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October 24th, 2017

ASEMV 2017— Exosomes from Ginger Plant Inhibit Activation of NLRP3 Inflammasome

On Thursday morning, October 12, Jiujiu Yu, PhD, Assistant Professor, Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, spoke on the inhibitory effects of ginger exosomes on NLRP3 inflammasome activation. She said that the aim of her group’s study was to screen and identify dietary exosomes that inhibit the activation of the NLRP3 inflammasome in macrophages. The NLRP3 inflammasome, a key sensor of the innate immune system, is a cytoplasmic complex consisting of the sensor NLRP3, the adaptor ASC, and the effector caspase-1. The NLRP3 inflammasome detects a variety of pathogens and cellular stress, mediates the release of inflammatory mediators, and, as such, its aberrant activation is implicated in many diseases, including metabolic syndromes and autoinflammatory diseases. Therefore, Dr. Yu said, identification of inhibitors that effectively dampen the activity of NLRP3 inflammasomes could be instrumental in controlling undesirable inflammatory conditions. In the work, the scientists purified exosomes from different plants using ultracentrifugation and screened their inhibitory effects on NLRP3 inflammasomes in bone-marrow-derived macrophages. The researchers determined that exosomes purified from ginger strongly inhibited activation of NLRP3 inflammasomes. The ginger exosomes were approximately 134 nanometers in diameter. The inhibitory effects of ginger exposure on NLRP3 inflammasome activation required long incubation (more than 12 hours), suggesting that the cargo inside of the vesicles mediated the inhibitory effects. Ginger exosomes had no effects on the protein levels of inflammasome subunits, including NLRP3, ASC, and caspase-1, or its cofactor Nek7. Based on these results, Dr.

October 23rd

ASEMV 2017—EV Secretion by Primary Cilia

On Monday evening, Octobe 9, Maxense Nachury, PhD, Stanford & UCSF, spoke on “Molecular Mechanisms and Functional Significance of EV Secretion by Primary Cilia.” He noted that the primary cilium, present on almost all cells, is a microtubule-based projection with its own complement of protein, lipids, and second messengers. The primary cilia have long been recognized for their roles in phototransduction and olfactory signaling. In recent years, cilia have been found to organize the developmental signaling pathway Sonic Hedgehog and various neuronal signaling pathways mediated by G-protein-coupled receptors (GPCRs). Dr. Nachury said that a hallmark of ciliary signaling pathways is the dynamic delivery and removal of ciliary proteins. Activated ciliary GPCRs are retrieved back into the cell through beta-arrestin2- and BBSome-mediated transport. (The BBSome is an octameric protein complex that is a component of the basal body and is involved in trafficking cargos to the primary cilium). Surprisingly, Dr. Nachury said, when retrieval is compromised, activated GPCRs become selectively concentrated into membranous buds at the tip of cilia, and actin-mediated scission releases extracellular vesicles (EVs) packaged with activated GPCRs. The microtubule-actin crosslinker Drebrin and Myosin VII localize to the site of ectosome release at the ciliary tip under ectosome-producing conditions and are required for the scission of EVs from the tip of cilia. Signal-dependent ectocytosis also applies to physiological context, where it removes, from cilia, specific GPCRs such as anorexigenic receptor NPY2R that lack retrieval determinants. Functionally, ciliary ectocytosis is required for the appropriate reulation of Hedgehog signaling in retrieval mutants. Dr.

October 22nd

ASEMV 2017—Exosomal miRNAs As Potential Biomarkers for Energy Expenditure by Human Brown Fat

