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Huge Potential of Exosomes Is Major Focus of Society's Annual Meeting October 2014

Over two hundred visionary scientists, pragmatic physicians, and savvy biotech sales reps from the United States and around the world gathered in California from October 10-13, 2014 to discuss the latest advances in research and technology related to exosomes, a new and extremely hot area of science with possibly huge potential for game-changing applications in clinical medicine. The occasion was the fourth annual meeting of the American Society for Exosomes and Microvesicles (ASEMV). The site was the magnificently beautiful Asilomar Conference Grounds bordering the Pacific Ocean on Northern California’s Monterey Peninsula approximately 100 miles south of San Francisco. This meeting was organized by Stephen Gould, M.D., President of the ASEMV, Professor of Biological Chemistry at The Johns Hopkins University School of Medicine, and an expert on exosome biogenesis; and by Douglas Taylor, Ph.D., Secretary-General of the ASEMV, formerly a professor at the University of Louisville, an exosome pioneer, and now the chief scientific officer (CSO) of a year-old start-up company called Exosome Sciences, Inc., located just outside Princeton, New Jersey, and a majority-owned subsidiary of Aethlon Medical, Inc. There was also notable organizational assistance from Sasha Vlassov, Ph.D., from Life Technologies (Thermo Fisher Scientific, Inc.), from Travis Antes, Ph.D., of System Biosciences, Inc. (SBI), and from many of the graduate students in Dr. Gould’s lab.

The meeting, which was described as “awesome” by more than one attendee, included 70 oral presentations and 80 posters on a very wide range of exosome-related topics, including the production of exosomes by cancer cells and the potential use of exosome molecular contents as easily accessible biomarkers for diagnosing and monitoring the course and treatment response of cancers and many other diseases and conditions, including Dengue fever, Crohn’s disease, heart damage, tuberculosis, neurodegenerative diseases such as Alzheimer’s, and prion-caused diseases such as Creutzfeldt-Jakob disease.

There was also much discussion of the various technologies—either already being used, currently in development, or now being suggested as improvements—that can be used to isolate and characterize exosomes, and to also analyze their diverse molecular cargo. In addition, there was significant emphasis on the urgent need to standardize these methods so that results can be readily compared among laboratories.

There were booth exhibits by a number of biotech companies and suppliers who are marketing materials to the new and exploding market of exosome research. The exhibitors, as well as other conference supporters, included System Biosciences, Inc. (SBI), headquartered in Mountain View, CA; Maverix Biomics, headquartered in San Mateo, CA; JSR Micro Life Sciences Materials Innovation, headquartered in Sunnyvale, CA; QIAGEN, headquartered in the Netherlands, with offices in Redwood City, CA, and around the world; Life Technologies (A Thermo Fisher Scientific Brand), headquartered in Carlsbad, CA; Exosome Diagnostics, Inc., headqurtered in Cambridge, MA; Malvern Instruments, headquartered in Worcestershire, UK; iZON Sciences Ltd., headquartered in Oxford, UK; CARIS Life Sciences, headquartered in Dallas-Fort Worth, TX; Particle Metrix, headquartered in Germany; HansaBioMed, headquartered in Tallinn, Estonia; PMDx (Precision Molecular Diagnostics) at the Moffitt Cancer Center in Tampa, FL; AG Scientific, headquartered in San Diego, CA; UNIFlow™ by UNIConnect, headquartered in Salt Lake City, Utah; and Cedarlane Corporation, with main offices in Burlington-Ontario, Canada, and Burlington, NC.

In addition to attendees from nearby Stanford, UCSF, and UC-Berkeley, as well as other California institutions and companies, scientists and physicians came from all over the United States and world. There were speakers and attendees from such far-off climes as New Zealand, Thailand, South Korea, Japan, Brazil, Switzerland, Germany, UK, and Finland. There were also two current US residents who came originally from Saudi Arabia and from southern Siberia (the warm part), respectively.

After a background description of exosomes, we will describe the key points of each of the oral presentations that was given during this four-day scientific event. We will describe the presentations in the order in which they were given. Unfortunately, we will not be able to summarize the many poster presentations that were also given during this signature meeting and which further demonstrated the already enormous and ever-increasing breadth and impact of exosome research.

Exosomes

Exosomes are tiny, subcellular, membrane-bound vesicles (approximately 30-100 nm in diameter) that are released by a wide variety of cell types and cancer cells and that can carry membrane and cellular proteins, as well as DNA, and various types of RNA, including mRNA and microRNA (miRNA), that are representative of the cell of origin. It is thought that exosomes may serve the purpose of shuttling information from one cell to another. For instance, it has been shown that exosomes can carry molecules from cancer cells that act to suppress the immune system and stimulate angiogenesis, thus encouraging cancer growth.

It is believed that profiles of the various biomolecules in exosomes may serve as useful biomarkers for cancers and other diseases. High-quality RNA, and even DNA, can be isolated from exosomes and profiles of these molecules can be determined. Exosomes are shed into biofluids by cells under both normal and pathological conditions. In pathological conditions, exosomes constitute a highly enriched, intact source of disease-specific nucleic acids and proteins. In cancer, it has been shown that exosomes are shed at a much increased rate relative to the shedding rate observed for normal, healthy cells.

Exosomes can be isolated from bodily fluids by ultracentrifugation and filtration, as well as by other approaches, and they offer a very attractive alternative to invasive biopsy for the diagnosis and monitoring of disease. They are said to represent the opportunity for the holy grail of the non-invasive “liquid biopsy.” While representative of the cell of origin, the cargo of exosomes contains certain molecules that are over-represented or under-represented when compared to the content of the originating cell. Consequently, it is believed that selective packaging mechanisms are involved in the loading of exosomes, but the nature of such mechanisms has yet to be elucidated. It has been suggested that it may be based, in part, on RNA “zip codes,” that are used by the cell to actively place RNAs in their proper positions in the cell, but this hypothesis is still under investigation.

A number of commercial companies, such as Exosome Diagnostics and Exosome Sciences for example, are now pursuing the development of diagnostic and disease-monitoring tools for cancer and other diseases that will make use of biomarkers found in exosomes. There is also significant potential for using exosome depletion as a way of treating disease—for example, cancer-generated exosomes can inhibit the immune response and stimulate angiogenesis—if these exosomes are removed, tumor growth might be inhibited and a window of opportunity for the effective use of other anti-cancer agents might be created. In addition, there is potential for exosomes to be used as targeted delivery vehicles of therapeutic molecules to cancer cells—e.g., delivering small interfering RNA (siRNA), also called silencing RNA, specific for a particular oncogene expressed in the cancer cell. There is significant interest by pharmaceutical companies in the development of such approaches.

It should be noted here that a significant terminology problem currently exists in the exosome field. Expert speakers vary in the terminology they employ, with some using “exosomes,” and others using “microvescicles” and/or “extracellular vesicles” and/or “microparticles” and it is not always clear precisely what the speaker is describing. In general, the latter terms are more general and more inclusive, but the fact is that the term “exosome” has yet to be rigorously defined, and we must await such a time before we can rigorously distinguish among these various terms. This problem was addressed by a number of speakers during the ASEMV meeting, but a solution has not yet been advanced.

FRIDAY

Meeting Opens Friday Evening

The fourth annual ASEMV meeting was opened early Friday evening by some brief remarks from ASEMV Secretary-General Dr. Taylor, a 35-year veteran of the exosome field. He highlighted some important developments for the ASEMV organization, including the development of a journal and of a website. He also hailed the efforts of ASEMV President Dr. Gould in organizing this year’s meeting and attracting a bumper crop of attendees from academia and industry, and also arranging for a number of big-league sponsors. Dr. Taylor extended a very warm welcome to the attendees and then introduced the first speaker, Xandra O. Breakfield, Ph.D., Professor of Neurology at Massachusetts General Hospital and Harvard Medical School, whose topic was “Extracellular RNA—Many Forms and Purposes.

Extracellular RNA—Many Forms and Purposes

Dr. Breakefield noted that there are many kinds of vehicles for extracellular RNA (EC RNA), including ribonucleic protein and various EC vesicles. Most of the RNA found extracellularly is small (<200 nucleotides), most is non-coding, and it is “never alone,” Dr. Breakefield said. Rather, it is found with protein or in vesicles that enhance its extracellular stability. The non-coding RNAs found extracellularly include microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), long noncoding (nc) RNA, piwi-interacting RNA (piRNA), riboswitches, and retrotransposons. There is little functional mRNA in EC RNA, but there are “tons of fragments,” she said. She noted the belief that EVs are involved in intracellular communication, but that it is still a challenge to prove this rigorously. Her lab focuses primarily on glioblastoma and she described recent experiments in mouse models showed that exosomes released by glioblastoma cells and containing large amounts of miR-451 and miR-21 were avidly taken up by microglia cells, leading to cell activation and down-regulation of the target c-Myc mRNA levels. Dr. Breakefield emphasized the need for increased standardization of methods for isolating and purifying EC RNAs, and she stated that she does not like either size or density as markers for extracellular vesicles and that this area needs significant work.

Polyspermy Block

The next speaker was Gavin Wright, Ph.D., of the Wellcome Trust Sanger Institute in the UK, who delivered the CARIS Discovery Lecture, sponsored by CARIS Life Sciences. Dr. Wright presented dramatically exciting new work describing the discovery of the two proteins—one from the sperm and one from the egg—that bind to each other to launch the process that creates a membrane block to polyspermy, i.e., the fertilization of the egg by more than one sperm. The protein from the sperm is called izumo1 (from the Japanese word for marriage shrine) and the protein from the egg was initially identified as folate receptor 4 (Folr4). This name was changed to “Juno” in light of the fact that Folr4 does not, in fact, bind folate. Rather amazingly, prior to the landmark work of the Sanger Institute team, no receptor-ligand pair for egg fertilization had ever been identified. Dr. Wright also showed that the Juno protein from the egg is packaged and released in exosomes after fertilization and noted that these exosome might serve as a “decoy eggs” to divert other sperm from going to the fertilized egg. He suggested that this exosome-based system might well have applications in fertility treatments and contraception.

Timp Function Harnessing the CAF State

The next speaker was Rama Khokha, Ph.D., Professor of Medical Biophysics at the Princess Margaret Cancer Centre at the University of Toronto, who described “Timp Function in the CAF Cell State.” Timps are a family of proteins known as tissue inhibitors of matrix metalloproteinases, a group of peptidases involved in degradation of the extracellular matrix (ECM). Cancer cells must orchestrate the breakdown of ECM for metastasis. Dr. Khokha described experiments in which her group generated Timpless fibroblasts in mice and demonstrated that these fibroblasts display molecular features of cell-associated fibroblasts (CAFs), and that they augment tumor growth and enhance spontaneous metastasis, as do CAFs, which are known to drive tumor progression. These timpless fibroblasts secreted exosomes that had distinct protein content and were shown to activate Notch signaling. Dr. Khokha concluded that Timps create a barrier to acquisition of the CAF state. She believes her group’s work has extended the biological function of metalloproteases and revealed an untapped target for cancer treatment.

Vesicle Isolation for Research and Clinical Applications

The final speaker of the evening was Dr. Taylor who focused on the isolation of vesicles for research and clinical applications. He began by noting the “catastrophe of nomenclature” that currently afflicts the exosome/microvesicle/extracellular vesicle field and the critical need to somehow resolve this problem. He noted that in peripheral circulation it is not possible to differentiate between exosomes and microvesicles and it is not yet clear that there is a difference. He emphasized the importance of the potential to use these vesicles for “liquid biopsies,” which could provide enormous benefits by allowing non-invasive diagnosis and monitoring of disease as well as much more frequent monitoring than is possible with typical biopsies. He noted one of the obstacles to this use is that the material in bodily fluids is a complex mixture. He outlined what are thought to be the functions of exosomes—i.e., normal homeostastis, regulation of immune recognition and response, promotion of wound healing, mediation of tumorigenesis, modulation of inflammation and chronic conditions, and a possible role in pregnancy (placental exosomes and key role in immune tolerance). He then described a number of methods that are currently used to purify exosomes and microvesicles, and described the pros and cons associated with each approach. These include ultracentrifugation, sucrose gradient separation, size exclusion chromatography, polymer-based precipitation, and immune-affinity chromatography. At his new company, Exosome Sciences, Inc., Dr. Taylor says they have started using a procedure that involves lectin-based selection of exosomes. The company developed ELLSA™, an enzyme-linked lectin-specific assay that has further demonstrated the ability to identify and quantify the presence of exosomes underlying human immunodeficiency virus (HIV), tuberculosis (TB), and all forms of cancer tested to date. The basis of the selection is the interaction of the lectins with specific miRNAs.

