal cord injury (SCI) healing still remain a great challenge due to the high morbidity, complex pathology and unclear targets. Human umbilical cord mesenchymal stem cells (hucMSC) play an important role in tissue regeneration. However, transplanting stem cells has a potential risk of teratogenicity. Recent studies have suggested that exosomes secreted by stem cells may contribute to tissue injury repair. We hypothesized that the application of hucMSC derived exosomes may be a potential way for SCI treatment. Our studies showed the hucMSC derived exosomes with a mean particle size of 70 nm could effectively trigger the bone marrow derived macrophage (BMDM) polarization from M1 to a M2 phenotype. In vivo studies demonstrated that the hucMSC derived exosomes could improve the functional recovery after SCI through down-regulation of the inflammatory cytokines, such as TNF-α, MIP-1α, IL-6 and IFN-γ. Collectively, our findings indicated that hucMSC derived exosomes could facilitate spinal cord injury healing via attenuating the inflammation of the injury region. Our results provided a new perspective and therapeutic strategy for the use of hucMSC derived exosomes in soft tissue repair., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The distinct role of low-level laser irradiation (LLLI) on endothelial exosome biogenesis remains unclear. We hypothesize that laser irradiation of high dose in human endothelial cells (ECs) contributes to the modulation of exosome biogenesis via Wnt signaling pathway. When human ECs were treated with LLLI at a power density of 80 J/cm 2 , the survival rate reduced. The potential of irradiated cells to release exosomes was increased significantly by expressing genes CD63, Alix, Rab27a, and b. This occurrence coincided with an enhanced acetylcholine esterase activity, pseudopodia formation, and reduced zeta potential value 24 h post-irradiation. Western blotting showed the induction of LC3 and reduced level of P62, confirming autophagy response. Flow cytometry and electron microscopy analyses revealed the health status of the mitochondrial function indicated by normal ΔΨ activity without any changes in the transcription level of PINK1 and Optineurin. When cells exposed to high power laser irradiation, p-Akt/Akt ratio and in vitro tubulogenesis capacity were blunted. PCR array and bioinformatics analyses showed the induction of transcription factors promoting Wnt signaling pathways and GTPase activity. Thus, LLLI at high power intensity increased exosome biogenesis by the induction of autophagy and Wnt signaling. LLLI at high power intensity increases exosome biogenesis by engaging the transcription factors related to Wnt signaling and autophagy stimulate.

Exosomes are circulating nanovesicular carriers of macromolecules, increasingly used for diagnostics and therapeutics. The ability to load and target patient-derived exosomes without altering exosomal surfaces is key to unlocking their therapeutic potential. We demonstrate that a peptide (CP05) identified by phage display enables targeting, cargo loading, and capture of exosomes from diverse origins, including patient-derived exosomes, through binding to CD63-an exosomal surface protein. Systemic administration of exosomes loaded with CP05-modified, dystrophin splice-correcting phosphorodiamidate morpholino oligomer (EXO PMO ) increased dystrophin protein 18-fold in quadriceps of dystrophin-deficient mdx mice compared to CP05-PMO. Loading CP05-muscle-targeting peptide on EXO PMO further increased dystrophin expression in muscle with functional improvement without any detectable toxicity. Our study demonstrates that an exosomal anchor peptide enables direct, effective functionalization and capture of exosomes, thus providing a tool for exosome engineering, probing gene function in vivo, and targeted therapeutic drug delivery., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Introduction: T cell and NK cell cytotoxicity can be mediated via the perforin/granzyme system and Fas Ligand (FasL, CD178). FasL is synthesized as a type II transmembrane protein that binds its cognate receptor Fas (CD95). Membrane-bound FasL is expressed on the plasma membrane of activated lymphocytes and is the main form of FasL with cytotoxic activity, but whether FasL is delivered to the immune synapse along with granzyme and perforin-containing granules is unclear., Methods: We stably expressed FasL-fluorescent fusion proteins into human NK cells and examined the localization of FasL relative to other intracellular markers by confocal and immunoelectron microscopy, and examined the trafficking of FasL during formation of immune synapses with HLA-deficient B cells., Results: FasL co-localized with CD63 more strongly than perforin or Lamp1+ in cytolytic granules. Electron microscopy revealed that FasL is enriched on intraluminal vesicles (ILVs) adjacent to the dense-core within cytolytic granules. In NK cells forming immune synapses with HLA-deficient B cells, a portion of FasL-containing granules re-localize toward the immune synapse, while a distinct pool of FasL remains at the distal pole of the cell., Conclusions: Localization of FasL to intra-luminal vesicles within cytolytic granules facilitates FasL trafficking to immune synapses and cytotoxic function in NK cells., (© 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