On Tuesday morning, October 10, Alexander Pfeifer, PhD, Institute of Pharmacology and Toxicology, University of Bonn, addressed the interesting topic of exosomal miRNAs, energy expenditure, and brown fat. He began by noting the brown adipose tissue (BAT) is essential for non-shivering thermogenesis and regulates whole-body metabolism. He said brown fat activity correlates with leanness and has, therefore, received significant attention as a potential target for anti-obesity therapies. However, he noted, there is presently a lack of BAT diagnostics, and that novel diagnostic tools are need to develop BAT-centered therapies. In the current work, Dr. Pfeifer focused on exosomes released from BAT in order to identify biomarkers of BAT activity. Exosomes were isolated from brown adipocytes, explanted brown fat depots, and also from serum of mice. Brown adipocytes released exosomes in an activation-dependent manner. Physiologically, norepinephrine (NE) induces BAT energy expenditure by activating the cyclic AMP signaling pathway in brown adipocytes. Increasing cyclic AMP levels resulted in activation of brown adipocytes and induced a five-fold increase in exosome release. In addition, BAT isolated from mice exposed to cold released nine-fold more exosomes than BAT from mice kept at room temperature. The conclusion drawn is that exosome release is increased after activation of BAT. In order to identify potential biomarkers of BAT activity, the scientists focused on miRNAs. They and other groups had previously identified miRNAs that regulat BAT function. Dr. Pfeifer’s group profiled the miRNAs contained in exosomes released from brown adipocytes and BAT.

ASEMV 2017—Possible Role for Natural Killer Cell-Derived Exosomes and Exosomal miRNAs in Cancer Therapy

On Tuesday evening, Octobere 10, Muller Fabbri, MD, PhD, Children’s Hospital Los Angeles, discussed the possible role of NK cell-derived exosomes and NK cell-derived microRNAs (miRNAs) in cancer therapy. He noted first that NK cell-derived exosomes exhibit cytotoxic activity against MYCN-amplified neuroblastoma cell lines. In addition, these exosomes carry tumor suppressor miRNAs whose expression in tumors correlates with NK activation markers (i.e., NKG2D and DNAM-1) and harbor prognostic implications. Importantly, the scientists found that NK-derived exosomal miRNAs target key driver oncogenes in neuroblastoma and the TGF pathway that is responsible for NK immune-suppression within the tumor microenvironment. Targeted delivery of NK-derived exosomal miRNAs by nanoparticles to MYCN-amplified neuroblastoma or NK cells, resulted in inhibition of neuroblastoma tumorigenic potential and prevented the TGF1-dependent inhibition of NK cells. Taken together, Dr. Fabbri said, these data support a role for NK-derived exosomes and exosomal miRNAs as a potential therapeutic option alongside NK cell-based therapy. The American Society for Exosomes and Microvesicles (ASEMV) 2017 Annual Meeting was held October 8-12 at the Asilomar Conference Grounds in Pacific Grove, California. The event was attended by 180 scientists from around the United States and world. This year’s meeting was organized by ASEMV President Dr. Stephen J. Gould of Johns Hopkins University, and sponsors of the meeting included Particle Metrix, CARIS Life Sciences, ReNeuron, Malvern, QIAGEN, Hitachi Chemical, iZON, System Biosciences, Beckman Coulter, Norgen Biotek Corporation, Hansa Bio Med Life Sciences, Lonza, and nanoView Diagnostics.

ASEMV 2017—Engineered Exosomes with Super Repressor IkB, a Potent Inhibitor of NF-kB Pathway, for Treatment of Septic Shock

On Monday morning, October 9, Chulhee Choi, MD, PhD, of the Department of Bio and Bioengineering, KAIST, Korea; and CEO of Cellex Life Sciences, Inc., described the potential for a new system to treat acute inflammatory diseases, including septic shock. To emphasize the need for improvements in the treatment of septic shock, Dr. Choi noted that there are 20 million cases of this disease each year and that it is the leading cause of death in intensive care units. Previously, Dr. Choi’s group had developed an opto-generated engineered exosome system, named “exosomes for protein loading via optically reversible protein-protein interaction” (EXPLOR) that can deliver soluble proteins into the cytosol via controlled reversible protein-protein interactions (PPI). Treatment with protein-loaded EXPLORs was shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in both a time- and dose-dependent manner. In the current study, the group sought to test the feasibility of using EXPLOR technology for the delivery of super repressor IkB (SRI), a potent inhibitor of the NF-kB pathway, as a potential treatment for acute inflammatory diseases such as septic shock syndrome. The group was able to load SRI into engineered exosomes by transiently or stably expressing fusion proteins in exosome-producing cells. The scientists further demonstrated the intracellular delivery of SRI as functional proteins in the target cells in vitro and target organs in vivo. Finally, the researchers observed a beneficial effect of SRI-loaded exosomes in septic shock animal models compared to what is seen with naïve exosomes. Dr.