SATURDAY

Transfer of Functional RNA from Blood to Brain by EVs

Saturday’s session began with Stefan Momma, Ph.D., principal investigator at the Institute of Neurology at Goethe-Universitat Frankfurt am Main, describing the transfer of functional RNA from blood to brain via EVs. Dr. Momma began by noting that the mechanisms by which the immune system communicates to the brain in response to systemic inflammation is not completely understood. He reported that his group has shown that reporter gene expression in neurons is caused by the intercellular transfer of functional Cre recombinase mRNA contained in secreted EVs, particularly exosomes. He presented evidence that EVs can communicate RNA-based signals directly from the hematopoietic system to the brain in response to peripheral inflammation. He noted that, in general, use of the Cre-lox system permits the tracing of EV-mediated RNA transfer in vivo without potentially confounding manipulations. He also suggested that these findings point to the possibility of using a related approach to move material into the brain for the treatment of neurodegenerative diseases.

MVs and Mycobacterium tuberculosis Infection

The next speaker was Pamela Wearsch, Ph.D., Assistant Professor of Pathology, Case Western Reserve University. She began by noting that the trafficking of Mycobacterium tuberculosis (Mtb) cell wall components is highly relevant to the regulation of infected and uninfected immune cells, but the molecular mechanisms of this trafficking have not been addressed. She noted that for over ten years it has been presumed that Mtb cell wall components insert into host membranes and traffic into exosomes. However, mycobacteria produce MVs in tissue culture that are similar in size (50-200 nm) and enriched for lipoglycans/lipoproteins. Therefore, her group proposed an alternative model that involves the intracellular production and extracellular release of Mtb MVs. The results suggest that Mtb MVs are released by infected macrophages and include EM data supporting the production of Mtb MVs within host cells. Together, Dr. Wearsch said, their data suggest that Mtb employs MVs as a mechanism to secrete cell wall components and impair effector functions within infected macrophages. In addition, the group proposes that MTB MVs, not exosomes, circulate bacterial components beyond infected cells to further modulate immune responses. These mechanisms may promote survival of Mtb and pathogenesis of infection.

The Landscape of Human Salivary Extracellular Non-Coding RNA

The following speaker, David Wong, Doctor of Dental Medicine, Doctor of Medical Science, Director of the UCLA Center for Oral/Head & Neck Oncology Research, and Professor at the UCLA School of Dentistry, presented evidence that exosomes released from pancreatic cancer cells actually traveled to the salivary glands, whence they could be swallowed and quite possibly travel to the small intestine to interact with an immune system control center consisting of the Peyer’s patches and other immune cells in order to suppress the immune response and thus enhance the survival of the pancreatic tumor. More generally, Dr. Wong believes that exosomes secreted from the salivary glands might be used as readily accessible biomarkers for a wide range of diseases.

Exosomes Regulate Cancer Cell Invasion by Controlling Invadopodia

The next speaker was Alissa Weaver, M.D., Ph.D., Associate Professor of Cancer Biology, Associate Professor of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center. Dr. Weaver’s laboratory studies all aspects of the metastatic process and has recently focused on how the secretion of exosomes promotes aggressive, invasive behavior and facilitates tumor growth and metastasis. In particular, she described her group’s focus on how exosomes play a role in regulating the activity of invadopodia, which are actin-based structures that function to degrade extracellular matrix (ECM) and thus facilitate cancer metastasis. She described experiments that her group had done to demonstrate that exosome secretion controls both the biogenesis and function of invadopodia. Dr. Weaver noted that exosome carry critical proteinases to invadopodia and, in particular, are essential for the delivery of the proteinase MT1-MMP to invadopodia and to its key involvement in matrix degradation. Her group also showed that multivesicular bodies (MVBs) localize to invadopodia and she noted that with docking sites for MVBs, the presence of invadopodia can control the degree of exosome secretion. Her group proposes that exosomes provide a potent positive feedback signal to promote cancer cell invasion by controlling the formation and function of invadopodia.

miR-146a Can Substitute for ApoE in Suppressing Inflammation and Atherosclerosis

The following speaker was Robet L. Raffai, Ph.D., Associate Professor in the Department of Surgery at UCSF. Dr. Raffai described studies exploring the impact of ApoE on the cellular release of endogenous miR-146a in exosomes and high-density lipoproteins from macrophages that can be communicated to neighboring cells in the vascular wall and the circulation to suppress inflammation and atherosclerosis. ApoE is known to be highly protective against atherosclerosis by regulating immunity and inflammation. Dr. Raffei described work in ApoE-deficient mice, where treatment with miR-146a substituted for ApoE control of atherosclerosis and acted by suppressing NF-kappa B signaling.

Thymic Exosomes Promote Maturation of Developing Thymocytes

Esbjorn Telemo, Ph.D., Professor of Immunology, Department of Medicine, Sahlgrenska Academy, University of Gothenburg, Sweden, spoke next, and discussed the use of a murine in vitro system to investigate the role of thymic exosomes in the maturation of single positive thymocytes into mature thymocytes, as well as the influence of thymic exosomes on natural regulatory T cell (nTreg) formation. Dr. Telemo reported that his group’s work confirmed that thymic exosomes promote the maturation of CD4+CD25- cells into mature thymocytes with CCR7+CCR9-, S1P1+Qa2+ or CCR7+QA2+ phenotypes. In addition, his group showed that thymic exosomes slow down the formation of CD4+CD25+FoxP3+ thymocytes and that these exosome effects are independent of dendritic cell co-stimulation. Furthermore, the group showed that there is an efficient direct uptake of exosomes by thymocytes and suggested that this may represent a new layer of communication within the thymus. The group also showed that thymic exosomes suppress the expansion of nTreg cells.

TMEM16F Lipid Scramblase Required for MV Formation in Hematopoietic Cells

Andrew Kim, Ph.D., Research Associate, Howard Hughes Medical Institute, UCSF, reported on the role of a calcium-activated ion channel called TMEM16F, a putative membrane lipid scramblase, in the generation of MVs. He noted that elevation and maintenance of high internal calcium levels is required for the activation of lipid scramblases resulting in phosphatidyl serine (PS) exposure. He also noted that lipid-scramblase-induced PS exposure correlates with the generation of membrane-derived MVs in normal platelets and red blood cells. He reported that platelets and RBCs lacking TMEMF do indeed show an impaired ability to produces MVs and membrane budding. Therefore, his group concluded that TMEM16F-mediated MV formation may prove to be an important step in pathologic conditions such inflammatory arthritis.

Propagation of the Unfolded Protein Response via EVs in Brain Tumor Cells

Jazmina Redzic, Ph.D., a postdoctoral fellow in the laboratory of Michael Graner, Ph.D., University of Colorado-Denver School of Medicine, Deparment of Neurosurgery, discussed work investigating the possible propagation of the unfolded protein response (UPR) by EVs in brain tumor cells. The UPR is a cellular mechanism related to the endoplasmic reticulum (ER) that is activated by various stressors such as hypoxia, oxidation, or changes in intracellular Ca+2 concentration. Dr. Redzik’s initial approach was to compare the proteomic profiles of stressed and unstressed cells derived from two different brain tumor types—U87MG cells are a glioblastoma cell line, while UPN933X96 cells are representative of an astrocytic oligodendroglioma. Mass spectrometry analyses were performed on lysates of stressed and unstressed cells, as well as from cells treated with EVs purified from stressed and unstressed cells. Changes in the proteome of stressed and unstressed cells were observed and, furthermore, changes in the proteome for unstressed versus stressed cells paralleled the changes observed for cells treated oth EVs purified from unstressed and stressed cells. The group concluded that proteomic changes associated with ER stress are propagated by EVs from stressed cells.

Y-Box Protein Is a Critical Sorting Factor in miRNA Packaging

Matthew J. Shurtleff, a graduate student in the laboratory of Nobel Prize winner Randy Schekman, Ph.D., Professor of Cell and Developmental Biology in the Department of Plant & Microbial Biology at UC-Berkeley, and Investigator with the Howard Hughes Medical Institute, described his efforts to identify and functionally validate exosomal miRNA sorting components by investigating miRNA packaging into exosomes directly in a cell-free biochemical reaction, without confounding variables. To identify selectively packaged miRNAs, Shurtleff’s group purified exosomes fromHEK293T conditioned media using a three-step strategy. First, Illumina-based small RNA sequencing revealed several miRNAs that are highly enriched in exosomes relative to cells. Next, the group developed a cell-free reaction to detect the selective incorporation of an exosomal miRNA. Cytosolic and crude membranes prepared from HEK293T cells were mixed with synthetic miRNA (miR-223) and ATP and incubated at 30 degrees C. miR-223, but not a cellular miRNA (miR-190), as incorporated into an RNase-protected membrane in a detergent-sensitive manner. Protection was dependent on cytosol proteins, membranes, ATP, and incubation at physiologic temperatures. To identify proteins that are directly involved in packaging miR-223, the group combined in vitro packaging with proteomics. They found that Y-box protein 1 (YBX1) co-precipitated with biotinylated miR-223 pulled down from a complete cell-free reaction. The group next generated at YBX1 knockout cell line (YBX1) by CRISPR/Cas9 genome editing and found that cytosol from deltaYBX1 cells did not support miR-223 packaging in vitro. However, the packaging was restored with cytosol from deltaYBX1 cells transfected with YBX1. Mr. Schurtleff noted that deltaYBX1 cells were only partially defective in YBX1 secretion due to overexpression of a germ-line-restricted YBX1 paralog (YBX2). However, secretion of miR-223 was found to be further diminished when YBX2 was depleted in deltaYBX1 cells. The group concludes that efficient packaging of YBX1 in vitro and in vivo requires Y-box protein with YBX1 as the dominant sorting factor in HEK293T.

Advantages of iZON qNano System; Trial of Aptamer-Based Exosome Isolation

The following speaker was Amy Phillips, Ph.D., a bionanotechnologist, who had journeyed all the way from New Zealand to represent her company, iZON Sciences. In particular, she emphasized the number of advantages conveyed by her company’s product, the qNano System, in work with exosomes. Dr. Phillips noted that recent developments in iZON’s qNano System have allowed it to determine exosome size and concentration, with particle-by-particle measurements, meaning that concentrations across specific size ranges can be quantified. Furthermore, she noted that iZON has developed a unique, charge-based method that permits the detection of exosome surface markers with high sensitivity. Dr. Phillps said that the qNano System is able to identify subpopulations with samples, even those of the same size. This is a crucial advantage as it permits the percentage of exosomes carrying a specific marker to be calculated—a fundamental deficiency of other current techniques. Finally, Dr. Phillips described an aptamer-based method of exosome isolation that is currently being verified in a worldwide trial and shows significant promise.

Characterization of Transplant-Organ-Specific MVs

Following lunch, the first speaker was Prasanth Vallabhajosyula, M.D., a cardiovascular surgeon at the Hospital of the University of Pennsylvania, who spoke on the critical need for a non-invasive biomarker platform for monitoring rejection in the field of transplantation. His group’s hypothesis is that all transplanted organs/tissues release donor-specific MVs into recipient bodily fluids and that the tissue-specific characterization of these MVs would prove to be a novel biomarker platform for monitoring transplant rejection. The experimental approach was study the concept in islet transplantation and renal transplantation. The conclusion was that transplanted tissues do release a stable and distinct MV signature into recipient bodily fluids that can be tracked over long-term followup. Transplant-specific MVs can be isolated with high purity, and the characterization of their protein and RNA cargo may serve as a novel biomarker platform for monitoring transplant rejection.

Suppression of HCV Replication by Liver-Derived Exosomes

Michae Kriss, M.D., at the University of Colorado School of Medicine, Division of Gastroenterology, next discussed his group’s evaluation of the anti-viral effect of exosomes derived from human liver sinusoidal endothelial cells (hLSECs) on hepatitis C virus (HCV)-infected hepatocytes. The group concluded that exosomes derived from hLSECs treated with either peg-IFNalpha or IFNA suppress viral replication in HCV-infected hepatocytes via shared and distinct anti-viral pathways.

EVs and Non-Alcoholic Steatohepatitis

The following speaker was Petra Hirsova, Ph.D., from the Liver Pathobiology Laboratory of Gregory J. Gores, M.D., at the Mayo Clinic in Rochester, Minnesota, who spoke on the possible process underlying non-alcoholic steatohepatitis (NASH). Although toxic lipids activate hepatocyte-expressed death receptor 5 causing lipotoxicity, how this process is linked to NASH is not clear. Dr. Hirsova and her group hypothesized that toxic lipids induce hepatotoxic release of EVs that induce the proinflammatory phenotype of macrophages seen in this condition. Experiment results indicated that lipotoxicity does induce release of EVs from hepatocytes that then activate macrophages toward a proinflammatory phenotype. Dr. Hirsova’s group speculates that inhibition of ROCK1-dependent EV release by hepatocytes may prove beneficial in steatohepatitis.