Extracellular vesicles (EVs) are mediators of intercellular communication by transporting cargo containing proteins, lipids, mRNA, and miRNA. There is increasing evidence that EVs have various roles in regulating migration, invasion, stemness, survival, and immune functions. Previously, we have found that EVs from Kaposi's sarcoma-associated herpesvirus (KSHV)-infected human endothelial cells have the potential to activate the complement system. Although many studies have shown that the physical properties of EVs can be changed by their storage condition, there have been few studies for the stability of biological activity of EVs in various storage conditions. In this study, we investigated various conditions to identify the best conditions to store EVs with functional stability for 25 d. Furthermore, the correlation between the function and other characteristics of EVs, including the expression of EV markers, size distribution, and particle number, were also analyzed. Our results demonstrated that storage temperature is an important factor to maintain the activity of EVs and would be useful information for basic research and clinical application using EVs.

This corrects the article DOI: 10.1038/bjc.2017.85.

Background: We have previously identified tissue inhibitor of metalloproteinases-1 (TIMP-1) as a prognostic marker in glioblastomas. TIMP-1 has been associated with chemotherapy resistance, and CD63, a known TIMP-1-binding protein, has been suggested to be responsible for this effect. The aim of this study was to assess CD63 expression in astrocytomas focusing on the prognostic potential of CD63 alone and in combination with TIMP-1., Methods: CD63 expression was investigated immunohistochemically in a cohort of 111 astrocytomas and correlated to tumor grade and overall survival by semi-quantitative scoring. CD63 expression in tumor-associated microglia/macrophages was examined by double-immunofluorescence with ionized calcium-binding adapter molecule 1 (Iba1). The association between CD63 and TIMP-1 was investigated using previously obtained TIMP-1 data from our astrocytoma cohort. Cellular co-expression of TIMP-1 and CD63 as well as TIMP-1 and the tumor stem cell-related markers CD133 and Sox2 was investigated with immunofluorescence. TIMP-1 and CD63 protein interaction was detected by an oligonucleotide-based proximity ligation assay and verified using co-immunoprecipitation., Results: The expression of CD63 was widely distributed in astrocytomas with a significantly increased level in glioblastomas. CD63 levels did not significantly correlate with patient survival at a protein level, and CD63 did not augment the prognostic significance of TIMP-1. Up to 38% of the CD63+ cells expressed Iba1; however, Iba1 did not appear to impact the prognostic value of CD63. A significant correlation was found between TIMP-1 and CD63, and the TIMP-1 and CD63 proteins were co-expressed at the cellular level and located in close molecular proximity, suggesting that TIMP-1 and CD63 could be co-players in glioblastomas. Some TIMP-1+ cells expressed CD133 and Sox2., Conclusion: The present study suggests that CD63 is highly expressed in glioblastomas and that TIMP-1 and CD63 interact. CD63 does not add to the prognostic value of TIMP-1. Co-expression of TIMP-1 and stem cell markers as well as the wide expression of CD63 might suggest a role for TIMP-1 and CD63 in glioblastoma stemness.

African American men in the United States have higher mortality due to prostate cancer (PCa) compared to other races. One reason for this disparity is the lack of in-depth understanding of the PCa biology in African Americans. For example, hypoxia in prostate tumor microenvironment is associated with adverse prognosis; still, no hypoxia-related studies have been reported in African Americans. Here, we compared African-American and Caucasian PCa cells for exosome secretion under normoxic (21% O 2 ) and hypoxic (1% O 2 ) conditions. All cell lines showed higher exosome secretion under hypoxia but it was clearly more prominent in African-American PCa cells. Further, under hypoxia, Rab5 (a biomarker for early endosome) was clustered in perinuclear region; and CD63 (a biomarker for exosomes and multivesicular endosomes) showed greater co-localization with actin cytoskeleton especially in African American PCa cells. Importantly, exosome biogenesis inhibitors GW4869 (10-20 µM) or DMA (10-20 µg/ml) significantly decreased cell viability and clonogenicity in PCa cells. Interestingly, we also observed higher level of lactic acid loaded in exosomes secreted under hypoxia. Overall, under chronic hypoxia, PCa cells secrete more exosomes as a survival mechanism to remove metabolic waste.