ASEMV 2017—STARD11-Mediated Ceramide Transport Necessary for Release of Lipotoxic EVs

On Monday morning, October 9, Harmeet Malhi, MD, of the Mayo Clinic, described her group’s work that has identified a novel pathway for the formation of lipotoxic extracellular vesicles (EVs). She noted that while lipotoxic hepatocyte-derived EVs are key mediators of cell-to-cell communication in nonalcoholic steatohepatitis, the cellular machinery for the biogenesis of lipotoxic EVs has remained undefined. Recently, her group has demonstrated that palmitate (PA)-induced EVs are enriched in C16:0 ceramide, are consistent with exosomes formed within the multivesicular body (MVB), and their release depends on the synthesis of ceramide at the endoplasmic reticulum (ER). She noted that STARD11, a ceramide transport protein, mediates the trafficking of newly formed ceramide from the ER to the Golgi, but that its role in the transport of newly formed ceramide from the ER to the MVB is unknown. The aim of the work reported here was to test the hypothesis that the ceramide transport protein, STARD11, is necessary for the transport of ceramide from the ER to the MVB, thus mediating the biogenesis of lipotoxic EVs. The results suggest that STARDT11 mediates the biogenesis of PA-induced ceramide-enriched EVs due to the transport of newly synthesized ceramide from the ER to the MVB. The group has identified a novel pathway for the formation of lipotoxic EVs, and future work will focus on the functional consequences of this pathway. The American Society for Exosomes and Microvesicles (ASEMV) 2017 Annual Meeting was held October 8-12 at the Asilomar Conference Grounds in Pacific Grove, California. The event was attended by 180 scientists from around the United States and world. This year’s meeting was organized by ASEMV President Dr. Stephen J.

October 21st

ASEMV 2017—Host and Storage Conditions May Influence Size, Cargo, and Function of Isolated Exosomes

In her Wednesday presentation, Chioma Okeoma, PhD, recently moved from the University of Iowa to Stony Brook University in New York, speculated initially that the clinical condition of the host that releases the exosome may influence “exosome speciation” (size, cargo, function) and that the environmental conditions in which the released exosomes are stored may affect exosomal physicochemical properties and function. Consideration of storage conditions is critical, she said, because biofluids are either used in the moment or frozen (especially in clinical settings) for future use. She then described her group’s work that bore on these points. The scientists demonstrated that exosomes isolated from the biofluids of donors who use illicit substances or who are exposed to infectious agents may differ in physicochemical properties from exosomes isolated from otherwise healthy donors. As an example, her group showed that semen exosomes from users of illicit drugs have decreased levels of mRNA, and CD63 and CD9, proteins, which are coincident with the loss of semen-exosome-induced HIV inhibition. Dr. Okeoma further noted storage conditions (temperature and time) can have significant effect on exosomes. Specifically, her group has found that prolonged freezing of semen exosomes at -80 degrees Centigrade may affect the activity of specific exosome cargos, such as acetylcholinesterase. Such exosomes are also impaired in anti-HIV activity in the context of exosome/HIV interaction but maintain potency in their ability to condition host cells. The American Society for Exosomes and Microvesicles (ASEMV) 2017 Annual Meeting was held October 8-12 at the Asilomar Conference Grounds in Pacific Grove, California.

ASEMV 2017—EV-Associated Tissue Factor Key to New Assay That May Predict Risk of Venous Thromboembolism in Cancer