Effects of Macrophage-Derived EVs and miRNAs on HIV Infection and Replication

Next to the podium was Ken Witwer, Ph.D., Assistant Professor of Molecular & Comparative Pathobiology at The Johns Hopkins School of Medicine, who addressed the effects of macrophage-derived EVs and miRNAs on HIV infection and replication. He began by remarking that noted overlap between the biogenesis and physical characteristics of retroviral particles and EVs has prompted investigations into the influence of EVs on the life cycle of retroviruses, especially of HIV-1. Dr. Witmer’s group compared the influence of miRNAs and EVs on HIV-1 replication primary monocyte-derived macrophages (MDM). The apparent anti-viral effects of several transfected miRNAs were confirmed, however in a least one case, these effects were due to toxicity. Unexpectedly, Dr. Witmer said, EVs purified from macrophages that had or had not been transfected with specific miRNA mimics, also inhibited viral replication, but this effect was independent of the miRNA content of the parent cell. Dr. Witmer concludes that, while the influence of several miRNAs has been confirmed in primary macrophages, the contribution of these miRNAs in the context of EV shuttling remains an open question.

Infectious Prion Proteins Co-Localize with Plasma EVs

The next speaker was Paula Saa, Ph.D. in Molecuar Biology, Scientist and Principal Investigator at the Red Cross. She began by noting that the recent development of variant Creutzfeldt-Jakob disease (vCJD) in three recipients of non-leukoreduced RBCs from asymptomatic donors, who subsequently developed the disease, has confirmed existing concerns regarding the possible spread of transmissible spongiform encephalopathies (TSEs) via blood products. Ms. Saa noted that the presence of disease-associated misfolded prion protein (PrPTSE), generally associated with infectivity, has been demonstrated in the blood of vCJD patients. However, its origin and distribution in this biological fluid are currently unknown. Various studies have identified the cellular prion protein (PrPC) and PrPTSE in exosomes from various TSE models; and exosomes released by TSE-infected cells have caused disease upon injection into mice. In addition, PrPC has been detected in human blood-circulating EVs, suggesting that they may also contain PrPTSE and serve as vehicles for the transfusion transmission of TSE. In this study, Ms. Saa and her group demonstrate the association of PrPTSE with plasma EVs from mice infected with human TSE agents. Ms. Saa reported that her group’s research had demonstrated detection of PrPTSE in plasma fractions containing exosomes obtained from pre-clinical and sick animals or the first time. Further confirmation of PrPTSE binding to eosomes by electron microscopy is presently underway. She asserted that biochemical identification of PrPTSE in blood-derived EVs provides an invaluable foundation for the development of new diagnostics and potential targets for TSE treatment, and the design of appropriate removal technologies to ensure blood safety. In addition, further characterization of PrPTSE-containing MVs will allow identification of the cellular origin of prion-containing exosomes and the site(s) of prion replication in blood. In closing, Ms. Saa issued the caveat that these findings and conclusions have not been formally disseminated by the FDA and should not be construed to represent any Agency determination or policy.

A Novel Role for Microparticles in Dengue Pathogenesis

Panissadee Avirutnan, Ph.D., Assistant Professor, Mahidol University, journeyed all the way from Thailand to discuss the role of microparticles (MPs) in the pathogenesis of dengue virus infection. Perhaps surprisingly, Dr, Avirutnam said that dengue is the most important, mosquito-transmitted viral disease in the world. Approximately half the world’s population is now at risk for this reemerging pathogen. Dengue virus (DENV) infection causes clinical symptoms that range from a self-limited mild flu-like illness, called dengue fever (DF) that usually resolves without any complications, to a life-threatening capillary leakage syndrome called dengue hemorrhagic fever or dengue shock syndrome (DHF/DSS). The latter is characterized by abnormalities in homeostasis and increased vascular permeability, leading to bleeding tendency and plasma leakage. During acute dengue disease, cellular activation and apoptosis directly induced by DENV, as well as inflammatory mediators and reactive oxygen species generated from the interaction between virus (or infected cells) and the host immune system may trigger many types of cells to shed MPs. Dr. Avirutnam and her group performed experiments to assess this possibility. Their work showed that infection of various susceptible cells by DENV led to apoptotic death an MP release. These DENV-infected cell-derived MPs had a viral envelope protein and a non-structural protein (NS1) on their surfaces and were capable of triggering the complement system. Further analysis showed that MPs generated from red blood cells and platelets are two major MP populations in the circulation of DENV-infected patients. Elevated levels of RBC-derived MPs correlated directly with DENV disease severity, whereas as significant decrease in platelet-derived MPs was associated with a bleeding tendency. Dr. Avirutnan concluded that her group’s results point to multiple roles of MPs in dengue pathogenesis and provide a potential novel biomarker that can distinguish severe DHF patients from mild DF patients.

Exosomes from EBV-Infected Cells Promote Phenotypic Modulation in Target Cells

Asuka Nanbo, Ph.D., Associate Professor, Graduate School of Medicine, Hokkaido University, noted that several lines of evidence suggest that exosomes from EBV-infected cells are internalized by other cells and transfer viral factors, including EBV-encoded latent membrane protein and microRNAs to the recipient cells. The detailed mechanism of this process is still poorly understood and Dr. Nanbo’s group sought to investigate this mechanism. Their work demonstrated that exosomes released from EBV-infected B-cells are internalized by caveolin-dependent endocytosis, which, in turn, contributes to phenotypic changes in the recipient cells by transferring one or more viral factors.

Neisseria gonorrhoeae Induces Production of Exosomes in Human Epithelial Cells

Kathleen Goodmon, Ph.D. candidate, Molecular and Translational Medicine, Boston University School of Medicine, Graduate Medical Sciences, began her talk by noting that N. gonorrhoeae is one of several bacterial pathogens that promote persistence in human cells by modulating host cell death mechanisms. Inhibitors of apoptosis proteins (IAPs) have previously been implicated in the establishment of microbial infection in host target cells due to their roles in both immune signaling and cell death. Dr. Goodmon said that her group, using female endothelial cells, had earlier shown that N. gonorrhoeae induces the expression of cIAP2, which, in turn, protects against host cell death during infection. Whil cIAP2 was detected early on in whole cell lysates, cIAP2 levels were not detected in whole cell lysates obtained 24 hours after N. gonorrhoeae stimulation. In addition, discrepancies between high cIAP2 mRNA levels initially and nearly absent cellular cIAP2 protein levels at later time points led the group to explore changes in cIAP2 localization in N. gonorrhoeae stimulated epithelial cells. These studies demonstrated that cIAP2 localization to exosomes was specific to bacterial infection and that this localization was due to both alterations in exosome content and increased production of exosomes. Dr. Goodmon noted that these studies are the first to show that a bacterial pathogen increases the production of exosomes released by human epithelial cells. In conclusion, she said her group’s studies reveal that delivery of cIAP2 by exosomes to uninfected cells may promote host cell survival and suggest a novel mechanism for bacterial induced intracellular persistence.

EVs Implicated in Propagation of Misfolded SOD1 in ALS Tissue

Sarah M. Fernando, Ph.D. candidate, Department of Pathology and Laboratory Medicine, The University of British Columbia, focused on work to determine if EVs are a mode of transportation for cell-to-cell propagation of misfolded superoxide dismutase 1 (SOD1) in amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, in ALS mouse models and in patient tissue, and to characterize any differences in the biology of EVs from these tissues. In summary, the scientists concluded that misfolded SOD1 is present on EVs isolated from ALS patients an murine neuronal tissues, and is abnormally localized to the surface of vesicles. Wild-type SOD1 is normally found in the lumen of EVs, which suggests that misfolded SOD1 may be sorted into EVs differentially from wild-type SOD1. Dr. Fernando also noted that data from post-mortem patient tissue also confirm a redistribution of SOD1 in the diseased state in humans. In adition, cell culture results indicate that the secreted EV-containing fraction is competent to induce misfolding of wild-type SOD1, implicating the EV transport system in the propagation of SOD1 misfolding seen in disease.

Exosomes As Predictive Biomarkers and Pathogenic Agents in Alzheimer’s Disease

Erez Eitan, Ph.D., Visiting Fellow in the Laboratory of Neuroscience at the National Institute on Aging Intramural Research Program, first described recent work to establish that neuronal marker (L1CAM and NCAM)-enriched exosomes isolated from blood samples of presymptomatic and symptomatic Alzheimer’s disease (AD) patients show significantly higher levels of Abeta and p-Tau (T181 and S396) than do samples from age-matched control subjects. In fact, these levels discriminated patients from controls with >96% accuracy. Next, the group sought to determine if exosomal Abeta and/or p-Tau can be neurotoxic. Exosomes produced by cells expressing any of the three different presenilin 1 (PS1) mutations contained elevated levels of Abeta and ubiquinated proteins. Exposure of rat cerebral neurons to exosomes from neuroglioma cells resulted in cell death. Lysosome inhibition with bafilomycin increased the release of Abeta-containing exosomes from neuroglioma cells and increased their neurotoxic effect. Imaging of neurons exposed to fluorescently tagged exosomes revealed that exosomes from PS1 mutant cells transfer Abeta aggregates into neurons, which is associated with caspase-3 activation and neurite degeneration. In conclusion, Dr. Eitan stated that his group has shown that levels of Abeta and p-Tau in plsma exosomes are elevated early in AD, and may provide a sensitive biomarker; and exosomal Abeta can be transferred between neurons and may play a role in neuronal dysfunction and degeneration in AD.

Purification and Characterization of EVs from HIV-1 Patient Plasma

Audrey Hubert, Ph.D. candidate, Department of Microbiology & Immunology, Laval University, reported that her group has shown, for the first time, that EVs in the plasma of untreated HIV-1-infected patients are heterogeneous in size and, on average, larger than in other subjects. Acetylcholinesterase-positive EVs (exosomes) are more abundant in these patients and contain considerable amounts of a mitochondrial protein involved in apoptosis (DAP-3). Perhaps more interesting, she noted, was the detection of vesicles bearing increased levels of miR-155 and miR-223, respectively, in conjunction with HIV-1 infectionand subsequent inflammatory episodes. In conclusion, Ms. Hubert said that this study offers new evidence for the value of plasma EVs as indicators of pathogenesis and inflammatory status. This will be helpful for long-term development of better and more accurate biomarkers in the form of miRNA or EVs or both.

miRNA Content of CSF EVs Derived from Glioblastoma Patients

Clark Chen, M.D., Ph.D., Associate Professor and Chief, Stereotactic and Radiosurgery, Department of Neurosurgery, UC San Diego Health System, began by stressing the urgent need for a minimally invasive biomarker for the grievous disease of glioblastoma. Toward this end, Dr. Chen and his group investigated the miRNA content of sera and CSF of patients afflicted with glioblastoma. Perhaps surprisingly, Dr. Chen reported finding less than one molecule of miRNA per 1,000-10,000 EVs. According to Dr. Chen, this suggests that most EVs in clinical samples carry little or no miRNAs and raise questions as to the biologic plausibility of cell-cell communication through EV miRNA. When sub-fractionated into exosomes (30-100 nm EVs) and MVs (>100 nm EVs), his group found that miRNAs reside in the exosome fraction. Finally, Dr. Chen asserted that, despite their relative rarity, CSF EV miRNA content, when quantitated in absolute levels, indicate the potential and challenges of using CSF EV as a platform for clinical biomarker development.

Exosomes from RNA-Virus-Infected Cells (HIV-1, HTLV-1, Rift Valley Fever)

Saturday evening’s talks began with Fatah Kashanchi, Ph.D., Director of Research, National Center for Biodefense and Infectious Diseases, George Mason University, explaining that while the role of exosomes in viral pathogenesis and disease states remains mostly unknown, it is now widely accepted that exosomes play key roles in intercellular communication, cellular inflammation, antigen presentation, programmed cell death, and pathogenesis. Dr. Kashanchi said that HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derivived miRNA, first reported by Dr, Kashanchi’s group, is the TAR (Trans-Activation Response element) miRNA. Prior exposure of naïve cells to exosomes from HIV-1-infected cells increased the susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. In addition, the group has found that TAR is able to activate cytokines in the recipient cells via the TLR (toll-like receptor) pathway. In a similar fashion, exosomes from human T-lymphotropic virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) also show the presence of viral components including Tax protein and pro-inflammatory mediator IL-6, as well as the viral mRNA transcripts tax, hbz, and env. Finally, the group has recently isolated resistant clones from Rift Valley Fever-infected cells and found that critical viral proteins such as N and NSS as wll as genomic RNA are present in these exosomes.