Mast cells and basophils play a pathogenetic role in allergic, inflammatory, and autoimmune disorders. These cells have different development, anatomical location and life span but share many similarities in mechanisms of activation and type of mediators. Mediators secreted by mast cells and basophils correlate with clinical severity in asthma, chronic urticaria, anaphylaxis, and other diseases. Therefore, effective biomarkers to measure mast cell and basophil activation in vivo could potentially have high diagnostic and prognostic values. An ideal biomarker should be specific for mast cells or basophils, easily and reproducibly detectable in blood or biological fluids and should be metabolically stable. Markers of mast cell and basophil include molecules secreted by stimulated cells and surface molecules expressed upon activation. Some markers, such as histamine and lipid mediators are common to mast cells and basophils whereas others, such as tryptase and other proteases, are relatively specific for mast cells. The best surface markers of activation expressed on mast cells and basophils are CD63 and CD203. While these mediators and surface molecules have been associated to a variety of diseases, none of them fulfills requirements for an optimal biomarker and search for better indicators of mast cell/basophil activation in vivo is ongoing., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Epstein-Barr virus LMP1 is an oncoprotein required for immortalizing B lymphocytes and also plays important roles in transforming non-lymphoid tissue. The discovery of LMP1 protein interactions will likely generate targets to treat EBV-associated cancers. Here, we define the broader LMP1 interactome using the recently developed BioID method. Combined with mass spectrometry, we identified over 1000 proteins across seven independent experiments with direct or indirect relationships to LMP1. Pathway analysis suggests that a significant number of the proteins identified are involved in signal transduction and protein or vesicle trafficking. Interestingly, a large number of proteins thought to be important in the formation of exosomes and protein targeting were recognized as probable LMP1 interacting partners, including CD63, syntenin-1, ALIX, TSG101, HRS, CHMPs, and sorting nexins. Therefore, it is likely that LMP1 modifies protein trafficking and exosome biogenesis pathways. In support of this, knock-down of syntenin-1 and ALIX resulted in reduced exosomal LMP1., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells. IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major oncoprotein, LMP1, into vesicles for secretion. We have recently described a role of the host cell protein CD63 in regulating intracellular signaling of the viral oncoprotein by shuttling LMP1 into exosomes. Here, we provide strong evidence of the utility of CD63-dependent EVs in regulating global intracellular signaling, including mTOR activation by LMP1. We also demonstrate a key role of CD63 in coordinating endosomal and autophagic processes to regulate LMP1 levels within the cell. Overall, this study offers new insights into the complex intersection of cellular secretory and degradative mechanisms and the implications of these processes in viral replication., (Copyright © 2018 American Society for Microbiology.)

It is known that endoplasmic reticulum (ER) stress in cells and extracellular vesicles (EVs) plays a significant role in cancer cells, therefore the evaluation of compounds that can regulate ER stress and EV secretion would be a suitable system for further screening and development of new drugs. In this study, we evaluated chemical chaperones derived from natural products based on monitoring Bip/GRP78 promoter activity during cancer cell growth, at the level of the single cell, by a bioluminescence microscopy system that had several advantages compared with fluorescence imaging. It was found that several chemical chaperones, such as ferulic acid (FA), silybin, and rutin, affected the activity. We visualized EVs from cancer cells using bioluminescence imaging and showed that several EVs could be observed when using CD63 fused with NanoLuc luciferase, which has a much smaller molecular weight and higher intensity than conventional firefly luciferase. We then examined the effects of the chemical chaperones on EVs from cancer cells by bioluminescence imaging and quantified the expression of CD63 in these EVs. It was found that the chemical chaperones examined in this study affected CD63 levels in EVs. These results showed that imaging at the level of the single cell using bioluminescence is a powerful tool and could be used to evaluate chemical chaperones and EVs from cancer cells. This approach may produce new information in this field when taken together with conventional and classical methods., (Copyright © 2017 John Wiley & Sons, Ltd.)