On Tuesday morning, Rienk Nieuland, PhD, a principal investigator of the Academic Medical Centre of the University of Amsterdam, presented a possible new approach to predicting the risk of venous thromboembolism (VTE) in cancer patients. He noted that VTE is effectively treated by prophylactic anticoagulation, but that because anticoagulation increases the bleeding risk, prophylactic treatment of all cancer patients is not desirable. Hence, there is a need to identify cancer patients at high risk of VTE in whom the benefits of thromboprophylaxis may outweight the risks. In describing his group’s work to meet this need, he first noted that saliva contains EVs exposing the protein tissue factor (TF). When we lick a wound, this brings together coagulation factors (blood) and TF (saliva), thereby promoting the clotting of blood. He added that blood from healthy humans does not contain EVs exposing TF, but that circulating EVs exposing TF are encountered during surgery and in several pathologic conditions, including sepsis and cancer. Dr. Nieuwand’s group developed a plasma clotting test (Fibrinogen Generation Test, FGT) in which the clotting time depends on the presence of endogenous EV-associated TF. The ability of the FGT to identify cancer patients at high risk of VTE was determined in a multicenter prospective cohort study. Dr. Nieuwand reported that the results were promising, particularly with regard to pancreatic cancer, and may provide clinically useful information, either as a stand-alone biomarker or when combined with other biomarkers and/or clinical prediction scores for VTE. The American Society for Exosomes and Microvesicles (ASEMV) 2017 Annual Meeting was held October 8-12 at the Asilomar Conference Grounds in Pacific Grove, California.

ASEMV 2017—Morphine-Mediated Potentiation of HIV-Associated Neurological Disorder (HAND)

On Monday morning, October 9, Shilpu Buch, PhD, Professor and Vice-Chair for Research, Director of the Nebraska Center for Substance Abuse Research, Nebraska Medical Center, University of Nebraska, presented on “Exosomal miRNA-Mediated Loss of Pericyte Coverage at the Blood-Brain Barrier: Implications for Morphine-Mediated Potentiation of HIV-Associated Neurological Disorder (HAND).” Dr. Buch began by noting that both in vitro and in vivo studies have shown that morphine potentiates the neurodegenerative effecs of HIV on the central nervous system (CNS). BBB breach by toxic stimuli is associated with the transmigration of monocytes and leukocyts into the brain, she said. Dr. Buch reported that three cell types are key to the blood-brain barrier (BBB). These are endothelial cells, astrocytes, and pericytes. She said that astrocyte-pericyte cross-talk is critical to BBB integrity. She said that pericytes regulate the BBB and that morphine reduces pericyte coverage of the BBB, resulting in cognitive impairment ant neuroinflammation. The underlying mechanism(s) by which morphine-exposed astrocytes regulate pericytes at the BBB, remains unknown. Previously, her group had identified a wide array of microRNAs (miRs) that are dysregulated in the CNS of SIV-infected, morphine-dependent macaques. In addition, her group has shown in vitro thatEVs released from morphine- and HIV-protein-Tat-exposed astrocytes can shuttle miRNAs, which, in turn, are taken up by neurons, leading to neuronal dysfunction. Here, Dr. Buch reported on the results of her group’s current study to examine the roles of miRs released from morphine-exposed astrocytes in mediating the loss of pericytes at the BBB.

ASEMV 2017—Gliomas Are Focus of Multiple Presentations

The American Society for Exosomes and Microvesicles (ASEMV) 2017 Annual Meeting was held October 8-12 at the Asilomar Conference Grounds in Pacific Grove, California. The event was attended by 180 scientists from around the United States and world. In the last talk on Tuesday, evening, Michael Olin, PhD, Assistant Professor in the Department of Pediatrics, Division of Hematology/Oncology, University of Minnesota, presented his group’s work on the identification of a peptide that activates a pathway that opposes the CD200 immune inhibition pathway induced by exosomes released by cancer cells, particularly glioblastoma (GBM) cells. Dr. Olin has shown that parenteral injection of the peptide and tumor lysate reduces or eliminates glioma in dogs with natural gliomas. Dr. Olin and colleagues have recently founded a company (OX2 Therapeutics, dedicated to bringing this therapeutic peptide to the clinic, hopefully within a year. Earlier on Tuesday evening, Xandra Breakefield, PhD, Professor of Neurology, Harvard Medical School; Geneticist, Massachusetts General Hospital, began by emphasizing the grim nature of glioblastoma multiforme which is fatal (15-month average lifespan after diagnosis) and for which there has been no change in treatment in 20 years. Dr. Breakefield noted that glioma-secreted extracellular vesicles (EVs) are taken up by microglia cells which typically constitute 30%-40% of the tumor microenvironment. She noted that normally microglia act as sentinels in the brain, actively sensing the milieu and performing “warrior” and “nurturer” functions as needed. The genes controlling this important sensory role have been termed the microglia “sensome.” Dr.