Urgent Need for Standardization in Extracellular RNA Studies

Louise Laurent, M.D., Ph.D., Assistant Professor, Department of Reproductive Medicine, UC San Diego School of Medicine, has recently done significant work investigating the possible relationships between exRNA and placental dysfunction. However, for her two talks at the ASEMV annual meeting, she moved away from her own research to focus on two areas of significant concern to the ASEMV. First, she addressed the enormous problem of standardizing work with exosomes and extracellular RNA (exRNA), in general, and then, in her second talk, she discussed improvements that could be made in incorporating exosome-related terminology into the Gene Ontology program. Dr. Laurent is a member of the Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium, which is a group of laboratories funded by the NIH to develop robust and standardized methods for collection and processing of biofluids, separation of different exRNA-containing particles, and isolation and analysis of exRNA. In her first talk, she basically outlined many of the enormous standardization problems currently confronting exRNA researchers. First of all, there are the different biofluid types that exRNA can be isolated from: serum, plasma, urine, CSF, saliva, etc. Some contaminants are specific to specific biofluids, she noted, and serum and CSF have EVs with different size ranges. Even the collection tubes are variables, Dr. Laurent noted, as they can include additives such as preservatives and anti-coagulants that can be confounding, and the cell removal steps can be very different. There are also many different particle types that contain exRNA, including EVs, ribonucleoproteins, and lipoproteins, among others, and many different methods to isolate them. The possibility of cross-contamination among particle types is significant. Also, there are the numerous ways in which exRNA is currently isolated, including precipitation, filtration, affinity purification, and ultracentrifugagtion, among many others. The yield and size of the particles obtained vary depending on the isolation method. There are now commercial kits for exRNA isolation, but the yield varies from kit to kit. Dr. Laurent also noted that use of proteinase K can also affect yield and that ethanol affects the size distribution of purified RNA. In summary, significant work must be done to standardize the methods used for exRNA purification if results are to be validly compared from lab to lab. Dr. Laurent’s second talk, on integrating exRNA work with the Gene Ontology project, was preceded by a talk by ASEMV President Dr. Gould, given for his Hopkins colleague, Dr. Florin Selaru, on the subject of EV-mediated transfer of miRNAs in vivo for the treatment of cancer.

EV-Mediated Transfer of miRNAs in Vivo for the Treatment of Cancer

ASEMV President Stephen Gould, M.D., Professor of Biological Chemistry at The Johns Hopkins University School of Medicine, gave the presentation originally scheduled to be given by his colleague Florin Selaru, M.D., Assistant Professor of Medicine at Johns Hopkins. The address focused on the vesicle-mediated inhibition of cholangiocarcinoma (CCA). CCA is the second most common primary liver cancer and the survival of CCA patients is dismal, usually measured in months. In preliminary studies, the Hopkins group identified miRNA species that are downregulated in fibroblasts, along the continuum of inactive to activated to cancer-associated fibroblasts (CAFs). In vitro studies showed that upregulation of these miRNAs in CAFs results in less cancer growth and less invasion, consistent with the hypothesis that CAFs play a key role. Next, the group investigated how the signaling between CAFs and cancer might take place. Experiments demonstrated that there was significant exchange of EVs between cancer cells and CAFs, constituting a rich signaling network. Finally, the group performed in vivo experiments that demonstrated that systemic delivery of EVs loaded with miRNAs concentrate in the liver tumors and are sufficient to decrease the cancer growth and increase the overall survival of treated animals. In conclusion, Dr. Gould reported that the group’s study had adduced evidence for the existence and functioning of EV exchange between cancr cells and CAFs, and also demonstrated that EVs can be utilized for therapeutic purposes, with no evidence of side effects. Further study will be required to determine the exact miR composition that needs to be delivered to human CCAs to elicit an optimal therapeutic effect.

Gene Ontology for the Extracellular RNA Field

Following Dr. Gould’s talk, Dr. Laurent devoted her second talk, and the last presentation of Saturday evening, to a discussion of the Gene Ontology (GO) project and the role that ASEMV members might play in advancing the cause of exosomes, exRNA, etc., in this project. The GO project is a collaborative effort to address the need for consistent descriptions of gene products across databases. Founded in 1998, the project began as a collaboration among three model organism databases, FlyBase (Drosophila), the Saccharomyces Genome Database (SGD), and the Mouse Genome Database (MGD). The GO Consortium (GOC) has since grown to incorporate many databases, including several of the world's major repositories for plant, animal, and microbial genomes. The GO Contributors page lists all member organizations. The GO project has developed three structured, controlled vocabularies (ontologies) that describe gene products in terms of their associated biological processes, cellular components, and molecular functions in a species-independent manner. There are three separate aspects to this effort: first, the development and maintenance of the ontologies themselves; second, the annotation of gene products, which entails making associations between the ontologies and the genes and gene products in the collaborating databases; and third, the development of tools that facilitate the creation, maintenance, and use of ontologies. The use of GO terms by collaborating databases facilitates uniform queries across all of them. Controlled vocabularies are structured so they can be queried at different levels; for example, users may query GO to find all gene products in the mouse genome that are involved in signal transduction, or zoom in on all receptor tyrosine kinases that have been annotated. This structure also allows annotators to assign properties to genes or gene products at different levels, depending on the depth of knowledge about that entity. Shared vocabularies are an important step towards unifying biological databases, but additional work is still necessary as knowledge changes, updates lag behind, and individual curators evaluate data differently. The GO aims to serve as a platform where curators can agree on stating how and why a specific term is used, and how to consistently apply it, for example, to establish relationships between gene products. Dr. Laurent noted efforts to incorporate exRNA terms appropriately into GO might help resolve the current terminology dilemma that afflicts exRNA work, and might also allow the true amount of work being done in this area to be rightly represented.

SUNDAY

Disease-Modifying Activity of Heart-Derived Exosomes

The first presentation on Sunday morning was delivered by Eduardo Marban, M.D., Ph.D., Director of the Cedars-Sinai Heart Institute in Los Angeles, who described experiments indicating that exosomes from cardiosphere-derived cells (CDCs) could be used to both decrease the scarring seen in myocardial infarctions (MIs) and to increase the formation of viable heart tissue after MIs. The doctor noted that an axiom in cardiology is that the scarring seen in MIs never goes away and the tissue that is lost is never regenerated. CDC-derived exosomes, however, have the potential to shatter this dogma and provide immense benefit to those who suffer MIs, an estimated 1.1 million people in the U.S. each year, for example. Dr. Marban is eager to move CDC-derived exosomes and their contents into clinical trials for the treatment of damage from MIs as soon as possible. He also believes that these exosomes and their contents may have non-ischemic cardiac applications in scleroderma and amyotropic lateral sclerosis (ALS). Previously, Dr. Marbán designed and completed the first-in-human NIH-funded CADUCEUS trial. This study was the first to show that cell therapy can repair "irreversible" tissue damage caused by heart attacks. Dr. Marban’s discoveries regarding allogeneic heart stem cells have led to a new clinical trial, ALLSTAR, now in phase 2 and being run by Capricor Therapeutics, a California-based biotechnology company. Dr. Marban counts the NIH and the California Institute for Regenerative Medicine, headquartered in San Francisco, CA, among the supporters of his work.

miRNAs and Neurodegenerative Diseases

Kendall Van Keuren-Jensen, Ph.D., Assistant Professor, Translational Genomics Research Institute (TGen), focuses on neurodegenerative diseases, particularly Parkinson disease and Alzheimer disease, and she followed Dr. Marban’s talk with a discussion of her recent work to determine if miRNAs might be indicative of neurodegenerative diseases. This is a particularly important question given the difficulty of taking biopsies of brain tissue and the relative ease of obtaining samples from bodily fluids such as serum. Dr. Keuren-Jensen began by describing her work to study CSF and serum from post-mortem-verified patient samples. Although she found significant numbers of miRNAs in both fluids (532 in serum, 486 in CSF), there was little overlap between the miRNAs in the two fluids. One microRNA, miR-34C, appeared to be a possible biomarker for PD with dementia, but more work is required to confirm this.

Functional Role for Cardiomyocyte-Derived EVs in Cardiac Remodeling

Saumya Das, M.D., Ph.D., Co-Director, Cardiovascular Genetics Institute at Beth Israel Deaconess Medical Center (BIDMC), Assistant Professor, Medicine, Harvard Medical School, began by outlining the significant problem of congestive heart failure and describing an implantable resynchronization device that can restore synchronous contractions, but which 30% of patients do not respond to. For these patients, the only option now is a heart transplant. A key goal of Dr. Das’s group is to find a way to identify responders and non-responders to this treatment. Here, he reported that his group had identified a specific miRNA, miR-30d, as being associated, when elevated, with beneficial cardiac remodeling, and implicated in cardiac remodeling—specifically, adaptive hypertrophy, anti-fibrosis, and anti-apoptosis—in models of HF. miR-30d was expressed in cultured primary cardiomyocytes and released in EVs in response to mechanical stress. Elevation of miR-30d was also associated with responders and not non-responders to the resynchronization device. Dr. Das hypothesized that miR-30d abrogates TNF-mediated deleterious effects and suggested that it may represent a therapeutic target space for non-responders to the resynchronization therapy. In conclusion, Dr. Das maintained that this study supports an emerging paradigm of extracellular EV-containing RNA biomarkers that are functionally implicated in disease pathogenesis.

A Generic Approach for Isolating and Characterizing EVs

Horst Vogel, Ph.D., Professor of Physical Chemistry, Institute of Chemical Sciences and Enginering ISIC, Ecole Polytechnique Federale de Lausanne, Switzerland, brought forward a new and different approach to isolating and characterizing EVs. Given the problems already described, this presentation was welcomed by many. Dr. Vogel outlined an approach incorporating ultracentrifugation and size -exclusion chromatography for isolation and a model-free fluorescence fluctuation analysis for the investigation of the physical and biochemical properties of EVs secreted by mammalian cells. Dr. Vogel maintained that his approach produces enriched samples of morphologically intact EVs, free of extra vesicular proteins, and allowing labeling of marker molecules on the vesicle surface for single-vesicle analysis with single-molecule sensitivity. Furthermore, he argued that this approach provides information on the distribution profile of both EV size and relative expression level of a specific exosomal marker, thus deciphering the overall heterogeneity of EV preparations. In addition, Dr. Vogel noted that, while no one is looking inside EVs for signaling, they comprise parts of the cell’s plasma membrane and cytosol and represent the smallest autonomous containers performing cell signaling reactions. Using single-molecule fluorescence microscopies, his group has measured in individual vesicles, the different steps of G-protein-coupled receptor-mediated signaling such as ligand binding to receptors, subsequent G-protein activation, and finally arresting translocation indicating receptor deactivation. Dr. Vogel suggested that the observation of cellular signaling reactions in individual vesicles opens new directions for the analysis of EVs.

Exosomal miRNAs of Cancer-Associated Fibroblasts (CAFs) in Breast Cancer

Elvira Donnarumma, Ph.D., IRCCS, SDN Foundation, Naples, Italy, followed and began by noting that a poor prognosis in breast cancer strongly depends on the contribution of cancer-associated fibroblasts (CAFs) to the breast cancer microenvironment. She highlighted recent evidence that exosomes and their cargo may traffic between cells and affect the biological behavior of recipient cells. The goal of this particular study was to identify oncogenic miRNAs involved in stroma-tumor communication. The first finding was that, in exosomes from CAFs, levels of miR-21-5p, miR-378e, and miR143-3p were increased compared to levels in normal fibroblast exosomes. In addition, a medium enriched in CAF exosomes was able to increase the levels of miR-21-5p, miR-378e, and miR-143-3p in T47D epithelial cells, suggesting that these miRNAs are transferred via exosomes. In addition, the group showed that TGF-beta, distinct from its direct role in activating normal fibroblasts to CAFs, increases the levels of these three miRNAs in normal fibroblast exosomes. The group also provided evidence for the role of CAF exosomes and their miRNA contents in the induction of “stemness” phenotype in breast cancer cells. Furthermore, T47D cells exposed to CAF exosomes showed an increased capacity to form mammospheres, and increased stem cell markers nanog and oct4. Dr. Donnarumma believes that further investigation will clarify the biological functions of CAF exosomes and their miRNA contents in the pathogenesis and progression of breast cancer.

Flow Cytometric Characterization of the Antigenic Spectra of Individual EVs

Anush Araklyn, Ph.D., Visiting Fellow, Margolis Lab: Section on Intercellular Interactions, National Institute of Child Health and Human Development, NIH, spoke next and described a nanoparticle-based flow technique for analyzing antigens on single nano-sized EVs from the blood. The novel technique consists of immuno-capture of EVs with 15 nm magnetic nanoparticles, staining captured EVs with antibodies against their antigens, and separating them in a magnetic field from unbound EVs and free antibodies, followed by flow analysis. Dr. Araklyn said the technique permits them to finely characterize EV populations in the blood according to their antigenic distribution, including minor EV fractions and combination of markers expressed by individual EVs. Using this technique, Dr. Araklyn’s group demonstrated that individual blood EVs carry different sets of antigens, none being ubiquitous, and quantitated their distribution. The physiological significance of antigenically different EVs and their correlation with different pathologies can now be directly addressed, Dr. Araklyn asserted.