Extracellular vesicles (EVs) can modulate microenvironments by transferring biomolecules, including RNAs and proteins derived from releasing cells, to target cells. To understand the molecular mechanisms maintaining the neural stem cell (NSC) niche through EVs, a new transgenic (Tg) rat strain that can release human CD63-GFP-expressing EVs from the NSCs was established. Human CD63-GFP expression was controlled under the rat Sox2 promoter (Sox2/human CD63-GFP), and it was expressed in undifferentiated fetal brains. GFP signals were specifically observed in in vitro cultured NSCs obtained from embryonic brains of the Tg rats. We also demonstrated that embryonic NSC (eNSC)-derived EVs were labelled by human CD63-GFP. Furthermore, when we examined the transfer of EVs, eNSC-derived EVs were found to be incorporated into astrocytes and eNSCs, thus implying an EV-mediated communication between different cell types around NSCs. This new Sox2/human CD63-GFP Tg rat strain should provide resources to analyse the cell-to-cell communication via EVs in NSC microenvironments., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Background/aims: Exosomal circulating long non-coding RNAs (lncRNAs) in blood are emerging as clinically useful and non-invasive biomarkers for tumor diagnosis. However, normal cells can also secrete exosomes, so it is a prerequisite to obtain tumor-derived exosomes for better understanding of their diagnostic impacts in cancer. In this study, a dual-antibody-functionalized immunoaffinity system was established to isolate exosomes and investigate their lncRNAs expression pattern and clinical significance in prostate cancer (PCa)., Methods: A commercially available kit was used to isolate total exosomes, which were then purified by a dual-antibody-functionalized immunoaffinity system. RT-qPCR was performed to detect the expression of exosomal lncRNAs. Receiver operating characteristic (ROC) curves were plotted to assess the diagnostic value., Results: Expression levels of two lncRNAs in tumor-derived exosomes were significantly higher than those in total exosomes. The levels of SAP30L-AS1 were upregulated in benign prostatic hyperplasia (BPH), and SChLAP1 levels were significantly higher in PCa than in BPH and healthy individuals. The area under the ROC curve indicated that SAP30L-AS1 and SChLAP1 had adequate diagnostic value to distinguish PCa from controls. Two lncRNAs separately combined with prostate specific antigen (PSA) possessed a moderate ability for discrimination. SAP30L-AS1 expression level was related to PSA values and tumor invasion. SChLAP1 expression was significantly higher in patients with higher Gleason scores, and was also effective in differentiating between BPH and PCa when the concentration of PSA was in the gray zone., Conclusion: The isolation of tumor-derived exosomes by dual-antibody-functionalized immunoaffinity systems and detection of their lncRNAs in plasma may lead to the identification of suitable biomarkers, with potential diagnostic utility., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background/aims: Lymphangiogenesis plays an important role in the pathogenesis of inflammatory bowel diseases (IBD), and vascular endothelial growth factor-C (VEGF-C) is a powerful lymphangiogenic factor. Adipose-derived stem cells (ADSCs) are a promising therapeutic modality for several diseases because ADSCs secret growth factors and exosomes, which modulate hostile microenvironments affected by diseases. However, the effect of exosomes on VEGF-C-dependent lymphangiogenesis and its mechanism remain unclear., Methods: ADSCs were cultured in media with or without recombinant VEGF-C and exosomes were extracted from conditioned medium (CM). Lymphatic endothelial cells (LECs) were treated with ADSCs-derived exosomes, then proliferation, migration and tube formation of LECs were assayed using cell counting Kit-8 (CCK-8), transwell chamber inserts and matrigel-based tube formation assay respectively., Results: We identified significantly higher levels of miR-132 in exosomes isolated from VEGF-C-treated ADSCs (ADSCs/VEGF-C) than in those from ADSCs control. miR-132 was directly transferred from ADSCs to the LECs by the mediation of exosomes. The exosomes from ADSCs/VEGF-C promoted LECs proliferation, migration, and tube formation more potently than the exosomes from ADSCs, whereas pretreatment of ADSCs with miR-132 inhibitor attenuates VEGF-C-dependent lymphangiogenic response. Finally we reveal that miR-132 promotes lymphangiogenic response by directly targeting Smad-7 and regulating TGF-β/Smad signaling., Conclusion: These data provide new insights into the role of ADSCs-derived exosomes as an important player in VEGF-C-dependent lymphangiogenesis., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Endothelial colony-forming cells (ECFCs) are a defined subtype of endothelial progenitors that modulate vascular repair and promote perfusion in ischaemic tissues. Their paracrine activity on resident vasculature is ill-defined, but mediated, at least in part, by the transfer of extracellular vesicles (EVs). To evaluate the potential of isolated EVs to provide an alternative to cell-based therapies, we first performed a physical and molecular characterization of those released by ECFCs. Their effects upon endothelial cells in vitro and angiogenesis in vivo in a model of proliferative retinopathy were assessed. The EVs expressed typical markers CD9 and CD63 and formed a heterogeneous population ranging in size from ~60 to 1500 nm by electron microscopy. ECFC EVs were taken up by endothelial cells and increased cell migration. This was reflected by microarray analyses which showed significant changes in expression of genes associated with angiogenesis. Sequencing of small RNAs in ECFCs and their EVs showed that multiple microRNAs are highly expressed and concentrated in EVs. The functional categories significantly enriched for the predicted target genes of these microRNAs included angiogenesis. Intravitreally delivered ECFC EVs were associated with the vasculature and significantly reduced the avascular area in a mouse oxygen-induced retinopathy model. Our findings confirm the potential of isolated EVs to influence endothelial cell function and act as a therapy to modulate angiogenesis. The functions associated with the specific microRNAs detected in ECFC EVs support a role for microRNA transfer in mediating the observed effects., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)