Differential Fates of Biomolecules Delivered to Target Cells by EVs

Masamitsu Kanada, Ph.D., Postdoctoral Fellow, Stanford University School of Medicine, described work he has done to demonstrate that exosomes and MVs are structurally and functionally distinct. Again, given the widespread confusion over this issue expressed throughout this meeting, these would certainly be welcome results if validated and accepted. First of all, he described exosomes as being 40-300 nm in size while MVs are 90-600 nm. He also said that the two types of vesicles differ in volume and in surface charge properties. Most MVs externalize phosphatidyl serine (PS), while less than 10% of exosomes do. Both types of vesicles are taken up similarly by recipient cells. Only MVs and not exosomes will induce luciferase expression in recipient cells. Dr. Kanada believes MVs do this mainly by the delivery of plasmid DNA (pDNA) to the recipient cells. He noted that exosomes encapsulate intact mRNA but do not deliver functionally deliver nucleic acids. MVs, on the other hand, encapsulate both intact mRNA and intact pDNA, and functionally deliver DNA, but not RNA. He presented data demonstrating that MVs functionally deliver Cre-expressing pDNA both in vitro and in vivo. Dr. Kanada said these results have significant implications for understanding the role of EVs in cellular communication and the development of delivery tools. In addition, the data suggest that studies from EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer.

miRNAs in Human CSF As Biomarkers for Alzheimer’s Disease

Julie Saugstad, Ph.D., Associate Professor, Director, Core Molecular Laboratories & Training for Research, Oregon Health & Science University, discussed her work to examine the utility of CSF-derived miRNAs as biomarkers for Alzheimer’s disease (AD). Clearly, the ability to find biomarker(s) that would allow the earlier diagnosis and treatment of AD would be of significant benefit. Dr. Saugstad’s approach was to examine miRNA expression in the CSF of living donors (47 healthy, 47 AD) obtained from the Oregon Alzheimer’s Disease Center using TaqMan Human MicroRNA Arrays. The group found 18 miRNA biomarker candidates: 17 with decreased presence in AD and 1 with increased presence in AD. In addition, they ran inferential tests based on the log-rank statistics and Kaplan Meier curves, which utilize Ct and Ct to reference: U6 small RNA. Of 643 miRNAs evaluated, 74 had log-rank p<0.05 and 28 had p<0.01. The top 20 most significantly differentially expressed miRNAs (p<0.006) had a false discovery rate of ~20%. The group also evaluated linear combinations of subsets of the top 20 miRNAs via best subsets logistic regression, and computed the area under the receiver operating characteristic (ROC) curve to ascertain classification performance. Top-performing linear combinations of 3, 4, and 5 miRNA have areas under the ROC of 0.80-0.87. Dr. Saugstad concluded that these studies highlight the potential of miRNAs in human CSF as clinical biomarkers for AD, and suggest that the use of multiple miRNAs improves the sensitivity and specificity of performance. She noted that future work would expand the pilot cohort, refine the statistical approach, and validate identical candidate miRNA biomarkers in an independent set of banked CSF samples.

Intercellular Transfer of Functional RNA Molecules through Exosomes

Sudipto Chakrabortty, a graduate student in the Laboratory of Thomas Gingeras, Ph.D., Cold Spring Harbor Laboratory, described his group’s work to perform detailed RNA profiling of twelve tumor- and primary cell-derived exosomes and nine body fluids. RNA-Seq analysis revealed two specific classes of non-coding RNA, namely miRNA and Y RNA to be abundant in exosomes. [Y RNAs are components of the Ro60 ribonucleoprotein particle, which is a target of autoimmune antibodies in patients with systemic lupus erythematosus. They are also necessary for DNA replication through interactions with chromatin and initiation proteins.] In addition, the ability of exosomes to transfer its RNA cargo as confirmed by both RNA-Seq and microscopy. The subcellular localization of the exosomal RNA upon transfer to recipient cells was found to be primarily cytoplasmic. The role of tumor-derived exosomes and exosomal RNA to induce apoptosis in multiple primary cells was investigated in vitro. The associated molecular phenotype in these cells was identified by differential expression analysis. Finally, the functional RNA molecule in exosomal RNA cargo determined to be responsible for inducing apoptosis was a small non-coding RNA RNY5. In particular, a specific 8-nucleotide motif in RNY5 was found to be responsible for the apoptotic phenotype.

Exploring the Path of Exosome Formation and Secretion with Next-Gen siRNA Tools

Alexander “Sasha” Vlassov, Ph.D., Senior Staff Scientist, Group Leader, Thermo Fischer Scientific, spoke next and discussed the use of RNA interference as a very useful tool for studying gene functions as it enables sequence-specific gene suppression in a variety of organisms and cultured cells. Dr. Vlassov noted that first-generation small interfering RNA (siRNA) technologies can yield confusing results in cell-based RNAi screens due to incomplete knockdown of the intended mRNA target, knockdown of unintended mRNAs, and siRNA toxicity. In its work, Dr. Vlassov’s group investigated the impact of 21 bp siRNA sequence design and LNA (locked nucleic acid), 2’-OMe, 2’-F chemical modifications on siRNA potency and off-target effects. Using a Support Vector Machine (SVM)-based learning paradigm, a novel hyperpotent siRNA designer was generated on the basis of over 3,000 individual siRNA knockdown test results. In parallel, the group screened over 100 siRNA modification patterns on more than 30 siRNA designs through a series of assays. A unique sequence-independent configuration of chemical modifications on the siRNA was identified that did not compromise siRNA knockdown potency, but efficiently reduced off-target effects as measured by microarray analysis and by high-content analysis in two cell-based assays. Thus, Dr. Vlassov stated, novel chemical modification, coupled to a hyper-potent siRNA designer provides an ideal technology, Silence® Select siRNAs, to enable more robust performance in RNAi screening.

Bovine miRNAs Are Bioavailable and Affect Gene Expression in Humans and Mice

After Sunday’s lunch, Janos Zempleni, Ph.D., Professor of Molecular Nutrition, University of Nebraska-Lincoln, began the afternoon session with a fascinating discussion of how miRNA from cows can affect gene expression in humans and mice. When healthy adult humans consumed nutritionally relevant doses of cow’s milk, postprandial concentration time curves suggested that meaningful amounts of miR-29b and miR-200c were absorbed, while the plasma concentration of miR-1 (negative control) did not change. The expression of RUNX2, a known target of miR-29b, increased by 31% in blood mononuclear cells following milk consumption compared with baseline. When milk exosomes were added to cell culture, mimicking postprandial concentrations of miR-29b and miR-200c, reporter gene decreased by 44% and 17%, respectively, compared with vehicle controls in HEK-293 human kidney cells. Dr. Zempleni said that his group’s bioinformatics analysis predicted that 175 miRNAs in bovine milk target approximately 10,000 human genes. When C57BL/6J mice a milk diet depleted of miRNA for four weeks, plasma levels of miR-29b were decreased by 61% compared with miRNA-sufficient controls, i.e., endogenous synthesis did not substitute for dietary depletion. Dr. Zempleni noted that when humans consumed a broccoli sprout meal, no postprandial increases were observed for plant-specific miR-167a or brassica-specific miR-824 in plasma. Dr. Zempleni said that, based on his group’s studies, he concludes that exosomes from bovine milk are bioavailable and elicit changes in gene expression in humans.

Cellular Communication via EVs During Stress

Antonio De Maio, Ph.D., Professor, Department of Surgery, UC San Diego School of Medicine, followed with a discussion of how EVs are involved in inter-cellular communication during conditions of stress. Dr. De Maio noted that stressed cells can release vesicles that contain information the type of injury they have experienced and this information can be targeted to healthy cells and prime them to develop the necessary mechanism to resist the insult. He said that the mechanism of communication during stress conditions has been termed the “Stress Observation System” (SOS). Heat-stressed cells release EVs containing heat-shock protein 70 (Hsp70), which is the major stress-inducible form of the heat shock family of proteins. EVs containing Hsp70 released by parenchymal cells have been shown to produce an inflammatory response when exposed to macrophages, which has been thought to be a mechanism for avoiding the propagation of the initial insult. Dr. De Maio noted that Hsp70 appears to be on the membranes of EVs rather than in the lumen, and remarked that this observation coincides with a large body of evidence for the presence of Hsp70 on the surface of cells. He further pointed out that Hsp70 does not have any consensus hydrophobic domains that would predict its insertion into lipid membranes. Using a liposome insertion assay, Dr. D Maio and colleagues have shown that Hsp70 is inserted into the liposome membrane by a process that is highly dependent on the negativity of the lipid within liposomes and is enhanced by a decrease in membrane fluidity. Hsp70 is inserted into the lipid membrane via the C-terminal end of the molecule, which contains the peptide-binding domain, and the process is regulated by the presence of nucleotides.

Role of Tumor-Derived Exosomes in Cancer Immunotherapy

Michael Olin, Ph.D., Assistant Professor, Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Minnesota Medical School, began by stating that in his recent studies, something appeared to be secreting CD200 (a type-1 glycoprotein) in order to suppress the powerful immunosuppressive response induced by glioma tumor cells. He hypothesized that this might be being done by exosomes and he is currently pursuing an investigation of this possibility with the hope that it might lead to identification of a therapeutic suppressor of the immune suppression induced by glioma and other tumor cells. Initially, his group had sought to determine if GL261 glioma-derived exosomes could inhibit the ability to mount an antigen-specific immune response in tumor-draining lymph nodes. Non-tumor-bearing mice were primed with OVA + Poly:ICLC +/- GL261-derived exosomes. In these experiments, inoculation with tumor-derived exosomes significantly suppressed an OVA-specific T-cell expansion and cytokine production. Additional experiments revealed that exosome activation was toll-like receptor (TLR)-mediated, potentially working through inflammasomes. The same response was observed in human glioma-derived exosomes. In addition, these responses were restricted as tumor-derived exosomes elicelicited them and non-tumor-derived exosomes did not. Based on these results, Dr. Olin and his group hypothesize that tumor-derived exosomes serve as a mechanism or immune suppression inhibiting the ability of the immune system to respond to neoplasms.

Reactive Oxygen Species, Exosomes, and Adult Cardiac Myocytes

Anne Knowlton, M.D., Professor, Department of Pharmacology, UC Davis Health System, described her work to determine if highly differentiated adult cardiac myocytes produce exosomes and she reported finding that they do, in response to brief hypoxia/reoxygenation, too short to cause necrosis. Additional work showed that the exosome cargo and the number of released exosomes vary with the stimulus. She noted that proteomic studies showed that exosomes released by cardiac myocytes after ethanol treatment with a concentration seen in humans consuming alcohol, contained more mitochondrial protein than seen in exosomes released after brief hypoxia/reoxygenation. Ethanol causes increased production of reactive oxygen species (ROS) by cells, and treatment with a combination of three different antioxidants reduced exosome production after ethanol exposure. Studies of the effect of these exosomes on other cardiac myocytes demonstrated mild to moderate injury, which was concentration-dependent. Dr. Knowlton and her group concluded that complex cell types, such as cardiac myocytes produce exosomes and the exosome content is specific to the inducing agent. Also, exosomes produced by healthy cardiac myocytes in response to mild injuries can have a mild adverse effect on other cells.

Programming Exosome Cargo and Targeting Cell Delivery

Travis Antes, Ph.D., Director of Product Management, Systems Biosciences, Inc. (SBI), described the development of new tools at SBI that permit the creation of “designer exosomes” that can carry any desired protein or RNA cargo for delivery to specific cell types. The XPack technology is an N-terminal protein tag sequence that allows for the packaging of protein fusions into the interior of exosomes for efficient delivery to target cells. RNAs can also be packaged into exosomes using a unique XMotif RNA 3’ sequence tag that traffics them into exosomes for secretion. XStamp technology from SBI involves the C-terminal fusion of a protein sequence that presents the fused protein on the surface of exosomes. Together, Dr. Antes maintained that this complete system now enables the engineering of exosome cargo and display of surface markers to program cell-specific delivery steps toward realizing the potential of using exosomes as therapeutic shuttles. Dr. Antes noted that SBI has already been cited in over 170 peer-reviewed journal articles and that this is strong evidence for the effectiveness of the tools the company has and is developing for the community.

Exosomes and MVs miRNAs in Human Bile for Assessment of Transplanted Liver in the Early Post-Operative Period

Daiki Yoshii, M.D., Department of Transplantation and Pediatric Surgery, Graduate School of Medical Sciences, Kumamoto University, stressed the current need for better ways to assess the condition of the transplanted liver in the early post-operative period and to make an accurate diagnosis that will aid the formulation of an appropriate therapeutic plan. He noted that liver biopsies are now the last resort for diagnosis, but are invasive and often inconclusive. In this presentation, Dr. Yoshii described his group’s work to determine if exosomes and MVs found in bile might prove useful in making assessments of transplanted liver, particularly in the early post-operative period. In their current work, they showed that beta-actin mRNA was detected from the majority of bile samples, suggesting that bile contained EVs and mRNAs were securely present inside EVs. Similarly, liver-specific albumin mRNA was also detected in the bile, suggesting, Dr. Yoshii said, that the bile contained liver-derived EVs. Interleukin-8, TNFproteoglycan 2, and defensin , were detected in cases of acute cellular rejection, and their expression decreased in response to therapy. In conclusion, Dr. Yoshii believes that bile-derived EVs are promising biomarkers for the assessment of pathological conditions of transplanted livers.

Platelet Microparticles and Derived miRNAs Reprogram Primary Human Macrophage Gene Expression and Function

Aurelie Corduan, Graduate Student, Center Hospital University of Quebec (CHUQ) Research Center/Center Hospital of Laval (CHUL), provided some initial background, describing platelets as anucleate cells in the blood that contain an abundant an diverse array of miRNAs, which are known as master regulators of mRNA translation. Previously, Dr. Corduan’s group had shown that activated platelets can release miRNA-containing microparticles (MPs) that can be internalized by cultured human umbilical vein endothelial cells (HUVEC). In the current study, Ms. Corduan’s group demonstrated that platelet MPs can be internalized by primary human macrophages and deliver functional miR-126. The increase in macrophage miR-126 levels was not prevented by actinomycin D, suggesting that is was not due to de novo gene transcription. Platelet MP-derived miR-126 downregulated expression of four predicted gene targets at the mRNA level. These effects were abrogated by expression of a miR-126 sponge, implying that the mRNA downregulatory effects of platelet MPs are mediated by miR-126. Platelet MPs induced a significant upregulation of 34 miRNAs and a simultaneous downregulation of 227 RNAs, including mRNAs encoding for cytokines/chemokines CCL4, CSF1, and TNF, which is consistent with the effective delivery of mRNA-regulatory platelet miRNAs to macrophages. These changes in macrophage gene expression were associatedwith a significant reduction in macrophage CCL4, CSF1, and TNF mRNA levels and cytokine/chemokine release , and accompanied by a marked increase in the phagocytic capacity of macrophages. Ms. Corduan and her group believe that these findings suggest that platelet MPs and derived miRNAs may modify the miRNA and mRNA transcriptomes of macrophages and reprogram macrophages towards a phagocytic phenotype. Platelet MPs may thus act as a natural vehicle for miRNAs, contribute to intercellular signaling, and condition the circulatory system under specific health conditions associated with platelet activation.

Exosomes Are Surface-Active Nanoparticles That Self-Assemble at Interfaces: Evidence and Applications

Mikhail Skliar, Ph.D., Professor, Chemical Engineering, University of Utah, described his group’s work to investigate and quantitate the surface activity of exosomes using several different analytical techniques. His group found that exosomes are surface active and migrate to the interfaces. Their surface activity is comparable, Dr. Skliar said, to that of strong surfactants in both their concentration dependence and their preferential migration to an interface. Direct visualization of the motion of individual exosomes by nanoparticle tracking analysis showed their preferential migration to an interface. Their interfacial dwelling was directly imaged by environmental SEM. Together, these results unambiguously show, Dr. Skliar asserted, that interfaces may act as exosome sinks. Measurements of the interfacial surface energy indicate that, once absorbed on the interface, the exosomes accumulate there and perhaps even saturate interfacial surfaces, given enough time and sufficiently high concentration in the bulk solution. Dr. Skliar noted several potential applications of these findings, including inexpensive and field-deployable measurements of exosome concentration; a novel technique for exosome isolation; and a method for loading exosomes on passive and targeted drug delivery systems.

Prostate Cancer Cell-Derived Exosomes in Oncogenic Reprogramming of Stem Cells

Asim Abel-Mageed, D.V.M., Ph.D., Professor of Urology, Director of Molecular Oncology Research Laboratories in Urology, Tulane University School of Medicine, began by noting that approximately 240,000 men are diagnosed with prostate cancer each year in the United States, and 29,900 die. Emerging evidence suggests that mesenchymal stem cells (MSCs) are often recruited to tumor sites, but their functional significance in tumor growth and disease progression has not been elucidated. Here, Dr. Abel-Mageed reported that prostate cancer (PC) cell microenvironment subverts PC patient adipose-derived stem cells (pASCs) to undergo neoplastic transformation. Unlike normal ASCs, the pASCs primed with PC cell conditioned media formed prostate-like neoplastic lesions in vivo and reproduced aggressive tumors in secondary recipients. The pASC tumors acquired cytogenic aberrations and mesenchymal-to-epithelial transition and expressed epithelial, neoplastic, and vasculogenic markers reminiscent of PC tumor xenografts. Mechanistic studies showed that PC-cell-derived exosomes are sufficient to recapitulate formation of prostate tumorigenic mimicry generated by CM-primed pASCs in vivo. In addition to downregulation of the large tumor suppressor homolog2 and the cell death protein 4, a neoplastic transformation inhibitor, the tumorigenic reprogramming of pASCs was associated with trafficking by, PC-cell-derived exosomes of oncogenic factors, including H-ras an K-ras transcripts, the oncomiRNAs miR-125b, miR-130b, and miR-155, as well as the Ras superfamily of GTPases Rab1a, Rab1b, and Rab11a. Dr. Abel-Mageed said that his group’s results suggest a new role for PC-cell-derived exosomes in the clonal expansion of tumors through neoplastic reprogramming of tumor-tropic ASCs in cancer patients. He referenced a recent paper on the subject (“Neoplastic Reprogramming of Patient-Derived Adipose Stem Cells by Prostate Cancer Cell-Associated Exosomes,” Stem Cells 32(4): 983-997, April 2014). [http://onlinelibrary.wiley.com/doi/10.1002/stem.1619/abstract].

A Positive miRNA Sensor Circuit for in Vivo Detection of Small RNA Activity

Vanille Greiner, Ph.D., Postdoctoral Scholar, Diabetes Center, UCSF School of Medicine, introduced here a highly responsive system for determining the expression of miRNAs in living single cells using an artificial genetic circuit, producing a positive readout for miRNA expression. To create a positive miRNA reporter, Dr. Greiner said that her team created a novel system that produces a positive signal when an miRNA enters a cell and is active. The system detects both moderately and highly expressed miRNAs, giving a proportional change in the EGFP expression level. In addition, changes in miRNA expression are detected rapidly and tracked by fluorescent analysis by flow cytometry anand microscopy in an in vitro cell culture system. Dr. Greiner said that the system is robust in that it produces a readily detectable signal in an in vivo xenograft model system that can be tracked during tumor progression. In conclusion, she maintained that their novel positive miRNA sensor system enables the detection of changes in a single miRNA both in cellular in vitro and in vivo models, constituting a superb tool for the study of extracellular miRNAs functionality and miRNA expression changes in mammalian development and disease.

Characterization of Circulating MV-Associated CD39 Family Ecto-Nucleotidases in Human Plasma

Linda Feldbrugge, M.D., Post-Doctoral Researcher, Laboratory of Simon Robson, Ph.D., Beth Israel Deaconess Medical Center/Harvard Medical School Teaching Hospital, began by providing some key background on purinergic signaling, noting that phosphohyrolysis of extracellular ATP and ADP to AMP and eventually adenosine is an essential step in this signaling that serves to regulate key pathophysiologic processes, including coagulation and inflammation. Dr. Feldbrugge noted that ecto-nucleoside triphosphate 1 (NTPDase1/C39) and adenylate kinase 1 (AK1) activities have previously been shown to be present in plasma, partly in soluble form, partly bound to circulating MVs. Other ecto-nucleotidases, however, have not been characterized in depth. The focus of the current study was to examine the ADPase activity in plasma and assess the relative contributions of the different enzymes and to determine if the enzymes are soluble or membrane-bound. An in vitro ADPase assay as developed to probe the ecto-enzymes responsible for the ectonucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating MVs. Based on the results, the group concluded that MV-bound NTPases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane-expressed NTPDases were also found in circulating MVs in human plasma, inclusive of CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members were found on distinct MV populations. A significant proportion of the MV-associated ecto-nucleotidase activity is sensitive to polyoxymethylene-related species 6 (POM6), implying the presence of NTPDases, either NTPDase 2 or NTPDase 3. Dr. Feldbrugge reported that they found that human plasma CD39, NTPDase 2, NTPDase 3, and NTPDase 8 are associated with circulating MVs, whereas only AK1 is present in soluble form. In conclusion, she stated that, using NTPDase-specific inhibitors, her group had developed a refined assay that can differentiate the activities of AK1 and those of various NTPDases in plasma samples. The group has also demonstrated the presence of CD39, NTPDase 2, NTPDase 3, and NTPDase 8 in circulating MVs. Together with AK1, these ecto-enzymes account for the majority of nucleotidase activity in the plasma of healthy individuals, suggesting a broader implication for the CD39 family in the regulation of extracellular nucleotide metabolism. In her presentation, she noted, interestingly, that CD39, NTPDase 2, NTPDase 3, and NTPDase 8 are all expressed and active in Crohn’s disease and found on the surfaces of MVs.

Efficient Exosome Capture Strategy for Disease Biomarker Discovery Using Engineered Peptides

Brandon Cook, Graduate Student, Chemistry, Washington State University, began his talk by emphasizing the need for a simple, direct, and global strategy for exosome capture, and this was certainly a dominant theme repeated throughout the meeting. Mr. Cook stated that his group’s effort to develop such a method (proprietary) was based on a hypothesis that peptides with selective affinity for highly curved and anionic phospholipid nanovesicles could be immobilized on a solid support to capture exosomes, independent of protein markers. A mini-library of peptides as engineered and synthesized using Biotage Initiator plus microwave reactor and characterized by mass spectrometry. Biotin was conjugated to peptides and liposomes were used as model systems to mimic exosomes. The liposomes (30 nm. 100 nm, and 400 nm; 0-20% phosphatidylserine) were designed to simulate exosome size and surface potential. Analyses showed that the liposomes were morphologically spherical and mimicked exosomes, with a polydispersity index of 0.07 to 0.02 that indicates negligible variation between batches. Zeta potential measurements indicated that the surface potential for each batch had the desired anionic strength of the particular liposome models. In addition, FRET measurements showed that the peptides were not just surface-localized, but insert into the lipid bilayer. BLI measurements indicate that cationic and aromatic amino acid residues play a significant role in lipid nanovesicle recognition and binding, with dissociation constants in the nanomolar range. The peptides show selection for liposome models that closely resemble exosomes in size and surface potential, while large liposomes and those that have neutral surface potential showed negligible binding. Going forward, Mr. Cook hopes to translate these findings into a useful method for isolating cancer-derived exosomes and comparing the capture efficiency of his peptide method with that of immunoaffinity methods.

Transcriptional Regulator Proteins in EVs: The Potential to Mediate Downstream Pathways

Michael Graner, Ph.D., Associate Professor, Department of Neurosurgery, University of Colorado School of Medicine, reported on an examination of his group’s previously published data, new (unpublished) data, and a publicly-available database combined with Gene Ontology (GO) applications to propose a novel concept: that EVs transport protein-based transcriptional and translational machinery that may have direct impacts on gene expression in recipient cells. His group examined the previously published proteomic contents of medulloblastoma-derived EVs, focusing on transcriptional regulators; they found that there are numerous proteins that may have potential roles in transcriptional and translational regulation with putative influence on downstream, cancer-related pathways. They included more of their own data from EVs of glioma cells and canine carcinoma cells, as well, with similar results. Then, they expanded the search to include all of the proteins in the Vesiclepedia database using GO approaches and found that these regulatory factors are implicated in many of the processes involved in cancer initiation and progression, including numerous cancer-related signaling pathways. This suggests, Dr. Graner said, that some of the effects of EVs on recipient cells may be due to the delivery of protein factors that can directly and fundamentally change the transcriptional landscape of the cells. The passage of these factors in a tumor setting, could, he said, clearly tilt the advantage toward the cancer cells.

EVs in Cancer: The Emerging Role of Large Oncosomes

Delores Di Vizio, M.D., Ph.D., Associate Professor and Research Scientist, Surgery, Cedars-Sinai, noted at the outset of her talk that the possibility that EVs preserve the stability of extracellular nucleic acid in the bloodstream is stimulating efforts to use EV fraction(s) in blood as a non-invasive source of personalized markers of disease aggressiveness, and as a means of following cancer progression and regression in real time. She then reported that her team had recently reported that the silencing of the gene encoding the cytoskeletal regulator Diaphanous related formin-3 (DIAPH3) in tumor cells results in a transition to a rapid migratory phenotype characterized by dynamic membrane perturbations and increased metastatic potential. In addition, the team discovered that DIAPH3 silencing results in the export of large (1-10 micrometer in diameter) bioactive EVs (large oncosomes) that originate from the shedding of bulky membrane protrusions from the plasma membrane. Dr. Di Vizio said that her group has demonstrated that the abundance of large oncosomes in the circulation and in tissue correlates ith advanced disease in mouse models and human subjects. Large-scale profile analyses (mass spectrometry, miRNA arrays, and next-generation sequencing reveal that large oncosomes represent a novel population of EVs, enriched in tumor-derived molecules. According to Dr. Di Vizio, DNA and RNA sequencing data of exosomes and large oncosomes suggest that large oncosomes represent valuable candidates that may permit new biomarker profiles to be developed using tissue- and blood-based assays in combination.

Non-Canonical Notch Signaling via ARMMS

Quan Lu, Ph.D., Associate Professor of Environmental Genetics and Pathophysiology, Harvard School of Public Health, began his presentation by noting that mammalian cells are capable of secreting nanoscale vesicles (50-200 nm) such as arrestin domain-containing protein 1 (ARRDC1)-mediated MVs (ARMMS) into extracellular space. ARMMS have the capacity to carry signaling molecules and thus may represent a new form of intercellular communication. Here, Dr. Lu reported that secreted ARMMS contain the Notch2 protein and mediate a non-canonical intercellular Notch signaling. Mass spectrometry of purified ARMMS from multiple types of mammalian cell lines identified Notch2 as a protein component of the vesicles. The Notch protein in ARMMS was not full length, but did contain the critical intercellular region (NICD) that is required for its transcriptional activity in the nucleus. Dr. Lu said that the incorporation of Notch into ARMMS is likely facilitated by the ITCH E3 ligase, which is also secreted into ARMMS. ARRDC1 overexpression increased the NICD incorporation into ARMMS without affecting the cellular Notch2 protein level. Conversely, CRISPR-mediated knockout of ARRDC1 dramatically reduced the amount of NICD in secreted ARMMS. Re-expression of ARDC1 in ARRDC1-knockout cells returned NICD in ARMMS to normal levels. Using luciferase-tagged Notch, Dr. Lu said his group showed NICD in ARMMS can be delivered into recipient cells to activate target gene expression. He concluded by stating that his group’s findings revealed non-canonical Notch signaling that is mediated by secreted MVs and is independent of direct cell-cell contact.

Profiling of Serum- and Plasma-Derived EVs miRNA and mRNA

Leonora Balaj, Ph.D., Research Fellow in Department of Neurology and Program in Neuroscience, Laboratory of Xandra Breakefield, Ph.D., Massachusetts General Hospital/Harvard Medical School, opened the last presentation Sunday evening by noting, as many others had, the potential power of liquid biopsies because they may permit the characterization of different conditions in a minimally invasive way. She further noted the caveat that disease-specific EVs are a minor component in a pool of normal cell-derived EVs, so detecting individual tumor markers is a major challenge. In addition, she said that there are major differences between the number of EVs and their cargos in different biofluids, so it is generally difficult to compare data among studies using different sources of biofluids, collected under different conditions. In her current work, Dr. Balaj and her colleagues sought to compare the mRNA and miRNA content of matched plasma and serum from healthy donors and characterize their EV RNA content. Many pathways become deregulated during tumor malignancies, Dr. Balaj said, and their differential presence in serum- or plasma-derived EVs c can reliably inform investigators as to how the primary tumor is evolving. For this purpose, Dr. Balaj’s group also compared the mRNA content of plasma-derived EVs from glioblastoma (GBM) patients and healthy controls. The analysis was performed using the miScript miRNA PCR Arrays (Qiagen) for serum and plasma comparisons, and the RT2 Profiler™ PCR array Human Cancer Pathway Finder™ for the comparison of mRNA profiles in the plasma of GBM patients and normal controls. The data was analyzed using PCR Array Data Analysis Software (Qiagen). The pathways included in the Human Cancer Pathway Finder™ array (Qiagen) contain genes from nine different pathways commonly involved in carcinogenesis, including apoptosis, DNA damage repair, angiogenesis, metabolism, and epithelial-to-mesenchymal transition. Dr. Balaj reported finding mRNA and miRNA signature differences between plasma and serum samples from healthy controls, as well as different signatures in plasma EV mRNA from GBM patients and normal controls. She said that the mRNA and miRNA signatures provide important information on physiological an pathological changes that are reflected in these biofluid EVs.

Breakefield, Not Exosome, Trafficks Tasty Cargo to Surprised Recipient

As Dr. Balaj closed her presentation, the irrepressible session moderator Dr. Michael Graner led the packed audience in a rousing chorus of “Happy Birthday” and Dr. Balaj’s mentor, Dr. Xandra Breakefield, strode forward, precariously balancing an enormous white birthday cake in her hands. It suddenly dawned on everyone in the erudite crowd that this was actually Dr. Balaj’s birithday. “We do this in the lab,” Dr. Breakefield said, “so why not here?” Why not, indeed? Dr. Balaj was clearly surprised and very moved, and the audience was particularly delighted as the cake was quite large enough for everyone to have a piece. It was a wonderful end to another fantastic day of science and community for the ASEMV.

MONDAY

Exosomes: From Tissue to Liquid Biopsy

Johan Skog, Ph.D., Chief Scientific Officer and Founding Scientist, Exosome Diagnostics, Inc., gave the opening presentation on Monday morning, the final day of the ASEMV meeting. He began by noting that biofluid molecular diagnostics have generated significant interest recently, particularly with the new emerging platforms that enable low-abundance tumor tanscripts and mutations to be detected even from biobanked plasma and urine. Using biofluids as a surrogate for tissue to monitor the progression of disease processes is highly desirable for diseases like cancer, where the genetic status of biopsies taken prior to treatment may not be relevant later in the disease. He noted that RNA can be found in different compartments of the biofluids. Some are protected within vesicles, allows for recovery of high-quality nucleic acid for analysis of tumor-derived mutations, splice-variants, and enables RNA profiling to stratify patients and monitor treatment efficacy on longitudinal samples. while others are found within lipid or protein complexes. These fractions are released from cells by different mechanisms and have vastly different RNA profiles. To harness these benefits, however, Dr. Skog emphasized that it is imperative to develop optimal standard operating procedures for exosome collection and processing. He noted at the outset that it is important to appreciate that exosome results are usually based on averages of billions of vesicles. The approach to pre-processing is critical, Dr. Skog said, noting that low g spin and filtration can produce highly variable and skewed data. There is an urgent need to develop standard operating procedures, he maintained, as did many others throughout the conference. He noted that the pelleting efficiencies of different rotors can be very different and need to be taken into account. While most blood collection tubes are compatible with exosome extraction, they all have different biases and these also must be considered. He also said that exosome workers should always include a control for DNA as small amounts of RNA are easily biased by the presence of DNA. He mentioned that for exosome isolation, Exosome Diagnostics had found the exoRNeasy Serum/Plasma Maxi Kit (Qiagen) to be “nice and scalable.” He added that high volumes of biofluids are required for high-sensitivity applications. In closing, he cited data obtained by Exosome Diagnostics in exosome RNA profiling to monitor the treatment response in a brain cancer patient. Dr. Skog said that unique expression changes were identified in responders, but not in non-responders. In particular, a decline in miR-10b was seen in responders, but not in non-responders. It is this kind of work, to advance the utility of liquid biopsies in cancer diagnosis and the monitoring of treatment effectiveness, that Exosome Diagnostics is focused on.

Human Mesenchymal Stem Cell-Derived MVs Modulate T-Cell Response to Islet Antigen Glutamic Acid Decarboxylase in Patients with Type 1 Diabetes

Maria Zanone, M.D., Ph.D., Department of Internal Medicine, University of Turin, Italy, began her presentation by noting that mesenchymal stem cells (MSCs) have been shown to potentially abrogate, in vitro, the proinflammatory response in type 1 diabetes, which is associated with several abnormalities of T-cell immunophenotype and function. The mechanism involves paracrine factors, which may include MVs. Dr. Zanone reported that her group evaluated whether MVs derived from heterologous bone-marrow MSCs exert an immunomodulatory effect on T-cell responses against the islet antigen glutamic acid decarboxylase (GAD) in type 1 diabetes. MVs were internalized by peripheral blood mononuclear cells (PBMCs), as assessed by confocal microscopy and flow cytometry analyses. MVs significantly decreased IFN-gamma spots and levels in GAD65-stimulated PBMCs, and significantly increased transforming growth factor-beta (TGF-), IL-10, IL-6, and PGE2 levels. In addition, MVs significantly decreased the proportion of Th17 cells and the levels of IL-17, and increased FoxP3+ regulatory T-cells in GAD65-stimulated PBMCs. Furthermore, Dr. Zanone noted, levels of miR-21, known to enhance TGF- signaling, were increased in stimulated PBMCs in the presence of MVs. Blockade of MV internalization or pre-treatment of MVs with RNase reduced the levels of IL-10 and TGF-1 transcripts in treated PBMCs. Dr. Zanone concluded that these results indicate that MSC-derived MVs mimic the immunomodulatory effects of MSCs and can inhibit, in vitro, a proinflammatory to an islet antigenic stimulus in type 1 diabetes. The action of MVs, she said, depends, at least in part, on their internalization and on the transfer of mRNA or miRNA, and involves PGE2, TGF-b signaling pathways and IL-10 secretion, suggesting a switch to an anti-inflammatory response of T-cells.

Luminal EVs in Patients with Inflammatory Bowel Disease

Masato Mitsuhashi, M.D., Ph.D., Chief Technology Officer, NanoSomiX, Irvine, California; previously Chief Scientific Officer, Hitachi Chemical Research Center, Los Angeles, California, prefaced his discussion by remarking that Crohn’s disease (CD) and ulcerative colitis (UC) are the two main forms of inflammatory bowel disease (IBD), and that there is an unmet need for non-invasive biomarkers of disease activity for these conditions. Toward this end, Dr. Mitsuhashi’s group sought to determine if EVs obtained from the intestinal lumen of patients with active inflammation show differential gene expression patterns compared to those from healthy controls. Intestinal luminal fluid (ILF) was obtained from 11 healthy controls, 13 patients with CD, and 21 patients with UC. Isolated EVs had a mean size of 150 nm (range: 50-400 nm) at a concentration of 5 X 1011 particles/mL. Expression of mRNA for epithelial defense genes (EPCAM, MUC2, TFF1, DEFA3) and leukocyte activation markers (CD45, TGFB1, S100A9) was significantly different between EVs from healthy controls and those from IBD patients. Using multivariate discriminant analysis (Minitab), mRNA levels were capable of distinguishing healthy controls from UC patients (83% predictability) and from CD (75% predictability). To determine if these EV mRNAs in fecal samples could be detected non-invasively, the group also quantified EV mRNAs in fecal samples, and confirmed that EV mRNA was present in both ILF and stool supernatant, but naked mRNA was not detected, presumably due to digestion by endogenous ribonucleases. In conclusion, Dr. Mitsuhashi said that EVs can be isolated from luminal fluid of patients with IBD, with different gene expression patterns relative to those from healthy controls. The mRNA levels in these EVs associate with the degree of inflammation. Additional studies to evaluate EVs as potential biomarkers of inflammation in IBD warranted. Relevant references cited by Dr. Mitsuhashi include the following: “Development of Glomerulus-, Tubule-, and Collecting Duct-Specific mRNA Assay in Human Urinary Exosomes and Microvesicles,” PLOS ONE 9(10): e109074 (October 2, 2014) and “Posttransplantation Bone Marrow Assessment by Quantifying Hematopoietic Cell–Derived mRNAs in Plasma Exosomes/Microvesicles,” Clinical Chemistry 60(4):675-687 (April 2014).

EVs Secreted During Human Myoblast Differentiation Regulate Myotube Formation of Human Adipose-Derived Stem Cells

Young Chan Choi, Ph.D. Candidate, Hanyang University, South Korea, first reminded everyone that skeletal muscle is a secretory organ that plays a part in energy homeostasis and whole-body metabolism, as well as locomotion. During skeletal muscle development, the skeletal muscle secretes exosomes containing signaling molecules and the inter-cellular cross-talk between skeletal muscle cells is mediated by the exosome transfer. Skeletal muscle-derived exosomes have been identified in recent profiling studies, but key issues as to how these exosomes regulate skeletal muscle development remain unresolved. In this study, Mr. Chan and colleagues investigated whether or not exosomes secreted during human myoblast (hSkM) differentiation can trigger the differentiation of human adipose-derived stem cells (hASCs) toward a myoblast phenotype. The isolated exosomes were approximately 40-100 nm in diameter and expressed exosomal markers such as Alix, TSG101, and CD63. The exosomes also contained various proteins and soluble factors. The exosome-treated hASCs fused with neighboring cells and exhibited the myotube-like phenotype with increased expression of myogen, myosin heavy chain, and desmin. Dr. Choi said that these results suggest that hSkM-derived exosomes can act as a biochemical cue for control of hASC fate toward myogenic lineage.

EV Biogenesis During Neuronal Differentiation of Mouse Embryonic Stem Cells from Ultrastructural Analysis to Cargo Protein Identification

Lilian Cruz, Ph.D., Department of Cell and Developmental Biology, Biomedical Science Institute, University of Sao Paulo, Brazil, noted the EVs have gained increasing importance for their possible role(s) during embryonic development. In the context of development, she said, embryonic stem cells are considered a model for the molecular study of events that drive stemness maintenance and cell commitment. Consequently, to better understand the possible role(s) of EVs in embryonic development, Dr. Cruz and her colleagues decided to investigate how EVs might be able to modulate the neuronal differentiation of mouse embryonic stem cells (mESCs). Her group first demonstrated that mESCs (E14Tg2A cell line) do contain structures like multivesicular bodies, with numerous intraluminal vesicles and several membrane events related to EV release/uptake. In addition, the scientists showed that expression of LBPA (lysobisphosphatidic acid), a lipid marker of intraluminal vesicles, concentrated in specific regions of the cytoplasm of several stem cells, but not all. The Cruz group also demonstrated that mESCs secrete vesicles of different sizes that express flotillin and Hsp90, and also contain a co-chaperone protein known as stress-inducible protein 1 (STI1). STI1, when engaged to normal prion protein (PrPc) has been reported to play a critical role neural progenitor/stem cells self-renewal, leading to the control of the stemness capacity of these cells during neural system development. The presence of STI1 in mESCs indicates, Dr. Cruz said, that secretable STI1 takes part of the embryonic stem cells niche and could modulate the biology of these cells. In conclusion, she believes that the study of EVs and their components could contribute to an elucidation of the importance of the microenvironment to stemness maintenance and also to a differentiation state.

WNT Cutter (Wnc) Is Required for Cleavage of Exosomal Wingless (Wg) and Enhances Wg Signaling

Go-Woon Kim, Ph.D., Center for Theragnosis, Korea Institute of Science and Technology, South Korea; Department of Biological Sciences, Korea Advanced Institute of Science and Technology, South Korea, began her talk by reminding everyone that Wnt/Wingless (Wg) proteins are secreted morphogens that are essential for embryonic development and adult homeostasis in all metazoans. Nevertheless, the mechanisms for Wnt secretion and the composition of extracellular Wnt remain largely unknown. Dr. Kim and her group decided to further investigate these current mysteries and reported some interesting results. First of all, the group showed that a Drosophila ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) named Wnt cutter (Wnc) promotes Wg signaling by generating an active form of soluble extracellular Wg. By immunohistochemistry of larval imaginal wing discs with several markers of endosome and exosomes, the scientist showed Wnc and Wg specifically co-localized in early endosomes and exosomes. Also, the enrichments of active Wnc and Wg on exosomes were revealed by western blotting of exosome pellets through serial centrifugation of Drosophila S2 wnc-HA and S2 GFP-wg cells cultured media. Interestingly, Dr. Kim noted, according to western blotting results, it appeared that Wg-cleaved fragments were generated on exosomes only in the presence of protease-active Wnc, demonstrating that Wnc’s protease activity is essential for cleaving Wg. The cleavage occurred in the linker region of Wg, by which the C-terminal domain (CTD) is released from the membrane-bound N-terminal CTD has full Wnt activity and that both of Wg and Wnt co-expressed larva of Drosophila showed increased Wg signaling phenotype,Dr. Kim and her colleagues propose that Wnc enhances Wg signaling by generating the free CTD form of Wg.

Osteoclast-Derived Exosomes Regulate Calcium-Stimulated Osteoclast Differentiation in Vitro

Shannon Holliday, Ph.D., Associate Professor, Orthodontics, Biochemistry and Molecular Biology, University of Florida, began by noting that osteoporosis costs the United States approximately $20 billion each year and that the disease involves an imbalance between the formation and resorption of bone. Most current treatments are aimed at reducing the loss of bone by inhibiting resorption rather than by efforts to actually fix the damage. Osteoclasts are cells that are specialized to perform this bone resorptive function. Osteoblasts, on the other hand, are cells that work together to synthesize bone. Together, osteoclast and osteoblasts control the amount of bone tissue. Dr. Holliday and his colleagues hypothesized that osteoclast-derived exosomes might be involved in the regulation of bone remodeling. As an initial test of this hypothesis, he said that the scientists isolated exosomes from osteoclast-like cells in culture and applied them to calcitrol-stimulated mouse marrow cultures to determine to determine their effects on osteoclast differentiation. Osteoclast-like cells were produced by stimulating RAW 264.7 cells with recombinant RANKL. Exosomes were isolated using ExoQuick™, negative-stained, and visualized by TEM. Exosomes (1 microgram/mL), from unstimulated RAW 264.7 cells or RANKL-stimulated RAW 264.7 cells, were applied to calcitrol-stimulated mouse marrow cultures during a six-day incubation. Osteoclasts were detected by staining for tartrate-resistant acid phosphatase (TRAP) activity. ANOVA and T-tests were used to analyze data. TEM results revealed that the vesicles isolated using ExoQuick™ were fairly homogeneous with an average diameter of approximately 40 nm. TRAP staining indicate that there were significantly fewer giant cells produced by mouse marrow that had been treated with exosomes from RANKL-stimulated RAW 264.7 osteoclast-like cells. In contrast, exosomes from unstimulated RAW 264.7 cells enhanced osteoclast formation by more than two-fold compared with controls. The data suggest, Dr. Holliday said, the hypothesis that exosomes may regulate the response of bone cells to a hormonal signal for increased osteoclastic bone resorption to release calcium to respond to a systemic need. Exosomes from mature osteoclast-like cells (which are already capable of resorbing bone) reduced new calcitrol-stimulated osteoclast formation. Exosomes from osteoclast precursors triggered increased formation of osteoclasts in otherwise identical marrow cultures. These data are consistent, Dr. Holliday said, with the exosomes derived from osteoclasts and their precursors being vital regulators of bone resorption, capable of fine-tuning the local resorptive response to a hormonal signal.

Presence of Oncogenic Genomic DNA Sequences in EVs

Elisa Lazaro-Ibanez, Ph.D. Student, Division of Biopharmaceutics and Pharmacokinetics, University of Helsinki, Finland, introduced her presentation by stating that prostate cancer (PCa) is the most frequent malignancy in men in developed countries and the sixth leading cause of cancer mortality worldwide. EVs have been suggested as a source of more specific and sensitive biomarkers for PCa. The goal of her current study was first to determine if different PCa EV subpopulations (apoptotic bodies, MVs, and exosomes) carry double-stranded genomic DNA (gDNA) fragments and then to assess whether these fragments encode specific regions that could be mutated in PCa. In the work, EV subpopulations were isolated from PCa cell lines, the plasma of PCa patients, and healthy donors by serial centrifugations. EVs were characterized by TEM, nanoparticle tracking analysis (NTA), western blotting, and total protein content. Primers specifically targeting human gDNA mutations of TP53, PTEN, and MLH1 genes were designed, and DNA fragments were detected by real-time quantitative PCR. Ms. Lazaro-Ibanez said that double-stranded gDNA was found in all cell-derived and plasma-derived EV types. Analysis of gDNA fragments of MLH1, PTEN, and TP53 genes from the PCa cell line-derived EV subpopulations showed that different EVs carry different gDNA content, which also harbored specific mutations. EV sub populations also differed from each other in terms of protein content and particle number. Although the particle concentration of MVs and exosomes were similar by NTA, the total protein content was significantly different in both populations. Particle concentration and total protein content correlated with each other for some, but not all PCa cell-derived MVs and exosomes. Finally, with respect to the plasma samples, the concentration of EVs was higher in the PCa patients than in the healthy donors. Based on these results, Ms. Lazaro-Ibanez concluded that EVs derived from PCa cell lines and human plasma samples contain double-stranded gDNA fragment that may be used to detect specific mutations, making the EVs potential biomarkers for cancer diagnostics and prognostics. Future work will focus on improving EV isolation and characterizing total EV DNA content by next-generation sequencing. Ms. Lazaro-Ibanez was the lead author on the following recent article on the possible role of EVs in the diagnosis of prostate cancer: “Different gDNA Content in the Subpopulations of Prostate Cancer Extracellular Vesicles: Apoptotic Bodies, Microvesicles, and Exosomes,” published in the October 2014 issue of The Prostate [74(14):1379-1390, October 2014].(http://onlinelibrary.wiley.com/enhanced/doi/10.1002/pros.22853/)

Intercellular Exosome Transfer in HGF-Induced Invasive Epithelial Growth

Sang-Ho Kwon, Ph.D., Associate Specialist, Department of Anatomy, UC San Francisco, began his presentation by noting that hepatocyte growth factor (HGF) plays a crucial role in invasive epithelial growth, tubulogenesis, and metastasis. In expression profiling of HGF-driven in vitro tubule formation, his group found that a significant fraction of temporally regulated genes were classified as secretory proteins expressed in exosomes. Dr. Kwon reported here that GPRC5B, an orphan G-protein-coupled receptor, is localized in exosomes produced by HGF-treated cysts, and released in the lumen of cysts through a Rab35-dependent pathway. Exosomal GPRC5B is taken up by nearby cells and, in combination with HGF, boosts ERK1/2 activation, thereby stimulating tubulogenesis, even under conditions where tubulogenesis would otherwise not occur. Exogenously added GPRC5B in exosomes augmented migration of recipient cells in an in vitro wound-healing assay. Significantly, he said, his team found that GPRC5B is elevated in urinary exosomes from patients with acute kidney injury. In conclusion, Dr. Kwon said that their finding of the unexpected role of GPRC5B-containing exosomes in epithelial morphogenesis and wound recovery represents an important step in understanding and improving the limited regenerative capacity of the kidney, such as after acute injury. Dr. Kwon referenced a recent article on this topic: “Intercellular Transfer of GPRC5B via Exosomes Drives HGF-Mediated Outward Growth,” Current Biology 24(2): 199-2014 January 20, 2014. [http://www.cell.com/current-biology/abstract/S0960-9822%2813%2901529-7].

Exosome Reduction in Vivo Is Associated with Lower Amyloid Plaque Load in the 5FAD Mouse Model of Alzheimer’s Disease

Michael Dinkins, Ph.D., Post-Doctoral Fellow, Department of Neuroscience and Regenerative Medicine, Georgia Regents University, gave the final oral presentation of the ASEMV 2014 Annual Meeting, and remarked at the opening that his group had previously reported that cultured astrocytes secrete exosomes in response to challenge with amyloid beta (Abeta), the peptide that is hypothesized to be a causative agent of Alheimer’s disease. Here, Dr. Dinkins reported that more recent work has indicated that exosomes stimulate aggregation of Abeta1-42 in vitro and in vivo and interfere with uptake of Abeta by primary cultured astrocytes and microglia in vitro. Exosome secretion is prevented by the inhibition of neutral sphingomyelinase 2 (nSMase2), a key regulatory enzyme generating ceramide from sphingomyelin, with GW4869. Using the 5XFAD mouse, the Dinkins team showed that intraperitoneal injection of GW4869 reduces the levels of brain and serum exosomes, brain ceramide, and Abeta1-42 plaque load. Reduction of total Abeta1-42, as well as number of plaques in brain sections, was significantly greater (40% reduction) in male versus female mice. Dr. Dinkins concluded that these results suggest that GW4869 reduces amyloid plaque formation in vivo by preventing exosome secretion and identify nSMase2 as a potential drug target in AD by interfering with exosome secretion.

Close of ASEMV 2014 Annual Meeting

At the end of Dr. Dinkins’ talk, ASEMV President Dr. Stephen Gould stepped forward and declared the year’s annual meeting officially closed and bade all the attendees a safe trip home.

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AUTHOR. This article was written by Michael D. O’Neill, who is a freelance science writer. You may contact him at logophile2000@yahoo.com. Mr. O’Neill is also editor and publisher of BioQuick™ Online News, an award-winning bionews web site located at http://www.bioquicknews.com. BioQuick has readers in over 160 countries and includes a Japanese-language edition. BioQuick Online News has won a number of publication excellence awards and currently includes over 1,900 articles on major life science advances. All these articles are readily accessible by means of a powerful search engine. Mr. O’Neill holds an M.A. degree in Immunology & Microbiology from Duke University and worked for 15 years as a science writer for Applied Biosystems.